Sinh Hoc Phan Tu

SINH HOC PHN T
LNG PHM NGC LM
Bi 1. SAO CHP DNA

1. Phn bi !"# $h%&' &hi(n )* + ,i-i )*. S"# $h%& C. $h/ 01 23 S-n &h4) 53$h h67$ !-n &h4) S8 96:n, &hn ; $#n <# 2" !"= )> 9?n !"# $h%&' &hi(n )*' ,i-i )* @nAB) $h3nh h") ,i" 2#n, C D=E 2Fnh N,=B(n 9i= Nhn i Nhn ADN (con Di b%ng gen m& Phi(n )* Truyn thng tin di truyn t ADN n ARN Nhn !"c lo#i ARN mARN ' gen Gi-i )* Truyn thng tin di truyn t mARN n polypeptide T bo cht $olypeptide $olypeptide ng(n h)n mARN /0 polypeptide b%ng 10 ribo1om tr23t tr4n , 13i -Amino5cyl tARN 1ynthet51e -$eptidyl tr5n16er51e
T#o * phn t+ ADN con
T , gen -.,mARN
ADN polymer51e
ARN polymer51e
7 lo#i dNT$8 AT$9 7 lo#i dNT$8 AT$9 Acid 5min TT$9 :T$9 !T$ ;T$9 :T$9 !T$ -$ro<5ryote8 - BC8 D55?giy =>> b51e?giy $ro<5ryote8 *A T8$ G> - Ci <huEn8 -@u<5ryote8 mARN?giy *>55?giy A> b51e?giy H. IJ$ GiK) !i(= L#En + M*n L#Nn O ADN +i Ph=4n. - /i4u Fo(n8 G Trong tH nhi4n9 phn t+ ADN 23c Ing gIi rt chJt K d#ng 1i4u Fo(nL G ADN MNng MOn K d#ng 1i4u Fo(n <hi 23c chit t"ch M tinh 1#ch -. hiPn t23ng 1i4u Fo(n l tr#ng th"i tH nhi4n cQ5 ADNL G Nghi4n cRu hiPn t23ng 1i4u Fo(n nhS topology (ngnh <ho5 hTc nghi4n cRu tUnh cht Vn Wnh cQ5 0i t23ng lun bin Vi hXnh d#ng cho phYp hiZu bit 1u h)n M cu tr[c M chRc n\ng ADNL G ADN MNng th5y Vi tUnh cht topology <hi cI 1H Rt hoJc n0i , hoJc c] * 13i ADNL - D^n Fo(n8 G $h_n l`n ADN t bo K d#ng d^n Fo(n chR5 10 Fo(n Ut h)n 1o Mì d#ng d^n <hng t#o Fo(n rel5Fed K d#ng cu tr[c ADN Fo(n aL G C nguy4n t(c9 , Fo(n bW gb 1c t"ch <ho]ng ,> bp9 Jc biPt thZ hiPn K 13i ADN di Mì mRc d gb Fo(n c5oL G D#ng gb Fo(n cI t"c deng8 df dng cho 1H Ing gIi K d#ng 1i4u Fo(n t"ch 13i phec Me gu" trXnh phi4n m^ G !hh Vn Wnh K d#ng ADN MNng h5y 23c g(n Mì protein gii cho c"c 13i <hng tH do Fo5y gu5nh nh5u
G j"c Wnh b%ng chh 10 li4n <t (k< 8 l 10 l_n mdt 13i ADN no I Fuy4n gu5 mJt phlng cQ5 13i <i59 l 10 nguy4nL G jo(n hXnh thnh K d#ng gu5y ph]i thX chh 10 li4n <t l d2)ng (G 9 ng23c l#i l m (- L !hh 10 Fo(n m <hng phV binL G m5i d#ng ADN MNng <h"c nh5u chh 10 k< 23c gTi l topoi1omerLG D#ng ADN gb Fo(n gi[p t"ch 13i M hXnh thnh nhing o#n ng(n ADN d#ng n gu5y tr"i (d#ng a gu5y ph]i C. ChQ$ nRn, !inh hS$ + GiT= 21 $U" T#&#i!#)V2"!V. - !hRc n\ng 1inh hTc8 G Duy trX M Vn Wnh tr#ng th"i 1i4u Fo(nL G B5 10 c) thZ 10ng (tr Mi <huEn chWu nhiPt M Arch5e5 9 ADN K d#ng 1i4u Fo(n mL G o Mi 1inh Mpt9 mRc d 1i4u Fo(n m 23c iu hN5 bq tr bKi ADN gyr51e M topoi1omer51e rL G o Mi <huEn chWu nhiPt M Arch5e59 reMer1e gyr51e t#o 1i4u Fo(n d2)ng b]o MP ADN <hng bW bin tUnh K nhiPt d c5oL - $hec Me iu trW8 G Topoi1omer51e l 0i t23ng t"c dng cQ5 thu0c chi5 bPnhL G ADN gyr51e M topoi1omer51e rC l 0i t23ng t"c dng cQ5 * nhIm thu0c <h"ng Mi <huEn G Topoi1omer51e r M rr l 0i t23ng t"c dng cQ5 nhiu lo#i thu0c chi5 ung th2L G Thu0c coum5rin l 1]n phEm tH nhi4n cQ5 nm Streptomyces Rc ch c#nh tr5nh Mì AT$ g(n l4n gyr51e M topoi1omer51e rCL G Thu0c guinolone l thu0c tVng h3p cI t"c dng b5o My phRc ensyme Mì ADN ng\n <hng cho ensyme gb Fo(n M phRc 15o chYp di chuyZn tr4n ADN9 gy Rt 13i i ADN M gy cht t boL G Thu0c ch0ng ung th28 Thu0c t"c dng l4n topoi1omer51e Rc ch ph"t triZn t bo n4n cI thZ 1+ deng cho mec Uch iu trW bPnh ung th2L tuinolone t#o li4n <t hI5 trW hoJc n%m Fen c"c g0c cQ5 ANDL W. 0F !"# $E$ b"!V ATGC $X P3$h h67$ PhE$ nh"= nh6n, &hn ; ADN +Yn $X hK Z GiT= $h[nh $\= hFnh G8i LQn, D=" 2]$. - m5i m#ch )n <t h3p Mì nh5u nhS c"c li4n <t hydro hXnh thnh gii5 c"c b51e bV 1ung n%m tr4n h5i m#ch8 A bV 1ung Mì T M ! bV 1ung Mì :L - uUch th2`c phn t+ cQ5 A . T M :. ! n4n bq tr cho nh5uL ^. Nh_ +# G= Phi !"# $h%& ADN <# 2" h"i )<$h )7i ,i8n, )<$h ,8$. - uhi 15o chYp9 h5i m#ch cQ5 phn t+ ADN 23c t"ch r5 do c"c li4n <t hydro gii5 c"c cJp b51e bW Rt9 mvi m#ch lm <hun c_n thit cho MiPc tVng h3p m#ch cJp mì t2)ng tH Mì m#ch cJp tr2`c IL - CX A ph]i b(t cJp Mì T M : ph]i lun b(t cJp Mì ! n4n trXnh tH c"c nucleotide tr4n mdt m#ch 1c F"c Wnh chUnh F"c trXnh tH Jc hiPu c"c nucleotide tr4n m#ch bV 1ung Mì nIL /5u I n0i c"c nucleotide mì l#i thnh m#ch mì bV 1ungL - Do I9 mdt phn t+ ADN b5n _u t#o r5 h5i phn t+ con gi0ng hPt nh5uL `. T3nh 2<n, Mi 2=BTn G6:$ D=" ADN' AaN h"B &2# Vinb Gi-i h3$h. TUnh tr#ng di truyn 23c gu5 ADN h5y ARN (0i Mì Miru1 MX8 - Theo hTc thuyt trung tm t5 bit wThng tin <hi ^ chuyZn 15ng protein thX <hng thZ ly r5 l#i 23c ni5xL Nghy5 l thng tin <hng thZ chuyZn ng23c t protein n 5cid nucleicL Thng tin K y l trXnh tH chUnh F"c c"c nucleotide cQ5 5cid nucleic gui Wnh trXnh tH 5cid 5min cQ5 proteinL Do I9 protein <hng gi[p tUnh tr#ng di truyn 23cL - Thng tin cI thZ chuyZn gii5 c"c phn t+ ADN thng gu5 1H 15o chYp9 hoJc gii5 phn t+ ADN M ARN thng gu5 1H phi4n m^9 hoJc t 5cid nucleic n protein nhS 1H dWch m^L - Ngoi r5 thng tin cNn cI thZ di chuyZn gii5 ARN n ARN trong gu" trXnh 1inh 1]n cQ5 c"c ARN Miru1 h5y gu" trXnh F+ lz c(t n0i cQ5 ARN hoJc ng23c l#i t ARN n ADN trong gu" trXnh phi4n m^ ng23c cQ5 retroMiru1L c. ChQn, )inh $. $h/ )inh hS" !"# $h%& ADN D=" h3 n,hi) MV!V9!#n + S "h9 9 $. $h/ bEn b-# hU. - Ci <huEn 23c cho ph"t triZn Mi th hP trong mi tr2Sng {ng MW nit) nJng (N,A Z ]m b]o cho tt c] ADN Mi <huZn u l lo#i nJngL - /5u I9 chuyZn t bo Mo mi tr2Sng chR5 nit) nh& N,79 nh2 l ngu{n cung cp nit) duy nht M Z cho phn chi5 trong mi tr2Sng N,7L
uhi <h0i l23ng ADN t\ng l4n gp i9 ADN 23c chit r5 <h|i t bo M 23c ly tm tr4n th5ng n{ng d ce1ium clorid8G Nu c) ch b]o t{n 23c thHc hiPn9 * b\ng ADN ph]i 23c thy r} 15u <hi ly tm8 mdt b\ng nguy4n thQy (N,A M mdt b\ng chR5 N,7 G Nh2ng <t gu] thu 23c chh cI mdt b\ng cI t~ trTng trung gi5n chR5 N,79A Fut hiPnL Tip tec9 em ADN l5i un nIng r{i lm ngudi nh5nh Z t"ch * m#ch9 ly tm 1c thy * b\ng t2)ng Rng Mì ADN N,A M N,7L Do I c) ch 15o chYp ADN theo c) ch b"n b]o t{n8 * m#ch cQ5 phn t+ AND m& lm <hun Z tVng h3p n4n m#ch con bV 1ung9 phn t+ AND con cI , 13i N,A M , 13i N,7L
d. CE$ B/= 8 $?n hi/ $h# !"# $h%& ADN O E.coli - uhun mOu (phn t+ b5n _u 8 !"c 13i AND t"ch r58 G /3i AND cI h2`ng -.A 1c 23c 15o chYp trHc tip bKi ADN-polymer51e rrr t#o r5 13i con bV 1ung gTi l 13i 1`mL G /3i AND cI h2`ng A-. <hng 23c 15o chYp li4n tec t#o r5 b]n 15o gTi l 13i mudnL - @nsyme ADN-polymer51e8 u cI ho#t tUnh polymer hI5 A-.9 g{m lo#i8 ADN-polymer51e rrr l ensyme 15o chYp chUnh cQ5 cQ5 E.coli8 G g{m D )n MW nh|8 9 9 9 9 9 M 9 c_n cho 1H 15o chYp ADN G 25 nhiPt n4n <hng 15o chYp 23c K nhiPt d 7*>!L G cI ho#t tUnh eFonucle51e -.A M A-. ADN-polymer51e r8 G jI5 m{i M l(p <Un o#n m{i bW c(t G /+5 chi5 c"c ADN h2 h|ng G !I ho#t tUnh eFonucle51e -.A M A-. ADN-polymer51e rr8 ch25 r}9 chh cI ho#t tUnh eFonucle51e -.A Ngoi r5 cI c"c ensyme <h"c8 $rim51e8 TVng h3p m{i ARN melic51e8 Th5m gi5 t"ch m#ch t#o ch#c b5 15o chYpL melic51e 1+ deng n\ng l23ng t thQy phn AT$ Z c(t Rt li4n <t hydroL :{m cI * lo#i8 Rep protein8 :(n Mo AND g0c tVng h3p 13i 1`m M di chuyZn theo h2`ng -A Z th"o Fo(n melic51e thR h5i8 :(n Mo AND g0c tVng h3p 13i mudn M th"o Fo(n Topoi1omer51e8 F[c t"c th"o Fo(n AND g{m * lo#i8 Topoi1omer51e r8 th"o d#ng 1i4u Fo(n b%ng c"ch g(n Mo phn t+ AND M c(t , trong * m#ch Z th"o Fo(n 15u I n0i l#iL Topoi1omer51e rr8 T#i MW trU "nh du bKi protein a9 gyr51e 1c t"c dng9 c(t AND lm th"o Fo(n r5 * phU5 protein aL :yr51e c(t t#m thSi * chuvi cQ5 AND t#o r5 c"c d#ng 1i4u Fo(n mL kyg51e8 N0i c"c o#n <5s5<i 15u <hi th5y th c"c o#n m{i ARN b%ng c"ch F[c t"c hXnh thnh li4n <t pho1phodie1ter gii5 c"c polynucleotideL !"c protein <h"c th5m gi5 15o chYp8 $rotein a8 Nhpn bit iZm <hKi 1H 15o chYp ri N-protein8 T#o phRc h3p Mì prim51e Z t#o thnh primo1ome chRc n\ngL N-protein nhpn diPn iZm ri M cho phYp prim51e ho#t dng //a-protein8 :(n Mo AND 13i )n Z gii cho c"c 13i AND <hng chpp l#i Mì nh5u Z MiPc 15o chYp difn r5 df dngL - 7 lo#i deoFyribonucleotide tripho1ph5t (dNT$ 8AT$9 :T$9 !T$ M TT$L DeoFyribonucleotide tripho1ph5t g(n Mo _u -m cQ5 chuvi AND 5ng 15o chYp M gi]i phIng pyropho1ph5tL $yropho1ph5t thQy phn thnh pho1ph5t M c)L - g*G c_n thit cho ho#t dng cQ5 ADN-polymer51eL e. IJ$ GiK) $U" ADNf&#9B)V2"!V O E.coli +T h#< 3nh &#9B)V2 hX" ^gfhCg + h#< 3nh VL#n=$9V"!Vb - E.coli cI lo#i ADN-polymer51e r9 rr M rrrL P#9B)V2"!V I II III P#9B)V2 hX" ^gfhCg i i i
H#< 3nh VL#n=$9V"!V ^gfhCg i f i Cgfh^g i i i mo#t tUnh polymer hI5 A-.8 th4m mdt nu t , deoFyribonucleotide-A- tripho1ph5t Mo nhIm hydroFy cQ5 chuvi ADN mo#t tUnh eFonucle51e8 thQy phn m#ch )n ADN cQ5 chuvi t _u theo h2`ng -.A (ho#t tUnh eFonucle51e -.A h5y t _u A (ho#t tUnh eFonucle51e A-. 3nh &#9B)V2 hX" ^gf p Nhn /5o chYp ADN nhifm 1(c thZ
1j. IJ$ GiK) $U" ADN &#9B)V2"!V $U" / b# nhn hk +T +1 23 + h#< hCg. - o nhn thpt cI A lo#i ADN-polymer51e8 9 9 9 M L P#9B)V2"!V l m n o 01 23 Nhn Nhn Ty thZ Nhn H#< 3nh /5o chYp /+5 chi5 /5o chYp /5o chYp &#9B)V2 ADN nhifm ADN ty thZ ADN nhifm hX" ^gfhCg 1(c thZ (13i 1(c thZ (13i mudn 1`m S# !Enh ADNf&#9B)"2V!V $U" / b# nhn hk + nhn n,=B(n hUBq - :i0ng nh5u8 GcI cu tr[c 5 phn t+ - uh"c nh5u T/ b# nhn n,=B(n hUB cI lo#i ADN-polymer51e8 r9 rr M rrr8 - AND-polymer51e r8 ho#t tUnh polymer hI5 A-M eFonucle51e -.A M A-. - AND-polymer51e rr8 ho#t tUnh polymer hI5 A-M eFonucle51e -.A - AND-polymer51e rrr8 ho#t tUnh polymer hI5 A-M eFonucle51e -.A M A-.
trong t bo cht 11. IiK) PhOi G?= !"# $h%& ADN. - /H tVng h3p ADN cQ5 N/T Mi <huEn lun lun b(t _u K cqng mdt iZm 23c gTi l MW trU rigin h5y ri9 g{m H^W cJp b51eL - CU de8 K E.coli9 gu" trXnh 15o chYp b(t _u <hi protein a nhpn bit 23c iZm <hKi 1H 15o chYp ri!L 1H. SZ !"# $h%& ADN O E.coli. :{m b2`c8 - $rotein a nhpn bit 23c iZm <hKi 1H 15o chYp ri! - @nsyme gyr51e (mdt lo#i ensyme topoi1omer51e c(t ADN lm th"o Fo(n c] * phU5 cQ5 protein a Z * phn t+ melic51e th5m gi5 t"ch m#ch t#o ch#c b5 15o chYpL melic51e 1+ deng n\ng l23ng AT$ Z c(t c"c li4n <t hydroL$rotein lm c\ng m#ch //a g(n Mo m#ch )n ADN Z Vn Wnh * m#chL - TVng h3p m{i ARN t A-,> nu b%ng c"ch8 protein N (Dn5 ! g(n prim51e t#o r5 m{iL c"c o#n m{i ARN bW ADN-polymer51e r c(t b|9 l(p c"c nu cQ5 ADN Mo chv tr0ng M thHc hiPn polymer hI5 h2`ng A-.L - T#o m#ch ADN li4n tec M c"c o#n o<5s5<i8 GADN-polymer51e rrr g(n Mo m#ch <hun cI _u M tVng h3p m#ch li4n tec gTi l 13i 1`m (le5ding 1trADN L
T/ b# nhn hk cI A lo#i ADN-polymer51e8 9 9 9 M - AND-polymer51e 8 trong nhn M 15o chYp AND nhifm 1(c thZ (13i mudn - AND-polymer51e 8 trong nhn M cI M5i trN 1+5 chi5 - AND-polymer51e 8 trong ty thZ M 15o chYp AND ty thZ - AND-polymer51e 8 trong nhn M 15o chYp AND nhifm 1(c thZ (13i 1`m - AND-polymer51e 8 trong nhn M 15o chYp AND nhifm 1(c thZ trong nhn M ty thZ
G ADN-polymer51e rrr g(n Mo m{i ARN M tVng h3p c"c o#n ng(n ,>>>-*>>> nu gTi l o#n <5s5<iL - T"ch m{i bKi ADN-polymer51e r9 15u I l(p c"c nu cQ5 ADN Mo chv tr0ng M thHc hiPn polymer hI5 h2`ng A-.L - N0i lin c"c o#n <5s5n. :i0ng nh5u8 - B23c tVng h3p t * m#ch cQ5 cqng , phn t+ ADN - #ch li4n tec 15u <hi <t th[c gu" trXnh 15o chYp - B23c tVng h3p bKi ADN-polymer51e rrr uh"c nh5u8 S:i !7) S:i )=>n M<$h Ph=rn - #ch <hun cI _u - #ch <hun cI _u A H67n, sn, h:& - #ch A-. h2`ng Mo - #ch -.A h2`ng r5 ch#c b5 15o chYp ngoi ch#c b5 15o chYp Mti - uhng c_n - !_n m{i ARN u=E 2Fnh sn, h:& - TVng h3p ng5y m#ch li4n - B_u ti4n tVng h3p c"c o#n tec t m#ch <hun <5s5n G6:$ sn, h:& $vn, $hiT=. - !"c 13i ADN 23c t"ch r5 M mdt 13i ADN cI h2`ng -.A 23c 15o chYp trHc tip bKi ADNpolymer51eL /3i con bV 1ung M5 t#o 23c gTi l 13i dOnL /3i g0c cNn l#i cI h2`ng A-.9 <hng 23c 15o chYp cho n <hi mdt ph_n cQ5 13i g0c 23c th"o Fo(nL a]n 15o ny 23c gTi l 13i mudnL /3i mudn 23c tVng h3p b%ng mdt gu" trXnh 15o chYp <hng li4n tecL - /H 1inh tVng h3p 13i mudn M 13i dOn 23c iu hN5 bKi 1H t#o n[t th(t (loop cQ5 13i mudn9 nhS I 13i mudn cng 23c tVng h3p cqng h2`ng Mì 13i 1`mL 1^. 53$h h67$ G#<n OP"A"Pi O / b# nhn n,=B(n hUB + / b# nhn hk . - Nhn thpt8 7>->> nu - Nhn nguy4n thQy8 ,>>>-*>>> nu 1`. 0"i 2w $U" $E$ VnAB)V 2#n, !"# $h%&. STT , * 7 A D = @nAB)V $rim51e Rep protein melic51e AND polymer51e rrr AND polymer51e r AND topoi1omer51e r AND topoi1omer51e rr AND lig51e ChQ$ nRn, TVng h3p m{i ARN Th"o Fo(n 13i 1`m Th"o Fo(n 13i chpm TVng h3p AND jI5 m{i M l(p <Un o#n m{i bW c(t !(t <hU5 mdt 13i )n AND !(t <hU5 c] h5i 13i )n AND N0i c"c _u cQ5 polynucleotide ^ hon thnh
1c. Mti L=\ &hE Phi n#b Nh_ +# G=b x=\ LQb uhi N-protein nhpn bit MW trU c_n tVng h3p 13i 1`m h5y 13i mudnL $rimo1ome gi]i phIng prim51e Z tVng h3p m{iL 1d. P2# Vin nhkn Min +1 23 ADN $h# &2i)"!V h#< G>n,. $rotein nhpn diPn l N-protein9 chTn c"c iZm (origin tr4n ADN9 t#i I prim51e cI thZ ho#t dngL 1e. B-n $h\ + P3$h h67$ $U" G#<n )ti. - {i ARN 23c tVng h3p bKi , ensyme Jc hiPu gTi l prim51e - !I thZ <Yo di 1H <hKi dng chuvi de novoL - uUch th2`c8 <ho]ng A-,> b51e Hj. T8$ G> Mi $h=BKn $U" $h<$ b" !"# $h%& O E.coli + 8$ G> hE# L#Nn $U" ADN ,8$. - T0c d di chuyZn cQ5 ch#c b5 15o chYp K E.coli: =>> b51e?giy - T0c d th"o Fo(n cQ5 ADN g0c8 => MNng?giy H1. Phn bi !i(= L#Nn M6.n, + !i(= L#Nn ). /H th"o Fo(n ADN gy r5 hiPn t23ng 1i4u Fo(n8 G Th"o Fo(n 13i <Yp dOn n 1i4u Fo(n m (Fo(n ng23c chiu M lm gi]m 10 MNng G jo(n th4m 1c dOn n 1i4u Fo(n d2)ng (Fo(n <Yp cqng chiu M 10 MNng t\ng HH. 0"i 2w $U" #&#i!#)V2"!V I + II. - Topoi1omer51e F[c t"c gb Fo(n M t#o Fo(n AND lm th5y Vi chh 10 k<L - Topoi1omer51e cI M5i trN gu5n trTng trong 15o chYp M Ing gIi ANDL G !) ch 15o chYp b"n b]o t{n c_n cQ5 AND Ni h|i c"c iZm c(t K , 13i hoJc K c] * 13i AND Z t"ch chuviL G /H Ing gIi AND c_n d#ng 1i4u Fo(n Jc hiPuL - !I * nhIm8 G Topoi1omer51e r8 c(t , trong * 13i ADN9 cho 13i cNn l5W di chuyZn gu5 13i bW c(t M n0i l#i 13i M5 bW c(t lm th5y Vi k< (chh 10 li4n <t , gi" trWL G Topoi1omer51e rr8 c(t * 13i lm th5y Vi k< * gi" trWL - CU de8 G o E.coli8 cI 7 lo#i topoi1omer51e (r n rC 8 ko#i r M rrr thudc nhIm topoi1omer51e r F[c t"c t#o th4m Fo(n b%ng c"ch gb 1i4u Fo(n m lm t\ng k<L Topoi1omer51e rr h5y ADN gyr51e F[c t"c gb Fo(n lm gi]m k<9 1+ deng n\ng l23ng do AT$ cung cp M c(t {ng thSi * 13i AND M cho phYp * 13i cNn l#i di chuyZn gu5 <he hK M5 t#o r5L G T bo eu<5ryote8 cng cI * lo#i topoi1omer51e r M rrL HC. 0"i 2w $U" $E$ ADN &#9B)V2"!V O / b# nhn hk . o t bo nhn thpt cI A lo#i ADN-polymer51e8 9 9 9 M L P#9B)V2"!V l m y o p 01 23 Nhn Nhn Ty thZ Nhn Nhn H#< 3nh /5o chYp /+5 chi5 /5o chYp /5o chYp /5o chYp &#9B)V2 ADN nhifm ADN ty thZ ADN nhifm ADN nhifm hX" ^gfhCg 1(c thZ (13i 1(c thZ (13i 1(c thZ mudn 1`m HW. 0"i 2w $U" HV9i$"!V. - mv tr3 cho 1H th"o Fo(n ADN g0c MX h5i 13i )n ADN <hng thZ tH nhi4n t"ch r5 23cL - !_n AT$ nh2 mdt co65ctor Z i dTc theo 13i ADN lm t\ng t0c d t"ch h5i 13i ADNL - !I * lo#i8 G Rep-protein g(n Mì ADN g0c9 trHc tip tVng h3p 13i 1`m M di chuyZn theo h2`ng -.AL G melic51e thR h5i8 g(n Mì 13i <hun <i5 Z tVng h3p 13i chpm9 t#o phRc h3p Mì prim51e Z t#o m{i ARNL
Bi H. CzC LOI AaN

1. 0F !"# ADN )"n, hrn, in Mi 2=BTn' nh6n, b-n hn nX 9<i Phrn, 2Z$ i/& $h[ h=B D=E 2Fnh !inh sn, h:& &2# Vinb ADN nhifm 1(c thZ chh m5ng mdt b]n 15o duy nht cho mvi gen9 trong <hi t bo cI hng nghXn phn t+ protein cqng lo#i t{n t#iL uhng ph]i tt c] c"c gen u biZu hiPn thnh protein cqng l[c m c"c gen <h"c nh5u 1c biZu hiPn <h"c nh5u t#i mvi thSi iZm trong MNng Si cQ5 t bo M tqy theo iu <iPn mi tr2SngL Do Mpy c_n cI c) ch trung gi5n Z <huch #i thng tin di truyn t ADN n protein9 {ng thSi <iZm 1o"t 1H biZu hiPn cQ5 gen theo nhu c_u cQ5 t boL H. CE$ +"i 2w $hU B/= $U" AaNb - ARN t#o n4n bd m"y 1inh tVng h3p protein8 Trung gi5n truyn thng tin di truyn n bd m"y 1inh tVng h3p proteinL T mvi gen cI thZ cI nhiu b]n 15o ARN Z chh huy gu" trXnh tVng h3p protein9 {ng thSi MNng Si cQ5 ARN ng(n n4n 1H biZu hiPn cQ5 gen gu5 trung gi5n ARN df dng 23c <iZm 1o"t bKi nhu c_u cQ5 t bo M iu <iPn mi tr2SngL Th5m gi5 cu t#o ribo1omL Cpn chuyZn 5cid 5min n ribo1om trong gu" trXnh 1inh tVng h3p proteinL - ARN cI thZ K d#ng tH do hoJc g(n Mì protein thnh c"c phRc h3p nucleoprotein gii nhiu M5i trN gu5n trTng trong ho#t dng 10ng cQ5 t boL - !I tì D lo#i ARN nh| <h"c nh5u9 trong I cI mdt 10 1nARN cI li4n gu5n n MiPc F+ lz hnARN thnh mARN chRc n\ngL - dt Mi lo#i ARN cI c] K t bo nhn nguy4n thQy M t bo nhn thpt l thnh ph_n cQ5 ensyme cI chRc n\ng F[c t"c trHc tipL CL CE$ 9#<i AaN 2#n, / b#b - mARN ARN thng tin - rARN ARN ribo1om - tARN ARN Mpn chuyZn - pre-rARN (tin rARN l ARN 23c tVng h3p t ADN9 15u <hi ch[ng 23c c(t n0i 1c trK thnh rARN - pre- tARN (tin tARN l ARN 23c tVng h3p t ADN9 15u <hi ch[ng 23c c(t n0i 1c trK thnh tARN - hnARN ARN nhn <hng {ng nht9 1c t#o mARN 15u <hi 23c c(t n0iL - 1nARN ARN nhn nh| - 1cARN ARN t bo cht nh| W. AaN 2ib#!#)q $\= 2{$' +"i 2w. - rARN l thnh ph_n cu t#o ribo1om9 chim phn n+5 <h0i l23ng cQ5 ribo1omL - vi mdt ribo1om cI r ' *LA - 7LA F ,>L - !I lo#i ribo1om <h"c nh5u phn biPt dH5 Mo hP 10 l(ng <hi 1i4u ly tm8 G ribo1om cQ5 t bo nhn nguy4n thQy M lec l#p ' D>/ G t bo nhn thpt ' =>/
G ty thZ dng Mpt cI M[ ' A>/ Tt c] ribo1om u 23c cu t#o bKi * tiZu )n MW cI chRc n\ng M cu tr[c ri4ng biPt9 c"c tiZu )n MW ny cI thZ t"ch r5 M g(n l#i Mì nh5u mdt c"ch thupn nghWch tqy theo n{ng d cQ5 g*G Ribo1om cQ5 t bo nhn nguy4n thQy ' D>/ g{m8 G )n MW l`n A>/ chR5 ,rARN */ M ,rARN A/9 M 7 protein G )n MW nh| >/8 ,rARN ,/ M *,-* protein Ribo1om cQ5 t bo nhn thpt ' =>/ g{m8 G )n MW l`n >/8 ,rARN *=/9 ,rARN AL= M ,rARN A/ M 7A protein G )n MW nh| 7>/8 ,rARN ,=/ M protein
^. AaN 2ib#!#) $U" E.coli? - !I D Mqng m^ hI5 cho rARN tr4n N/T8 rrn - vi rrn m5ng c"c gen theo thR tH ,/-*/-A/ - , 13i pre-rARN 23c phi4n m^ t rrn - $re-rARN 23c c(t Z t#o thnh c"c rARN t2)ng RngL - !u t#o ribo1om8 A>/G>/8 G A>/8 * rARN (*/GA/ 7 protein 23c Jt t4n t k,-k7 G >/8 , rARN (,/ *, protein 23c Jt t4n t /,-/*, - !"c protein ribo1om cI <h0i l23ng t2)ng 0i nh| M chR5 nhiu 5cid 5min b51eL - !"c protein cQ5 ribo1om cI c"c trXnh tH bpc , <h"c nh5u9 ngo#i tr kD9 k,* M k=L G kD l mdt d#ng 5mino5cetyl hI5 k,* G k= l phRc h3p cQ5 mdt phn t+ k,> li4n <t Mì kD M k,*L `. SZ )V hB9 hX" n=$9V# iMV O 2AaN $U" E.coli. - ethyl hI5 nucleotide l mdt Jc tUnh cu tr[c bpc , cQ5 rARN - j]y r5 15u <hi phi4n m^ - j[c t"c bKi ARN-methyl51e - !) cht cung cp methyl l /-Adeno1yl ethionin (/A - rARN cQ5 t bo nhn nguy4n thQy M bo gu5n8 methyl hI5 b51e <hKi _u (A-methylcyto1in - rARN cQ5 t bo nhn thpt8 methyl hI5 K MW trU * cQ5 2Sng ribo1e (*--methyl5deno1yl - /H methyl hI5 Ing M5i trN bin Vi b]n 15o 1) cp cQ5 rARNL - CW trU methyl hI5 23c b]o t{nL c. SZ bi/n Gsi $U" 2AaN O / b# nhn hk . Th3 M] !Z bi/n Gsi $U" 2AaN O / b# HV9". - $re-rARN 7A/ l b]n 15o cQ5 c"c gen ARN-ribo1omL - rARN 7A1 23c bin Vi do 25 Mo <ho]ng ,,> nhIm methyl K c"c nucleotide Jc hiPuL Nhing ph_n methyl hI5 u 23c b]o t{n K rARN tr2Kng thnhL - mng lo#t difn bin trong M ngoi nhn ^ t#o r5 23c c"c tin thZ trung gi5n */ M *>/ t prerARN 7A/ G pre-rARN */ c(t n0i thnh *=/ M AL=/ G pre- rARN *>/ thnh ,=/ - :en m^ hI5 cho A/ n%m ri4ng d. SZ bi/n Gsi $U" 2AaN O / b# nhn n,=B(n hUB. - pre-rARN g{m rARN ,/*/-A/ - :en rARN chR5 * chuvi tARN h5y nhiu h)n - /H phn c(t _u ti4n cQ5 pre-rARN 23c F[c t"c bKi ARN1e rrr Jc hiPu cho ARN m#ch <YpL e. C\= 2{$ + +"i 2w $U" AaN +kn $h=BKn. 5L !u tr[c8 - Di D- nucleotid - !u tr[c g{m mdt m#ch cudn l#i nh2 hXnh l ch b58 G B_u tpn cqng A pho1ph5te G Nh"nh g(n 5cid 5min8 g{m D cJp b51e
!"c nucleotide _u tpn cqng cI m tH do Z g(n 5cid 5min !"c cJp b51e cI thZ <hng theo trXnh tH bV 1ung G Bui !!A8 trXnh tH !!A t#i gi[p ensyme nhpn bit tARN <hi dWch m^ G CNng D8 g{m 7 cJp b51e9 chR5 dihydrouridine G CNng 0i m^8 g{m A cJp b51e 0i m^L vi tARN m5ng , bd b5 0i m^ Jc hiPu cQ5 , h5y nhiu codon m^ hI5 cho , 55L G CNng T8 g{m A cJp b51e9 chR5 trXnh tH T! G !"c b51e bin Vi8 <h"c Mì A9 ;9 :9 !8 r ' rno1ine T ' Ribothymidine ' p1eudouridine m,: ' methyl gu5no1ine m**: ' dimethylgu5no1ine m,r ' methylino1ine D ' dihydrouridine bL !hRc n\ng8 !"c ensyme Jc hiPu l 5mino5cyl tARN 1yntret51e g(n mvi 5cid 5min Mì tARN t2)ng Rng nhS n\ng l23ng AT$ t#o r5 5mino5cyl-tARNL $hRc h3p ny n ribo1om g(n Mì mARNL tARN g(n Mì mARN b%ng 1H b(t cJp bV 1ung nhS 0i m^L 1j. SZ bi/n Gsi $U" AaN O E.coli. 0"i 2w $U" aN"!V P. - tARN 23c c(t r5 t phn t+ di h)n nhS RN51e $L - !"c ph]n Rng bin Vi8 Amino5cyl hI5 ormyl hI5 tARN mK _u (t bo nhn nguy4n thQy :(n nhing yu t0 n0i di :(n ribo1om Nhpn diPn codon-5nticodon - !"c gen cQ5 tARN th2Sng 23c ph"t hiPn trong c"c operon hvn h3p cqng l[c m^ hI5 cho 7 tARN (tARNT~9 tARNThr9 tARN:ly9 tARNThr M mARN cho 1H tVng h3p protein yu t0 n0i di TuL - ThQy gi]i _u ti4n cQ5 b]n 15o 1) cp 23c F[c t"c bKi ARN1e t#o c"c _u tpn cqng A cQ5 phn t+ tARNL - tARN cI trXnh tH cu0i l !!A m phi4n m^ t c"c b51e bV 1ung cI trong ADN cQ5 N/TL - RN51e $8 G k mdt ensyme9 g{m , ARN cI DA nucleotide M , protein cI trTng l23ng phn t+ *>>>> G Trong c"c iu <iPn dinh lz9 c] * thnh ph_n protein M ARN u c_n nhing yu t0 Z ho#t dng9 nh2ng <hi n{ng d g*G c5o thX chh cI thnh ph_n ARN cI ho#t tUnh F[c t"cL G Theo 15u ho#t dng cQ5 ARN1e $9 mdt 10 ph]n Rng trong nhn M bin Vi b51e 1c t#o n4n tARN hon chhnhL 11. CE$ &h-n Qn, L; 9| AaN. - Amino5cyl hI5 - ormyl hI5 tARN mK _u (t bo nhn nguy4n thQy - :(n nhing yu t0 n0i di - :(n ribo1om - Nhpn diPn codon-5nticodon 1H. C\= 2{$ + +"i 2w $U" )AaN O / b# nhn n,=B(n hUB + / b# nhn hk . :i0ng nh5u8 - $hn t+ mARN g{m ph_n8 G Bo#n A <hng m^ hI5 (A ;TR G Cqng m^ hI5 G Bo#n <hng m^ hI5 (A ;TR - Bo#n A M l trXnh tH <hKi _u M <t th[c <hng 23c dWch m^9 do I mARN chR5 trXnh tH nucleotide nhiu h)n 10 23c dqng Z m^ hI5 proteinL
- A;TR M ;TR cI t"c deng8 G km gi5 t\ng h5y gi]m d Vn Wnh cQ5 phn t+ ARN G :i[p phn t+ ARN t{n t#i lu h)n trong t bo cht9 do Mpy 1c tVng h3p 23c nhiu protein h)nL G Th[c Ey gu" trXnh dWch m^ hiPu gu] h)n uh"c nh5u8 T/ b# nhn n,=B(n hUB T/ b# nhn hk - mARN l polyci1tron - mARN l monoci1tron - DWch m^ F]y r5 ng5y <hi 5ng phi4n m^ - DWch m^ F]y r5 15u <hi phi4n m^ hon tt - ThSi gi5n b"n hQy ng(n trung bXnh * ph[t - ThSi gi5n b"n hQy di h)n t > ph[t n *7 giS - m_u nh2 <hng bW bin Vi - $re-mARN bW bin Vi8 G :(n chIp Mo _u A G :(n ui poly A Mo _u G ethyl hI5 1C. GNn $hX& + ,Nn G=ri )AaN $U" / b# nhn hk b 5L :(n chIp8 - @nsyme pho1phohydrol51e c(t li4n <t pho1phodie1ter9 lo#i b| M pho1ph5tL - Tip theo9 ensym gu5nylyltr5n16er51e g(n gu5nine M pho1ph5te Mo pho1ph5te cQ5 _u A b%ng li4n <t A-A tripho1ph5tL - /5u I9 MW trU N-D cQ5 gu5nine 1c bW methyl hI5 do ensyme gu5nin-D-methyltr5n6er51eL(!hIp > - !u0i cqng9 ensym *--methyltr5n16er51e 1c methyl hI5 MW trU * cQ5 2Sng ribo1eL( !hIp , - $h]n Rng methyl hI5 chIp cI thZ F]y r5 theo , trong c"ch8 G!hIp >8 nhIm methyl g(n Mo MW trU :D di ensyme gu5nine-D-methyl tr5n16er51e F[c t"cL $h]n Rng ny F]y r5 K tt c] mARN t bo nhn thptL G!hIp ,8 nhIm methyl g(n Mo MW trU *-m 2Sng ribo1e cQ5 nucleotide _u ti4nL $h]n Rng do ensyme *--methyl tr5n16er51e F[c t"c9 F]y r5 h_u ht mARN t bo nhn thpt G!hIp *8 ph]n Rng methyl hI5 F]y r5 K nucleotide thR * Mì th lP ,>-,AL GNgoi r5 cNn cI gu" trXnh methyl hI5 ndi phn t+ F]y r5 t#i MW trU N cQ5 5denine Mì t_n 1ut >9,L - !hRc n\ng8 G a]o MP phn t+ ARN <hng bW c(t li4n <t pho1phoe1ter do c"c phn t+ hydro ln cpnL G Du hiPu nhpn bit MW trU g(n cQ5 mARN tr4n ribo1om <hi dWch m^ bL :(n ui poly A Mo _u 8 - Bui poly A g(n Mo _u K dng Mpt cI M[8 <ho]ng A>-*A>9 nm men <ho]ng ,>> AdeninL - Bui poly A g(n Mo pre-mARN cI trXnh tH nhpn diPn AA;AAA - tu" trXnh ny do ensyme poly A polymer51e F[c t"cL - !hRc n\ng8 G :i[p mARN t nhn r5 t bo cht G j"c Wnh 10 l_n mARN 23c dWch m^ G a]o MP phn t+ ARN G :(n Mì ribo1om 1`. C\= 2{$ + +"i 2w $U" AaN nhn nh}' AaN / b# $h\ nh}. - 1nARN M 1cARN phRc h3p Mì c"c protein Jc hiPu t#o c"c h#t ribonucleoprotein (RN$ 23c gTi l 1nRN$ h5y 1cRN$ - 1nRN$ t#o thnh 1pliceo1om F[c t"c MiPc c(t n0i pre-mARN thnh mARNL - !I D lo#i 1nRN$ t ;, n ;D8 G ;, M ;*8 1+5 Vi hnARN thnh mARN hon chhnhL G ;8 1+5 Vi pre-rARN thnh rARN t bo chtL ARN cI <h] n\ng F[c t"c 23c gTi l rybosymeL - 1cARN cI M5i trN bit r} nht l D/k ARN8 G mdt thnh ph_n cQ5 phRc h3p nhpn diPn tUn hiPu Fut /R$L G /R$ g{m8 , 1cARN cI chR5 *7 nucleotide M polypeptide cI r ' >>>-D*>>>L G uhi mARN cQ5 protein Fut b(t _u 23c dWch m^9 /R$ t2)ng t"c Jc hiPu Mì o#n peptid tUn hiPu Fut cQ5 protein mì 1inh M Mì ribo1om9 ng\n chJn 1H dWch m^ n <hi ribo1om tip F[c Mì l2ì ndi cht9 <hi I
nI rSi <h|i ribo1om M tUn hiPu Fut cQ5 protein mì 1inh g(n Mo bd m"y chuyZn MW protein tr4n mng l2ì ndi cht M 1H tVng h3p protein 23c tip tec9 protein 1inh r5 23c chuyZn Mo trong l2ì ndi chtL HH. 0"i 2w L{$ E$ $U" !naNP 2#n, D=E 2Fnh $N n8i )AaN. G 1nRN$ ;, g(n Mo MW trU cho G 1nRN$ ;* t#o li4n <t hydro Mì MW trU nh"nh cQ5 intronL :i5i o#n ny 1+ deng AT$ thQy phn M wyu t0 tr3 gi[px 1nRN$ ( ;*A G 1nRN$ ;, t2)ng t"c Mì 1nRN$ ;* gi[p MW trU cho M MU trU nhpn tin l#i g_n nh5uL G $hRc h3p ;A?;7?; g(n Mì ;, M ;* 1+ deng AT$ thQy phn ;A g(n Mì MW trU cho cQ5 intron ; g(n Mì ;* y l 1pliceo1om hon chhnhL AT$ cung cp n\ng l23ng cho mvi gi5i o#nL ;7 t2)ng t"c Mì ; b%ng li4n <t hydroL G ;A di chuyZn t ph_n intron 15ng ph_n eFon cQ5 MW trU cho9 ;* th5y th ;7 li4n <t Mì ; nhS li4n <t hydroL G 1nRN$ ;A t2)ng t"c Mì ph_n intron cQ5 MW trU nhpn M ;*9 F[c t"c ph]n Rng c(t li4n <t introneFon M n0i * eFonL $h]n Rng chuyZn e1ter thR * F]y r5L G /pliceo1om 23c th"o rSi9 gi]i phIng ;,9 ;*9 ;79 ;A9 ;L G /3i i intron 23c th"o Fo(n M tho"i hI5L
Bi C. S~ PHIN M 0 M DI TaN
1. 0"i 2w M1 L{$ E$ $U" ADN. ADN thZ hiPn <h] n\ng dW F[c t"c tRc l 9) Ph=rn Z sn, h:& n4n mdt &hn ; PhE$L H. N,=B(n N$ $U" &hi(n )*. - !hh , trong * m#ch cQ5 phn t+ ADN dqng lm <hun Z tVng h3p ARNL - ARN polymer51e b"m Mo ADN lm t"ch m#ch M di chuyZn theo h2`ng -. A tr4n ADN Z cho wARNx 23c tVng h3p theo h2`ng A-.L - ARN polymer51e g(n Mo ADN gy r5 hiPn t23ng wch]yx t#i chv b4n trong chuvi Fo(n <Yp ADNL ut gu] lm Rt c"c li4n <t hydro gii5 c"c i b51e bV 1ungL - !) cht cho ARN polymer51e l c"c ribonucleotid A-tripho1ph5t AT$9 :T$9 !T$ M ;T$L - dt ion <im lo#i hI5 trW * hoJc l g*G hoJc l n*G Ing M5i trN co-65ctorL C. Ti/n 2Fnh &hi(n )*. 5hi n# G?= ^g $U" $h=i )AaN )7i !inh E$h Ph}i s h:& 9"i G?= i(n. - Tin trXnh phi4n m^8 G ARN polymer51e nhpn diPn wpromoterx G ADN 23c th"o Fo(n cec bd (t#m thSi G $olymer51e b(t _u Tc ADN M tVng h3p ARN G $olymer51e di chuyZn n MW trU <t th[c G ARN t"ch r5 <h|i polymer51e - B_u A cQ5 chuvi mARN mì 1inh t"ch <h|i tV h3p l5i _u ti4n <hi8 G !huvi ARN <Yo di theo h2`ng A- M ARN polymer51e cqng Mì Mqng 13i <Yp ADN duvi Fo(n ( cNn gTi l bong bIng phi4n m^ chuyZn dng dTc theo 13i <hun mOu theo h2`ng -AL G uhi tV h3p l5i ADN-ARN #t 23c chiu di ,* b51e9 _u tpn cqng A cQ5 chuvi ARN mì hXnh thnh 23c t"ch r5 t m#ch ADN bV 1ung Mì nI M * m#ch <Yp cQ5 ADN b5n _u phec h{i l#iL W. Mti &2i)V2 $X $?n hi/ $h# D=E 2Fnh &hi(n )* h"B Phrn,b 0F !"# ADN $X 3nh $h\ M1 L{$ E$. - {i <hng c_n cho gu" trXnh phi4n m^ MX ARN polymer51e cI <h] n\ng <Yo di chuvi de novoL - ADN thZ hiPn <h] n\ng dW F[c t"c tRc l lm <hun Z tVng h3p n4n mdt phn t+ <h"cL ^. SZ &hi(n )* G8i LQn,. Th3 M]. - Trong 5 10 tr2Sng h3p9 chh cI mdt trong * m#ch cQ5 ADN 23c phi4n m^ M 1H phi4n m^ ny 23c gTi l phi4n m^ bt 0i FRngL - !ng cI nhing tr2Sng h3p trong I 1H phi4n m^ F]y r5 K c] * m#ch ADN9 I l 1H phi4n m^ 0i FRngL - CU de8 /H phi4n m^ K ADN ty thZ t bo th[9 trong I c] * m#ch cQ5 bd gen MNng ,<b u 23c phi4n m^ r5 ARNL Tuy nhi4n 15u I mdt trong * b]n 15o ny bW tho"i hI5L `. 0F !"# AaNf&#9B)V2"!V Phrn, $X 3nh !;" !"ib - Nhing nucleotide no <t h3p <hng chUnh F"c th2Sng 23c th5y th ng5y b%ng nhing nucleotide phq h3pL - Nu cI lvi him hoi no F]y r5 thX cng <hng di truyn 23c MX ARN cI MNng Si ng(n M <hng ph]i l n)i l2u tri thng tin di truynL c. IJ$ GiK) &hi(n )* O / b# nhn n,=B(n hUB. - !hh mdt lo#i ARN polymer51e chWu tr"ch nhiPm tVng h3p tt c] c"c lo#i ARN - mARN th2Sng chR5 thng tin nhiu gen n0i tip nh5u (polyci1tron mARN
tu" trXnh tVng h3p mARN 23c tin hnh <hi ARN polymer51e b"m Mo Mqng <hKi dng (promoter L uhi g(n Mo9 polymer51e <hng tVng h3p mARN ng5yL /H tVng h3p chh b(t _u t du Fut ph"t th2Sng l TA! n%m c"ch D-= b51e phU5 15u chv b"m M phU5 _u A cQ5 m#ch <hunL o ph_n l`n t bo nhn nguy4n thQy9 gu" trXnh tVng h3p tip tec n <hi Tc gu5 du <t th[cL ut th[c phi4n m^9 ARN polymer51e M mARN t"ch rSi <h|i ADNL tu" trXnh phi4n m^ M dWch m^ F]y r5 {ng thSi Mì nh5uL
d. C\= <# $U" AaNf&#9B)V2"!V O E.coli. - k mdt protein phRc h3p chR5 A tiZu )n MW g{m 7 lo#i9 trTng l23ng phn t+ 7A<D5 - Ton bd ensyme gTi l holoensyme - dt ensyme duy nht K pro<5ryote - k}i ensyme cI chRc n\ng F[c t"c g{m 7 tiZu )n MW * - u t0 1igm5 ( nhpn diPn promoter - !"c tiZu )n MW cQ5 ARN polymer51e cQ5 E.coli8 TiK= G.n +1 GVn )* hX" S8 96:n, ChQ$ nRn, rpoA * :(n promoter rpoB , :(n nuclectid rpoC , :(n <hun mOu D> rpoD , uhKi _u e. C\= <# &2#)# V2 O E.coli. - $romoter g{m * trXnh tH8 G TrXnh tH TATAAT (hdp TATA h5y hdp $ribno 9 c"ch iZm Fut ph"t -= b51e M cI b51e trung tm l -,>L G TrXnh tH TT:A!A cI b51e trung tm -AL Cqng ny th5m gi5 Mo MiPc g(n trHc tip ARN polymer51e - * trXnh tH ny chWu tr"ch nhiPm cho phYp ARN polymer51e g(n Mo Mqng <hKi dng phi4n m^L - uhng ph]i promoter no cng cI cu t#o gi0ng hPt c"c trXnh tH ny9 m cI mdt Ut 15i lPch nh|9 do I ng2Si t5 gTi trXnh tH ny l trXnh tH chungL - $romoter <iZm 1o"t t_n 1ut <hKi _u hiPu gu]8 G uhKi _u nh5nh do promoter wm#nhx G k yu t0 iu hN5 phi4n m^ chUnh K Mi <huEn - $romoter cI trXnh tH cng g_n Mì trXnh tH chung thX cng ho#t dng m#nh 1j. C\= <# &2#)# V2 O / b# nhn hk . - $romoter n%m phU5 tr2`c iZm Fut ph"t cQ5 mARN - !I nhing trXnh tH chung trong tin hI58 G TrXnh tH K Mqng -> l TATA (hdp TATA cI b51e trung tm -*A h5y -> K dng Mpt cI M[ M t -> n -,*> K nm men9 Wnh h2`ng cho ARN polymer51e b(t _u phi4n m^ G TrXnh tH K Mqng -7> bp l !!AAT (hdp !AT G TrXnh tH K Mqng -,,> bp l trXnh tH giu :! (hdp :! - o t bo nhn thpt9 polymer51e <hng g(n promoter m l c"c protein iu hN5 - mdp !AT M :! cI thZ <iZm 1o"t MiPc g(n <hKi _u9 cI chRc n\ng li4n <t Mì c"c yu t0 phi4n m^ (T L - mdp TATA cI thZ <iZm 1o"t MiPc chTn iZm <hKi _u phi4n m^L S# !Enh &2#)# V2 $U" / b# nhn hk + / b# nhn n,=B(n hUB 8 - :i0ng nh5u8 G Cqng <hKi dng cQ5 phi4n m^ G !u t#o cqng cI hdp TATA - uh"c nh5u8 N,=B(n hUB Nhn hk $romoter g{m * trXnh tH8 $romoter g{m trXnh tH8 - TrXnh tH TATAAT (hdp TATA h5y - TrXnh tH K Mqng -> l TATA (hdp TATA $ribno 9 c"ch iZm Fut ph"t -= b51e9 cI cI b51e trung tm -*A h5y -> K dng Mpt cI b51e trung tm -,> M[ M t -> n -,*> K nm men9 Wnh h2`ng
cho ARN polymer51e b(t _u phi4n m^ - TrXnh tH TT:A!A cI b51e trung tm -AL Cqng ny th5m gi5 Mo MiPc g(n trHc tip ARN polymer51e - mdp TATA ho#t dng cI hiPu gu] cqng Mì * trXnh tH t2)ng Rng phU5 tr2`c <ho]ng -7> l !!AAT (hdp !AT M -,,> giu :! cI chRc n\ng li4n <t Mì c"c yu t0 phi4n m^ (T
11. ChQ$ nRn, $U" +vn, f1j + fC^ O E.coli. - m5i Mqng ny t#o thnh promoter l trXnh tH <hKi _u cho 1H phi4n m^ - Cqng -,> l nì g(n cQ5 yu t0 1igm5 - Cqng -A th5m gi5 Mo MiPc g(n <hKi _u ARN polymer51e 1H. 0vn, PhOi G>n, D=B/ G1nh 8$ G> &hi(n )*. Th3 M]. - Cqng <hKi dng guyt Wnh t0c d phi4n m^L - !ng g_n trXnh tH chung cng m#nh - CU du8 l5c repre11or chh phi4n m^ > ph[t?l_n gen rADN phi4n m^ * giy?l_n 1C. GVn h& a + AaNf&#9B)V2"!V O E.coli. - :en ny "p Rng Mì 1hoc< nhiPt M m^ hI5 cho chuvi polypeptide cI r * >>> - BIng M5i trN nh2 mdt yu t0 bin ViL u t0 ny (23c gTi * <t h3p Mì ph_n l}i bXnh th2Sng cQ5 ARN polymer51e Z t#o n4n holoensyme ** - @nsyme ny chh b"m Mo nhing promoter cQ5 gen 1hoc< nhiPt9 chR <hng b"m Mo promoter cQ5 c"c gen bXnh th2Sng M cI trXnh tH Mqng -A di h)n Mì mdt 10 <h"c biPt nh|L 1W. P#)# V2 SP`q N,=tn ,8$ + Qn, M]n,. /$ l mdt thHc <huEn thZ cQ5 Salmonella typhimurium9 m^ hI5 23c mdt ARNpolymer51e Miru1 Jc hiPuL !"c promoter cQ5 t bo chQ M c"c promoter cI trong pl51mid ly trUch t E.coli u <hng 23c /$ polymer51e nhpn bitL ng deng Z nhn b]n 15o m^ c_n thit8 G $romoter /$ 23c chn Mo Mector pl51mid phU5 tr2`c polylin<erL Bo#n ADN c_n 15o chYp 23c chn Mo polylin<er M pl51mid t"i tV h3p 23c bin n#p Mo E.coli. G $l51mid chR5 ADN l# 23c wlm duvi thlngx bKi mdt ensyme c(t h#n chL G $l51mid duvi thlng ny 23c Q Mì /$ polymer51e M c"c NT$L G !"c pl51mid-ADN 23c lo#i <h|i c"c b]n 15o mARN b%ng c"ch Q Mì DN51eL 1^. u=E 2Fnh PhOi G?= + n8i Mi $h=i AaN. - CiPc g(n cQ5 holoensyme ARN polymer51e Mo promoter t#o r5 phRc h3p <hKi dng Ing cI cu hXnh ADN Fo(n <YpL !"c iZm tip F[c chQ yu gii5 promoter M ARN polymer51e n%m K c"c Mqng -,> M -AL - $hRc h3p Ing nh5nh chIng bin thnh phRc h3p mK do c"c li4n <t hydro bW ensyme c(t tr4n mdt Mqng b5o g{m ,D bpL /3i ADN 23c t"ch t5 nhS 1H th"o Fo(n ,9 MNng cQ5 Fo(n <Yp a-ADNL - Nucleotide tripho1ph5t _u ti4n (th2Sng l AT$ hoJc :T$ g(n Mo tiZu )n MW cQ5 polymer51e9 h2`ng [ng Mo b51e bV 1ung K MW trU G, cQ5 13i <hun mOuL - dt nucleotide tripho1ph5t thR * tip theo 23c <t n0i M hXnh thnh li4n <t pho1phodie1ter _u ti4nL Nh2 Mpy9 chuvi ARN ph"t triZn m5ng tripho1ph5t K _u A M theo h2`ng A-.L 1`. 5/ h{$ D=E 2Fnh sn, h:& AaN G>$ 9k& +7i B/= 8 ah#. - ARN 23c phi4n m^ t trXnh tH giu :! tr4n AND t#o n[t w<&p tIcx MX trXnh tH ny 0i FRng * b4n - 7 ; 15u n[t li4n <t yu Mì ADN n4n ARN cI thZ t"ch <h|i ANDL - /5u I 13i <Yp AND 23c hXnh thnh trong wbong bIng phi4n m^x M ph_n l}i cQ5 ensyme polymer51e cI "i lHc thp Mì AND n4n 23c phIng thUch r5L 1c. 5/ h{$ D=E 2Fnh sn, h:& AaN 9 h=>$ B/= 8 ah#. - !_n 1H cI mJt cQ5 n[t M MNng ng5y tr2`c _u <Yo di cQ5 ARN9 nh2ng <hng cI trXnh tH oligo (; - u t0 Rho l protein g(n ARN8 G Nhpn diPn c"c trXnh tH giu ! M ngho : G !I <4nh Z ARN ch#y gu5 G mo#t dng nh2 melic51e Z <Yo ARN <h|i ADN 1d. IJ$ GiK) &hi(n )* O / b# nhn hk .
ARN polymer51e rr chWu tr"ch nhiPm tVng h3p mARN9 ARN polymer51e r tVng h3p rARN9 ARN polymer51e rrr phi4n m^ cho r5 c"c ARN cI <Uch th2`c nh|9 tARN9 ARN A/ - mARN chR5 thng tin cQ5 mdt gen (monoci1tron mARN - tu" trXnh phi4n m^ phRc t#p h)nL o _u A mARN cI g(n th4m wchIpx (!A$ l D-methylgu5no1ine9 cNn cu0i mARN phU5 cI th4m wuix poly5denin di ,>>-*>> nucleotid - a]n phi4n m^ _u ti4n cNn gTi l tin mARN <hng 23c 1+ deng trHc tip m ph]i gu5 bin ViL 1e. C\= 2{$ ,iEn G#<n $U" $E$ ,Vn O / b# nhn hk . - Nhiu gen cQ5 t bo nhn thpt cI tUnh gi"n o#n - !"c o#n m^ hI5 cho protein 23c gTi l eFon Fen <c Mì o#n <hng m^ hI5 23c gTi l intronL - a]n phi4n m^ _u ti4n tRc l tin mARN chR5 c] trXnh tH phi4n m^ cQ5 eFon M intronL - !"c intron <hng m^ hI5 protein 23c c(t rSi r59 cNn c"c eFon m^ hI5 protein 23c n0i l#i Mì nh5u Z t#o ARN tr2Kng thnhL tu" trXnh ny 23c gTi l c(t n0iL Hj. IJ$ GiK) &hi(n )* $E$ 2AaN M# AaNf&#9B)V2"!V I. - rARN 23c phi4n m^ rt m#nh - $romoter thHc g{m ,A> bp gii5 c"c MW trU -,7 M G - $romoter MP tinh n%m K -,*>> M -*>> l n)i Fp hng cQ5 polymer51e tr2`c <hi chuyZn dng n promoter thHc - Trong Mqng MP tinh cI nhiu b]n 15o > hoJc =, bp cQ5 promoter thHc - /]n phEm phi4n m^ _u ti4n l tin rARN 7A/ (pre-rARN 15u I 23c c(t thnh *=9 ,= M A9=/ H1. SZ &hi(n )* bOi AaNf&#9B)V2"!V II. - $hi4n m^ c"c ARN thng tin (mARN - $romoter g{m hdp TATA9 !AT M :! Z g(n yu t0 phi4n m^ chuy4n biPt h)n l g(n trHc tip Mì ARN-polymer51e rrL - TrXnh tH t\ng c2Sng <Uch thUch phi4n m^ m#nh9 cI tUnh chuy4n biPt M t"c dng t F5 - a]n 15o l hnARN 23c g(n !A$ M cI trXnh tH AA;AAA K _u Z c(t <h|i polymer51e M tVng h3p ui polyA HH. 01 23 + 2Fnh Z $h=n, $U" $E$ h>& TATA' CAAT + GC O / b# nhn hk . - TATA8 -*A h5y -> K dng Mpt cI M[ M -> n -,*> bp K ADN nm men - !AAT8 -7> bp h5y -=> - :!8-,,> bp hoJc th5y Vi tr2`c M 15u hdp !AT HC. 0"i 2w $U" 2Fnh Z Rn, $6_n, Vnh"n$V2. - !I <h] n\ng <Uch thUch phi4n m^ m#nh - !I t"c dng ci19 nh2ng cI thZ ho#t dng t F5 n Mi nghXn b51e9 theo bt <X h2`ng no - !I tUnh chuy4n biPt - !I thZ g(n c"c yu t0 phi4n m^ - CU de8 1teroid g(n yu t0 phi4n m^ thnh phRc h3p g(n 23c Mo enh5ncer lm gi5 t\ng ho#t dng c"c gen "p Rng 1teroid HW. SZ &hi(n )* bOi AaNf&#9B)V2"!V III. - $hi4n m^ rARN A/9 tARN M mdt 10 ARN nh| cQ5 nhn M cQ5 t bo cht (th2Sng >> nucleotid - !I 1H th5m gi5 cQ5 yu t0 phi4n m^ <h"c l TrrrA9 Trrra M Trrr!L TrrrA chuy4n biPt cho no^n boL H^. 0"i 2w $U" TIIIA 2#n, D=E 2Fnh sn, h:& ^S 2 AaN. - TrrrA cI r' 7><D59 chuy4n biPt cho no^n boL - :(n Mì Mqng ndi <iZm 1o"t cQ5 gen rARN A/ - Trrr g(n rARN A/ tH do thnh D/ RN$ <hng th5m gi5 phi4n m^ 23c9 1H tVng h3p rARN A/ bW gi]m Fu0ng cho n <hi TrrrA 23c gi]i phIng trK l#iL H`. Phi(n )* n,6:$. - RetroMiru1 l c"c Miru1 m Mpt liPu di truyn cQ5 ch[ng l ARN9 MU de Miru1 lm 1uy gi]m mifn dWch (mrC - /H tVng h3p ADN t <hun mOu ARN 23c F[c t"c bKi c"c ensyme phi4n m^ ng23c (reMer1e tr5n1cript51e8 g{m * tiZu )n MW A> 55 - @nsyme ny c_n m{i l mdt tARN MQ5 t bo chQ9 g(n <t Mo _u cQ5 retroMiru1L - !huvi <Yp _u ti4n l chuvi l5i ARN-ADNL ARN Miru1 trong chuvi l5i bW hQy bKi RN51emL
/5u I9 mdt m#ch ADN 23c tVng h3p Z t#o r5 chuvi <Yp ADNL !huvi <Yp tUch h3p Mì ADN <z chQL Hc. IJ$ GiK) $U" )* Mi 2=BTn. - B)n MW m^ hI5 h5y cNn gTi l codon g{m cI nucleotid < tip nh5u - 7 codon - codon <hng m^ hI5 cho 55 (;AA9 ;A:9 ;:A 23c gTi l codon M nghy5 h5y codon <t th[c (1top codon - ^ di truyn cI tUnh w1uy tho"ix8 mdt 55 cI nhiu codon m^ hI5 chh tr methionin M tryptoph5n chh cI mdt codon - !odon {ng nghy5 m^ hI5 cho cqng mdt 55 th2Sng cI * b51e _u ti4n gi0ng nh5u9 nh2ng <h"c nh5u K nucleotide thR - Tr mdt 10 ngo#i lP9 m^ di truyn cI tUnh phV bin cho tt c] 1inh MptL - dt codon chh m^ hI5 cho mdt lo#i 559 tr2Sng h3p ngo#i lP l A;: M5 m^ hI5 cho et ndi9 M5 m^ hI5 cho 55 mK _u N-6ormyl ethionin trong t bo nhn nguy4n thQy hoJc methionin trong t bo nhn thptL SZ PhE$ bi 2#n, &hi(n )* O / b# nhn n,=B(n hUB + / b# nhn hk T/ b# nhn n,=B(n hUB !hh cI , lo#i ARN polymer51e chWu tr"ch nhiPm tVng h3p tt c] c"c lo#i ARN T/ b# nhn hk !I lo#i ARN polymer51e8 - ARN polymer51e r tVng h3p rARN - ARN polymer51e rr tVng h3p mARN - ARN polymer51e rrr tVng h3p c"c lo#i ARN <Uch th2`c nh|9 tARN9 ARN A/ mARN chR5 thng tin cQ5 mdt gen (monoci1tron
mARN th2Sng chR5 thng tin nhiu gen n0i tip nh5u (polyci1tron $romoter g{m * trXnh tH8 - TATAAT (hdp TATA h5y $ribno 9 c"ch iZm Fut ph"t -= b51e9 cI b51e trung tm -,> - TrXnh tH TT:A!A cI b51e trung tm -AL Cqng ny th5m gi5 Mo MiPc g(n trHc tip ARN polymer51e
- mdp TATA cI b51e trung tm -*A h5y -> K dng Mpt cI M[ M t -> n -,*> K nm men - mdp TT ho#t dng cI hiPu gu] cqng Mì * trXnh tH t2)ng Rng phU5 tr2`c <ho]ng -7> l !!AAT (hdp !AT M -,,> giu :! cI chRc n\ng li4n <t Mì c"c yu t0 phi4n m^ (T $hi4n m^ M dWch m^ difn r5 {ng thSi DWch m^ b(t _u <hi phi4n m^ <t th[c a]n phi4n m^ 23c 1+ deng ng5y Mo dWch a]n phi4n m^ _u ti4n (pre-ARN ph]i gu5 m^9 <hng gu5 bin Vi c(t n0i (1plicing mì 25 Mo dWch m^ tu" trXnh hpu phi4n m^ <hng g(n chIp :(n th4m chIp (!A$ D-methylgu5no1ine (!A$ K _u A M ui polyA K _u L K _u A M cu0i mARN phU5 th4m ui polyA (,>>-*>>A S# !Enh AaNf&#9B)V2"!V $U" / b# nhn n,=B(n hUB + nhn hk q - :i0ng nh5u8 G !u tr[c 5 phn t+ - uh"c nh5u8 T/ b# nhn n,=B(n hUB T/ b# nhn hk !hh cI , lo#i ARN polymer51e chWu tr"ch !I lo#i ARN polymer51e8 nhiPm tVng h3p tt c] c"c lo#i ARN - ARN polymer51e r tVng h3p rARN - ARN polymer51e rr tVng h3p mARN - ARN polymer51e rrr tVng h3p c"c lo#i ARN <Uch th2`c nh|9 tARN9 ARN A/ T bo cht Nhn
Bi W. SINH TNG HP PaOT@IN

1. C\= <# $U" $E$ iK= G.n +1 2ib#!#) O / b# nhn n,=B(n hUB + / b# nhn hk . - Ribo1om K t bo nhn nguy4n thQy8 g{m * tiZu )n MW8 G TiZu )n MW l`n8 g{m * phn t+ rARN (*/ M A/ M 7 phn t+ proteinL G TiZu )n MW nh|8 cI , rARN ,/ M *, phn t+ protein G rARN cI cu tr[c <hng gi5n phRc t#p do nhiu o#n b(t cJp Mì nh5u nhS cI trXnh tH nucleotide bV 1ungL - Ribo1om K t bo nhn thpt8 g{m * tiZu )n MW9 cu t#o bKi rARN M protein8 G TiZu )n MW l`n8 g{m phn t+ rARN (*=/9 AL=/ M A/ M 7A phn t+ proteinL G TiZu )n MW nh|8 cI , rARN ,=/ M phn t+ protein H. u=E 2Fnh &hi(n )* + M1$h )* Min 2" nh6 h/ n# O E.coli. - uhi ribo1om _u ti4n dWch m^ mARN 23c mdt o#n9 c"c ribo1om <h"c cI thZ tip tec g(n Mo phU5 tr2`c Z dWch m^L - uho]ng ,A-*> ribo1ome cI thZ g(n cqng l[c tr4n mARNL - !"c ribo1ome Fp thnh chuvi tr4n mARN t#o n4n cu tr[c polyribo1ome h5y cNn gTi l poly1omeL C. C\= 2{$ $U" )AaN h#n $h[nh O / b# nhn n,=B(n hUB + / b# nhn hk . Thng tin di truyn l mdt trXnh tH thlng cQ5 cQ5 c"c b51e nu t#o thnh c"c codon tr4n mARN trong I l_n l23t chR5 c"c Mqng 15u8 G 0vn, Phrn, )* O G?= ^g 23c gTi l trXnh tH dOn b5o g{m mdt trXnh tH bV 1ung Mì rARN cQ5 ribo1om Z g(n ribo1om Mo mARNL !"c trXnh tH dOn _u A rt <h"c nh5u M d di K c"c lo#i mARN <h"c nh5u9 nhXn chung9 K c"c t bo nhn thpt u di h)n K t bo nhn nguy4n thQyL i ChX& CAPq Ngoi trXnh tH <hng m^ hI5 K _u A M 9 c"c mARN t bo cht nhn thpt cI g(n th4m wchIpx (!A$ D-methyl gu5no1ine tripho1ph5te K _u AL BJc iZm ny li4n gu5n n 1H t23ng t"c gii5 mARN M ribo1omL G Ph?n )O G?=8 chR5 mdt bd b5 mK _u (th2Sng l A;: G 0vn, )* hX" (coding region cQ5 mARN chR5 c"c codon gui Wnh trXnh tH 5cid 5min cQ5 proteinL TrXnh tH codon 23c Tc theo h2`ng A n tr4n mARNL G Ph?n P/ h{$8 l mdt trong c"c bd b5 <t th[c (codon 1top8 ;AA9 ;:A h5y ;A: G T2Fnh Z Phrn, )* hX" n%m t#i ph_n chm dRt cQ5 phn t+ mARNL !"c trXnh tH M A <hng m^ cNn 23c gTi l c"c Mqng <hng phi4n dWch9 Mit t(t l ;TR (untr5n1l5ted region1 L !"c Mqng <hng phi4n dWch K _u tpn cqng cI chiu di rt <h"c nh5uL G I=ri &#9BA8 m_u ht c"c phn t+ mARN t bo nhn thpt K _u cI th4m ui ,>>-*>> Adenin (polyA L !hRc n\ng cQ5 ui poly(A ch25 r} rngL !h[ng Ing M5i trN gu5n tTng trong MiPc b]o MP mARN <h|i bW thQy gi]i cQ5 eFonucle51e9 nh2ng <hng c_n thit cho 1H dWch m^L dt Mi lo#i mARN nh2 hi1ton-mARN M mARN Mi <huEn <hng cI ui poly(A L W. AaN +kn $h=BKn + $E$ VnAB)V sn, h:& AaNf")in#"$B9 !Bn hV "!V AaN M=B nh\ ' AaN Grn, nhkn. - AaNq G !"c tARN cI , 5nticodon t2)ng Rng Mì 5cid 5min m nI Mpn chuyZn G vi tARN Jc hiPu cho mdt lo#i 5cid 5min ri4ng biPtL G !I thZ cI Mi tARN gi]i m^ cho cqng mdt 5cid 5mimL !"c lo#i tARN chuy4n chK cqng mdt 5cid 5min thX 23c gTi l tARN {ng nhpnL
G !I Mi bd b5 trong m^ di truyn cqng gi]i m^ cho mdt lo#i 5cid 5min9 ngo#i tr methionine M tryptoph5nL Nhing bd b5 {ng lo#i ny 23c gTi l codon chYo9 y l iZm <h"c nh5u gu5n trTng nht gii5 loi ny M loi <h"cL G /0 l23ng c"c tARN bin dng theo loi8 nhn nguy4n thQy >-7>9 nhn thpt A>L G !"c tARN ph]i 23c nhpn bit chuy4n biPt {ng thSi bKi mARN M bKi ensyme g(n 5cid 5min Mo tARN t2)ng Rng (ensyme tARN-5mino5cyl 1ynthet51e L Biu ny gi[p gi]i guyt trK ng#i <hng gi5n trong gu" trXnh dWch m^L G uUch th2`c cQ5 codon l`n h)n 1o Mì 5cid 5min9 n4n nu mdt 5cid 5min nhpn bit M g(n trHc tip l4n mdt codon tr4n mARN thX nI 1c c"ch Mì 5cid 5min t2)ng Rng Mì codon < cpn Z hXnh thnh li4n <t peptidL f AaNf")in#"$B9 !Bn hV "!Vq G !hh cI mdt lo#i tARN-5mino5cyl 1ynthet51e cho mdt lo#i 5cid 5minL !I *> lo#i 5cid 5min <h"c nh5u cu tr[c n4n c_n ph]i cI tì *> lo#i tARN-5mino5cyl 1ynthet51e <h"c nh5uL G /H nhpn diPn chUnh F"c tARN cQ5 1ynthet51e thng gu5 MiPc nhpn diPn 5nticodonL @nsyme tARN-5mino5cyl 1ynthet51e ph]i phq h3p M chTn [ng tARN8 tARN chh [ng cho mdt 1ynthet51e thX 23c gTi l tARN duy nhtL !"c lo#i tARN li4n gu5n n cqng mdt 55 thX 23c gTi l tARN {ng nhpnL G a2`c Tc 1+5 15i chh 23c thHc hiPn K gi5i o#n 5mino5cyl hI5 bKi chUnh ensyme tARN5mino5cyl 1ynthet51eL ^. H"i b67$ ")in#"$B9 hX". B67$ 18 mXnh thnh 5mino5cyl-5denyl5te ho#t hI58 - Acid 5min ph]n Rng Mì AT$ cI ensyme F[c t"c Z t#o thnh 5mino5cyl 5denyl5te ho#t hI5L - $yropho1ph5t t#o r5 bW thQy gi]i thnh * phn t+ pho1ph5t M c)L $h]n Rng ny t#o r5 mdt n\ng l23ng thQy gi]i tH do t2)ng 0i c5o9 Ey ph]n Rng theo chiu t#o 5mino5cyl-A$ trung gi5nL B67$ H8 mXnh thnh 5mino5cyl-tARN8 Amino5cyl-A$ 23c chuyZn tì phn t+ tARN t2)ng Rng b%ng 1ynth51eL `. CE$ AaN + $E$ $#M#n PhOi G?=. TrXnh tH m^ cQ5 phn t+ mARN 23c <hKi _u Mì bd b5 A;:9 rt him hoi c"c bd b5 <h"c (A;;9 ;;: ho#t dng nh2 bd b5 <hKi _uL !hh cI mdt codon m^ hI5 cho et nh2ng trong t bo cht l#i hiPn diPn * lo#i tARN cI <h] n\ng m5ng et n <t h3p Mì codon I K nhn nguy4n thQy8 G tARNmet <t h3p Mì codon A;: n%m gii5 phn t+ mARN9 cI nhiPm Me g(n et Mo chuvi polypeptide G tARNiet <t h3p Mì codon A;: <hKi dng9 g(n et mK _u Mo chuvi polypeptideL o Mi <huEn9 tARNiet <hKi dng <hng chh Mpn chuyZn et m cNn Mpn chuyZn 6ormyl-methionin8 tARN ny gTi l tARN6et (h5y 6et 0i Ph=4n Nhn hk B> b" PhOi G?= A;: (:;: A;: AaN PhOi G?= tARN6et tARNiet AaN )"n, "" ormyl-methionin ethionin c. IJ$ GiK) $U" !Z PhOi G?= sn, h:& $h=i &V& iMV O +i Ph=4n. Nhpn diPn trXnh tH /hine-D5lg5rno9 n%m g_n iZm Fut ph"t uhng y4u c_u Jc biPt cho 1H <hKi _u ph]i n%m g_n Mì iZm <hKi _u A cQ5 mARNL /H phi4n dWch cI thZ b(t _u K bt cR iZm no tr4n phn t+ mARN9 chRng t| tr2`c bd b5 <hKi _u ^ cI mdt trXnh tH /hine-D5lg5rnoL !"c mARN t bo nhn nguy4n thQy cI chR5 tì Mi Mqng m^ hI59 [ng h)n l Mi <huEn th2Sng cI polyci1tron mARNL BJc biPt9 r} nht l mARN chR5 operon m^ hI5 cho lo#i protein <h"c nh5u K l5c operonL !"c phn t+ tARN lun lun g(n Mì ribo1om nh2 nhing phRc b51e cI chR5 :T$ M yu t0 proteinL mARN M phRc h3p tARN g(n tr2`c ti4n Mo b"n )n MW >/9 c] * b2`c ny u F]y r5 tr2`c <hi A>/ g(n Mo Z t#o r5 phR h3p D>/ cho MiPc n0i diL
- Difn bin cQ5 1H <hKi _u dWch m^8 G >/ g(n Mì r t#o thnh phRc >/-r,-r 15u I g(n Mo trXnh tH /D G r* t#o phRc yu t0 Mì :T$ M tARN <hKi _u l r*-:T$-6et-tARNi6et9 phRc ny 15u I g(n Mo phRc >/-r-mARN-r, G A>/ g(n Mo >/ Z b(t _u gu" trXnh dWch m^ d. IJ$ GiK) $U" !Z PhOi G?= sn, h:& $h=i &V& iMV O / b# nhn hk . TrXnh tH dOn mARN nhn thpt <hng 23c Fem nh2 trXnh tH /hine-D5lg5rno K nhn nguy4n thQyL B_u tpn cqng cQ5 rARN ,=/ (t2)ng 2)ng Mì rARN ,/ K Mi <huEn <hng cI trXnh tH !!;!!L ad b5 A;: cI thZ chh l bd b5 m^ hI5 bXnh th2Sng nh2ng cng cI thZ l bd b5 <hKi _uL ARN K nhn thpt l monoci1tron9 ch[ng chh chR5 mdt trXnh tH m^ hI5 M <hKi _u9 th2Sng F]y r5 K bd b5 <hKi _u A;: trong mARNL o nhn thpt et-tARNiet ph]i 23c g(n <t tr2`c Mo tiZu )n MW 7>/L Biu I cho thy r%ng 0i m^ (!A; tr4n tARN-<hKi _u ^ nhpn r5 A;: trong <hi 7>/ 5ng guYt tr4n mARNL uos5< Fy dHng m hXnh <hKi _u 1inh tVng h3p protein K t bo nhn thpt8 G Tr2`c ti4n )n MW 7>/ cQ5 ribo1om g(n Mo _u tpn cqng cQ5 mARN9 15u I di chuyZn dTc theo mARN tì <hi gJp bd b5 <hKi _u A;:L G N\ng l23ng cho 1H di chuyZn ly t 1H thQy phn AT$ M 1H t2)ng t"c <hKi _u Mì mARN cI thZ li4n gu5n tì cu tr[c chIp A (!A$ L G A c"c tr2Sng h3p <hKi _u nhn thpt F]y r5 t#i bd b5 <hKi _u A;:9 h_u nh2 <hng dqng tì :;:L e. Di/n bi/n D=E 2Fnh PhOi G?= M1$h )* O / b# nhn hk . uos5< Fy dHng m hXnh <hKi _u 1inh tVng h3p protein K t bo nhn thpt8 - 7>/ g(n Mì !A$ M g(n tip Mì c"c yu t0 <hKi _u er* M er - 7>/ g(n Mì et-tARNi - 7>/ guYt dTc theo mARN dN tXm A;: M c_n n\ng l23ng AT$ Z th"o Fo(n n[t <&p tIc - 7>/-er*-:T$-et-tARNiet-er t2)ng t"c Mì >/ Z b(t _u dWch m^L 1j. SZ n8i Mi $U" D=E 2Fnh M1$h )* O / b# nhn hk + nhn n,=B(n hUB. 11. Di/n bi/n P%# Mi $h=i &V& iMV O / b# nhn hk + nhn n,=B(n hUB. - tu" trXnh phi4n m^ tr4n mARN 23c n0i di theo h2`ng A-.9 chuvi polypeptide 23c b(t _u tVng h3p K _u tpn cqng NL tu" trXnh ny gi0ng nh5u K t bo nhn M nhn nguy4n thQyL - /H n0i di cI nhiu chu <XL - /H n0i di c_n n c"c yu t0 n0i diL G o Mi <huEn9 c"c yu t0 ny l @-Tu9 @-T19 @-:L G o t bo nhn thpt8 @-, (t2)ng Rng Mì @-Tu 9 @-* (t2)ng Rng Mì @-: - Tr4n ribo1om cI * MW trU A M $8 G CW trU A8 l MW trU 5mino5cyl (h5y 5cceptor G CW trU $8 l MW trU peptidyl9 n0i gii5 phRc h3p peptidyl-tARNL - Difn bin cQ5 gu" trXnh <Yo di dWch m^8 G o iZm <hKi _u mvi chu <X9 chh cI MW trU $ bW chim chv b%ng mdt peptidyl-tARN chR5 chuvi polypeptide mì 1inh g(n Mì tARN m5ng 55 M5 23c th4m MoL G CW trU $ 23c going hng Mì codon < 15u ^ 23c dWch m^9 trong <hi I MW trU A going hng Mì codon < tip cQ5 mARNL k[c ny9 MW trU A tr0ngL at <X 5mino5cyl-tARN (ngo#i tr tARN-<hKi _u u Mo MW trU A9 ch[ng l2u l#i y nu 5nticodon <h`p m^ Mì codon t#i AL a%ng c"ch ny9 5mino5cyl-tARN chUnh F"c 23c lH5 chTn M chTn 23c [ng 5cid 5min cho 1H hXnh thnh chuvi polypeptide mì 1inhL CiPc g(n ny l g(n phRc h3p gii5 @-Tu hoJc @-, Mì :T$ gi0ng nh2 tARN-<hKi _u g(n <t trong 1u0t gi5i o#n <hKi _uL G Tip theo l gu" trXnh thQy phn :T$ gi]i phIng :D$ M $i8 :D$ 23c phIng thUch <h|i ribo1om 1c g(n <t Mì nhn t0 n0i di t#o phRc h3p @-Tu:D$ h5y @-,-:D$L BZ nhn t0 n0i di l#i cI thZ th5m gi5 Mo gu" trXnh g(n <t 5mino5cyl-tARN tip theo Mo ribo1om thX :D$ ph]i 23c th5y bKi :T$L
o Mi <huEn9 nhn t0 @-T1 Ing M5i trN trung gi5n cho MiPc chuyZn Vi :D$?:T$9 t2)ng tH c"c nhn t0 cQ5 protein-: li4n gu5n tì 1H t]i n#p tUn hiPu hormoneL G :ii5 5cid 5min 15u cqng cQ5 chuvi mì 1inh Mì 5cid 5min tr4n tARN K MW trU A9 hXnh thnh li4n <t peptidL $h]n Rng ny 23c F[c t"c bKi ensyme peptidyl-tr5n16er51eL ut gu] l tARN K MW trU $ l[c by giS <hng cNn m5ng 5cid 5min hoJc l t"ch <h|i chuvi polypeptideL G a2`c chuyZn MW cu0i cqng cQ5 gi5i o#n n0i di c_n mdt yu t0 n0i di <h"c l @-: hoJc @-* M :T$L :T$ thQy phn thnh :D$ M $iL /H chuyZn MW cI hiPn t23ng F]y r5 {ng thSi8 tARN Mpn chuyZn Fong 23c t"ch <h|i MW trU $ $eptidyl-tARN di chuyZn t MW trU A 15ng MW trU $L Ribo1om chuyZn dWch theo tng bd b5 tr4n mARN9 do I bd b5 tip theo 23c 25 Mo giIng hng Mì MW trU AL - tu" trXnh chuyZn MW n0i di 1c 23c lJp l#i cho n <hi cI mdt bd b5 <t th[c Fut hiPn t#i MW trU AL - C5i trN cQ5 :T$ trong gu" trXnh n0i di ch25 r} rngL /H thQy phn :T$ cung cp n\ng l23ng cho MiPc t#o n4n li4n <t peptid M cho b2`c chuyZn MW9 nh2ng nI cI thZ li4n gu5n mdt c"ch <hng Vn Wnh n mRc d chUnh F"c cQ5 1H chuyZn MWL IJ$ GiK) $h=BKn +1 2#n, D=E 2Fnh n8i Mi M1$h )*. - a2`c chuyZn MW cu0i cqng cQ5 gi5i o#n n0i di c_n mdt yu t0 n0i di <h"c l @-: hoJc @-* M :T$L :T$ thQy phn thnh :D$ M $iL - /H chuyZn MW cI hiPn t23ng F]y r5 {ng thSi8 G tARN Mpn chuyZn Fong 23c t"ch <h|i MW trU $ G $eptidyl-tARN di chuyZn t MW trU A 15ng MW trU $L G Ribo1om chuyZn dWch theo tng bd b5 tr4n mARN9 do I bd b5 tip theo 23c 25 Mo giIng hng Mì MW trU AL - tu" trXnh chuyZn MW n0i di 1c 23c lJp l#i cho n <hi cI mdt bd b5 <t th[c Fut hiPn t#i MW trU AL - C5i trN cQ5 :T$ trong gu" trXnh n0i di ch25 r} rngL /H thQy phn :T$ cung cp n\ng l23ng cho MiPc t#o n4n li4n <t peptid M cho b2`c chuyZn MW9 nh2ng nI cI thZ li4n gu5n mdt c"ch <hng Vn Wnh n mRc d chUnh F"c cQ5 1H chuyZn MWL 1H. 5/ h{$ M1$h )* + $E$ B/= 8 P/ h{$ M1$h )*. - !hu trXnh dWch m^ thHc hiPn th4m <ho]ng ,A55?giy Mo m#ch polypeptideL !hu trXnh 23c chm dRt <hi tr]i gu5 codon <t th[c l ;AA9 ;A: M ;:AL - o b2`c <t th[c9 c"c m^ <t th[c <hng cI 5nticodonL Th5y Mo I l c"c yu t0 <t th[c (R lm <t th[c gu" trXnh dWch m^L - o Mi <huEn cI yu t0 phIng thUch8 R-, (cho ;AA hoJc ;A: 9 R-* (cho ;AA hoJc ;:A 9 R- (uUch thUch R-* M R, L o y cNn c_n yu t0 n0i di @-: M yu t0 phIng thUch ribo1om (RR chuy4n biPtL - o t bo nhn thpt chh cI mdt yu t0 phIng thUch eRL 0i Ph=4n E.coli T/ b# nhn hk R-, (cho ;AA hoJc ;A: eR (phq h3p Mì c] 1top R-* (cho ;AA hoJc ;:A codon R- (uUch thUch R-* M R, - /H <t th[c li4n gu5n n MiPc phn gi]i li4n <t gii5 5cid 5min cu0i cqng cQ5 chuvi peptid mì 1inh M tARN cu0i cqngL $h]n ny 23c F[c t"c bKi ensyme peptidyl tr5n16er51e cQ5 ribo1om M Ni h|i :T$L NhS I polypeptide hon chhnh 23c phIng thUchL - /5u I9 * tiZu )n MW ribo1om M mARN 23c phIng thUchL Tt c] c"c thnh ph_n th5m gi5 chu trXnh u 23c 1+ deng l#iL 1C. C. $h/ $h3nh LE$ hX" D=E 2Fnh M1$h )* ")in#"$B9 hX" AaN. - dt tARN cI thZ nhpn th4m mdt 5cid 5min <hng [ng hoJc ensyme 5mino5cyl-tARN 1ynthet51e dqng tARN cI 5nticodon <hng chuy4n biPt cho c"c 5cid 5min 23c g(n Mo nIL - dt 10 5cid 5min cI cu tr[c t2)ng tH Mì 5cid 5min c_n 23c 25 Mo cho n4n c"c ensyme 5mino5cyl-tARN 1ynthet51e ph]i cI c"ch thRc chTn [ngL - CU de8 @nsyme i1oleucyl-tARN 1ynthet51e phn biPt 23c c"c 5cid 5min nh| h)n i1oleucin M c"c 5cid 5min < n2`c9 c{ng <nh h)nL !) ch iu chhnh nh2 15u8 - @nsyme i1oleucyl-tARN 1ynthet51e cI * MW trU t"c dng8
G CW trU A8 F[c t"c thnh lpp 5mino5cyl-tARN G CW trU m8 K 1"t ng5y MW trU A9 thQy phn 5mino5cyl-tARN Z co r5 5cid 5min tH do M tARN - !"c b2`c 1+5 15i 5mino5cyl hI5 tARN cQ5 ensyme i1oleucyl-tARN 1ynthet51e8 G !"c 5cid 5min l`n h)n i1oleucin <hng Mo 23c MW trU AL ut gu]8 ph]n Rng <hng F]y r5L G !"c 5cid 5min nh| h)n i1oleucin Mo MW trU A M t#o 5mino5cyl-tARN9 nh2ng cng i Mo 23c MW trU m n4n bW thQy phn thnh * ph_n tARN M 5cid 5minL ut gu]8 ph]n Rng <hng F]y r5L G!hh cI i1oleucin g(n Mo MW trU A t#o i1oleucyl-tARN nh2ng <hng thZ g(n Mo MW trU m M do I <hng bW thQy phnL ut gu]8 Acyl hI5 chUnh F"c i1oleucinL 1W. N,=B(n nhn P/ h{$ !7)' P/ h{$ )=>n $U" D=E 2Fnh M1$h )*. - !"c 15i 1It cQ5 gi5i o#n <t th[c cI thZ F]y r5 do <t th[c 1`m hoJc <t th[c mudn do Tc gu"L - /H Tc gu" do8 G kvi cQ5 bd m"y dWch m^ <hng nhpn r5 codon dng9 5cid 5min 23c 25 Mo M dWch m^ li4n tec gu5 c] Mqng <hng m^ hI5 mARNL G dt dt bin K codon 0i m^ no I lm cho codon 0i m^ ny gi]i m^ 23c codon <t th[cL - /5i 1It 2`c tUnh K th lP ,?>>> g0c 5cid 5min 23c <t h3p M th2Sng nhing lvi ny 23c Wnh MW m <hng gu5n trTng 0i Mì cu tr[c hoJc chRc n\ng cQ5 proteinL S# !Enh !Z M1$h )* O / b# nhn n,=B(n hUB + / b# nhn hk q T/ b# nhn n,=B(n hUB aib#!#)8 g{m * tiZu )n MW G TiZu )n MW l`n8 g{m * phn t+ rARN (*/ M A/ M 7 phn t+ proteinL G TiZu )n MW nh|8 cI , rARN ,/ M *, phn t+ protein DWch m^ F]y r5 ng5y <hi 5ng phi4n m^ n4n t0c d tVng h3p protein nh5nh )AaN8 G <hng cI chIp K _u A G trXnh tH dOn _u A(A;TR ng(n h)n G cI nhiu Mqng m^ hI5 (polyci1tron G trXnh tH <hng dWch m^ K _u tpn cqng (;TR <hng cI ui poly(A AaNq G /0 l23ng8 >-7> G !odon <hKi _u8 A;: (:;: G Acid 5min mK _u8 ormyl-methionin G tARN <hKi _u8 tARN6et /= 8 PhOi G?=q !I yu t0 <hKi _u8 G r-,8 C5i trN ch25 r} G r-*8 :(n et-tARN (hoJc 6et-tARN Mo ribo1om thnh phRc h3p Mì :T$ G r-8 Ng\n c]n 1H <t n0i cQ5 c"c tiZu )n MW T/ b# nhn hk G TiZu )n MW l`n8 g{m phn t+ rARN (*=/9 AL=/ M A/ M 7A phn t+ proteinL G TiZu )n MW nh|8 cI , rARN ,=/ M phn t+ protein DWch m^ F]y r5 <hi phi4n m^ hon tt n4n t0c d tVng h3p protein chpm9 dWch m^ F]y r5 trong t bo cht cNn phi4n m^ difn r5 trong nhn G cI chIp K _u A l D-methyl :u5no1ine tripho1ph5t G trXnh tH dOn _u A(A;TR di h)n G cI , Mqng m^ hI5 (monoci1tron G trXnh tH <hng dWch m^ K _u tpn cqng (;TR cI ui poly(A G /0 l23ng8 A> G !odon <hKi _u8 A;: G Acid 5min mK _u8 ormyl-methionin G tARN <hKi _u8 tARNiet !I ,> yu t0 <hKi _u8 G er-,8 C5i trN ch25 r} G er-*8 :(n et-tARN (hoJc 6et-tARN Mo ribo1om thnh phRc h3p Mì :T$ G er-8 Ng\n c]n 1H <t n0i cQ5 c"c tiZu )n MW G er-7A9 er-7a9 er-7@9 er-78 NhIm yu t0 li4n gu5n "p Rng cho MiPc g(n chIp A Mo _u mARN M th"o Fo(n cu tr[c bpc * t#o iu <iPn cho ribo1om pu Mo codon <hKi _u G er-A8 :i]i phIng er-* M er- <h|i
ribo1om M gi[p cho tiZu )n MW >/ <t n0i G er-8 Th5m gi5 Mo MiPc t"ch ribo1om thnh c"c tiZu )n MW G :@8 u t0 bin Vi nu :u5nin8 t"i 1inh er-* bKi 1H bin Vi trung gi5n :D$ thnh :T$ trong gu" trXnh t2)ng tH Z t"i 1inh @Tu bKi @-T1 5hOi G?= $h=i &V& iMV8 G >/ nhpn diPn 23c [ng codon <hKi _u cQ5 mARN nhS trXnh tH /hine-D5lg5rno l trXnh tH polypurin ng(n K _u A cQ5 mARN bV 1ung Mì trXnh tH giu pyrimidin K _u cQ5 rARN ,/ G Difn bin cQ5 gu" trXnh <hKi _u dWch m^8 >/ g(n Mì r,M r t#o thnh phRc >/-r,-r 15u I g(n Mo trXnh tH /D r* t#o phRc yu t0 Mì :T$ M tARN <hKi _u l r*-:T$-6ettARNi6et9 phRc ny 15u I g(n Mo phRc >/-r-mARN-r, A>/ g(n Mo >/ Z b(t _u gu" trXnh dWch m^ N8i Mi8 !"c yu t0 n0i di8 G @-Tu8 5ng c"c 5mino5cyl-tARN tì MW trU A cQ5 ribo1om G @-T18 T"i h{i @-Tu G @-:8 Th5m gi5 Mo b2`c chuyZn MW cQ5 chu <X n0i di 5/ h{$8 !"c yu t0 <t th[c8 G R-,8 cho ;AA hoJc ;A: G R-*8 cho ;AA hoJc ;:A G R-8 <Uch thUch R-, M R-* Nh= $?= nRn, 96:n,q G Amino5cyl hI5 tARN8 AT$ -. A$ G $$i G uhKi _u8 :T$ -. :D$ G $i9 <t h3p Mì r-* G 7>/ nhpn diPn 23c [ng codon <hKi _u cQ5 mARN nhS n[t <&p tIc K _u A cQ5 mARN
7>/ g(n Mì !A$ M g(n tip Mì c"c yu t0 <hKi _u er* M er 7>/ g(n Mì et-tARNi 7>/ guYt dTc theo mARN dN tXm A;: M c_n n\ng l23ng AT$ Z th"o Fo(n n[t <&p tIc $hRc 7>/-er*-:T$-ettARNiet-er t2)ng t"c Mì >/ Z b(t _u dWch m^L G @-,8 5ng c"c 5mino5cyl-tARN tì MW trU A cQ5 ribo1om G @-,-Tu8 T"i h{i @-, G @-*8 Th5m gi5 Mo b2`c chuyZn MW cQ5 chu <X n0i di eR8 phq h3p Mì c] 1top codon
AT$ -. A$ G $$i :T$ -. :D$ G $i9 <t h3p Mì er-* AT$ -. AD$ G $i9 <t h3p Mì MiPc g(n chIp M th"o Fo(n mARN (ch25 r} cI b5o nhi4u phn t+ AT$ 23c 1+ deng :T$ -. :D$ G $i9 <t h3p Mì @-, :T$ -. :D$ G $i9 <t h3p Mì @-* :T$ -. :D$ G $i
G N0i di8 :T$ -. :D$ G $i9 <t h3p Mì @-Tu :T$ -. :D$ G $i9 <t h3p Mì @-: G ut th[c8 uhng c_n n\ng l23ng
Bi ^. II HA HOT ING G@N

1. CE$ ,i"i G#<n GiT= hw" D=E 2Fnh sn, h:& &2# Vin. - ThSi <X chuEn bW 15o chYp bd gen - $hi4n m^ nghy5 l 1inh tVng h3p c"c ARNL - ThSi <X <t th[c- dWch m^ t#o r5 nhing phn t+ proteinL H. I1nh n,h" GiT= hw" M6.n,' GiT= hw" ) $U" D=E 2Fnh !"# $h%&. - uiZm 1o"t d2)ng8 l 1H tUch ly cht ho#t hI5 cho n ng2bng Q Z <hKi dng mdt chu trXnh 15o chYp mì9 c_n thit cho 1H cn b%ng Mì MiPc nhn i cQ5 1inh <h0i t boL - uiZm 1o"t m8 uhi cht <im h^m c_n 23c tVng h3p mdt c"ch giì h#n tip ng5y <hi b(t _u chu trXnh 15o chYp tr2`c (MX cI thZ l 1]n phEm cQ5 gen iu hN5 n%m g_n MW trU b(t _u 15o chYp -.phi4n m^ theo 1H 15o chYp L C. O&V2#n 9 ,Fb Sinh +k n# $X #&V2#nb - peron l )n MW phi4n m^ b5o g{m t0i thiZu l Mqng <hKi dng M c"c gen m^ hI5 mARNcho mdt h5y Mi chuvi polypeptideL - /inh Mpt cI operon8 W. Phn bi &2# Vin Q$ $h/ + &2# Vin h#< hX". P2# Vin Q$ $h/ P2# Vin h#< hX" (Repre11or A$ i+" #2 GVn )* hX" :en cI thZ Rc ch :en cI thZ c]m Rng 01 23 ,Nn per5tor ActiM5tor-binding 1ite ChQ$ nRn, Ng\n c]n 1H phi4n m^ T\ng c2Sng phi4n m^ C. $h/ PiK) !#E m D2)ng ^. Phn bi PiK) !#E ) + PiV) !#E M6.n, 2#n, GiT= hw" &hi(n )*. 5iK) !#E ) 5iK) !#E M6.n, P2# Vin 9i(n D="n Repre11or (cht Rc ch ActiM5tor (cht ho#t hI5 0vn, 2Fnh Z ADN GK per5tor ActiM5tor-binding 1ite ,Nn &2# Vin T<# !-n &h4) )AaN uhng (phi4n m^ bW Rc !I (phi4n m^ t#o mARN ch Ts h:& PiK) !#E !Z - uiZm 1o"t Rc ch m - uiZm 1o"t Rc ch d2)ng &hi(n )* - uiZm 1o"t c]m Rng m - uiZm 1o"t c]m Rng d2)ng `. Phn bi $h\ $-) Qn, + $h\ Q$ $h/. CE$ &h-n Qn, !; M]n,. Ch\ Q$ $h/ BIng 1H phi4n m^ B{ng hI5 Ch\ $-) Qn, K gen Z phi4n m^ DW hI5
0"i 2w Th") ,i" &h-n Qn,
c. 5iK) !#E $-) Qn, ) h h8n, 9"$ #!V O E.coli. - k5c operon8 g{m promoter9 oper5tor9 gen cu tr[c9 termin5torL :en cu tr[c g{m8 l5cn9 l5cy M l5cAL G l5cn8 gen m^ hI5 ensyme -g5l5cto1id51e9 mdt ensyme ndi bo thQy phn di15cch5ride l5cto1e cho r5 gluco1e M g5l5cto1e G l5c8 gen m^ hI5 -g5l5cto1ide perme51e9 mdt protein chuy4n chK Ing M5i trN b)m l5cto1e gu5 mng t bo G l5cA8 gen m^ hI5 -g5l5cto1ide tr5n15cetyl51e9 mdt ensyme g(n nhIm 5cetyl cQ5 5cetyl-!oA Mo -g5l5cto1ideL G !hh l5cn M l5c cI M5i trN c_n thit cho MiPc chuyZn hI5 l5cto1eL - $rotein iu hN5 l nhn t0 <Xm h^m (repre11or cI "i lHc Jc biPt Mì trXnh tH oper5tor tr4n ADN n%m gii5 promoter M gen cu tr[c _u ti4nL !I thZ F]y r5 h5i tXnh hu0ng8 G uhi mi tr2Sng chh cI l5cto1e8 dt Mi phn t+ l5cto1e cI cu tr[c g5l5cto1e-(,-.7 -gluco1e bW bin Vi thnh 5llol5cto1e cI cu tr[c g5l5cto1e-(,-. -gluco1e nhS -g5l5cto1id51eL Allol5cto1e l cht c]m Rng cQ5 lac operon9 g(n Mo repre11or lm th5y Vi cu hXnh cQ5 repre11orL $hRc h3p 5llol5cto1e- repre11or <hng thZ g(n lu Mo oper5tor n4n oper5tor 23c gi]i phIng ARN-polymer51e cI thZ Tc oper5tor Z phi4n m^ c"c gen cu tr[c tr4n operonL G uhi mi tr2Sng <hng cI l5cto1e8 repre11or M5 tVng h3p 23c gi]i phIng <h|i 5llol5cto1e cI thZ g(n Mo oper5tor9 ng\n c]n 1H phi4n m^ c"c gen cu tr[c cQ5 lac operonL d. 0"i 2w $U" #&V2#n GiT= hw". - peron iu hN5 th2Sng Fuy4n 1inh r5 c"c protein Rc ch (repre11or K mRc d thp MX cI promoter <Ym hiPu 1utL - Repre11or cI thZ g(n h5y <hng g(n Mo oper5tor Z iu hN5 1H phi4n m^L - CU de8 G uhi mi tr2Sng <hng cI l5cto1e8 repre11or g(n Mo oper5tor9 ng\n c]n 1H phi4n m^ c"c gen cu tr[c cQ5 lac operonL G uhi mi tr2Sng cI l5cto1e8 repre11or g(n cht c]m Rng l 5llol5cto1e <hng cNn g(n 23c tr4n oper5tor n4n 1H phi4n m^ F]y r5L e. B-n $h\ $U" $h\ bi/n M1 9k& hK. - /H th5y Vi cu hXnh trong phn t+ protein do mdt phn t+ <h"c g(n Mo gTi l 1H bin hXnh dW lpp thZL - CU de8Allol5cto1e l cht c]m Rng cQ5 lac operon9 g(n Mo repre11or lm th5y Vi cu hXnh cQ5 repre11or M lm th5y Vi MW trU g(n cQ5 repre11or Mo oper5torL 1j. 5iK) !#E Q$ $h/ ). 0"i 2w $U" Q$ $h/ ,8$ + Gtn, Q$ $h/. uiZm 1o"t Rc ch m trong tVng h3p 5cid 5min tryptoph5nL Tryptoph5n 23c tVng h3p theo A b2`c9 mvi b2`c 23c F[c t"c bKi mdt ensyme Jc hiPuL !"c gen "p Rng cho A ensyme n%m tr4n mdt operonL!) ch <iZm 1o"t nh2 15u8 - :en iu hN5 1]n 1inh r5 mdt lo#i protein <hng chRc n\ng gTi l cht Rc ch g0c (5porepre11or L - uhi tryptoph5n 1]n Fut d2 th5 trK thnh cht {ng Rc ch (corepre11or g(n Mo cht Rc ch g0c (5porepre11or t#o thnh phRc h3p Rc ch chRc n\ng(repre11or L $hRc h3p Rc ch chRc n\ng g(n Mo oper5tor M Rc ch 1H phi4n m^ cQ5 A gen cu tr[c tr4n operonL - uhi n{ng d tryptoph5n gi]m Fu0ng9 tryptoph5n t"ch <h|i cht Rc ch g0c M cht Rc ch g0c rSi <h|i oper5tor thX ARN-polymer51e phi4n m^ t#o mARNL 11. Hi= Qn, ,9=$#!V ,9=$#!V VV$ . - uhi cI mJt gluco1e thX Mi <huEn E.coli <hng c_n thit ph]i phn gi]i mdt lo#i 2Sng no <h"c M c"c gen m^ hI5 cho c"c ensyme phn gi]i c"c 2Sng y cng Ing l#iL - miPu Rng gluco1e8 <hng cI gluco1e nh2ng cI l5cto1e -. l5c operon 23c c]m RngL Nu cI gluco1e thX 1H c]m Rng cQ5 l5c operon 1c <hng F]y r5L - miPn t23ng ny l Rc ch dW hI5L - $hRc hP cQ5 * cht ho#t hI5 Rc ch dW hI5 l cA$ M !A$L - ARN-polymer51e g(n cI hiPu gu] Mo promoter nu phRc h3p cA$-!A$ 23c g(n Mo MW trU nyL - uhi mRc gluco1e t\ng thX l23ng cA$ gi]m M cI Ut phRc hP cA$-!A$ thX ARN-polymer51e <hng g(n 23c nhiu M l5c operon Ut 23c ho#t hI5L 1H. 5iK) !#E $-) Qn, M6.n,.
!"c ensyme cQ5 gu" trXnh tr5o Vi 5r5bino1e K E.coli nhS 1H <iZm 1o"t cQ5 repre11or cI <h] n\ng thHc hiPn iu hN5 d2)ng tUnh M m tUnhL - Ar5bino1e l 2Sng m 1H chuyZn hI5 c_n ensyme m^ hI5 bKi c"c gen 5r5a9 5r5A9 5r5DL - uhi mi tr2Sng <hng cI 5r5bino1e8 :en 5r5! n%m tr2`c promoter cQ5 nhIm aAD t#o r5 cht Rc ch cQ5 nhIm aAD g(n Mo oper5torL - uhi mi tr2Sng cI 5r5bino1e8 5r5bino1e g(n Mì repre11or lm bin Vi cu hXnh thnh phRc h3p ho#t hI5 rSi <h|i oper5tor M g(n Mo ini5tor9 t#o iu <iPn cho ARN-polymer51e g(n 23c Mo oper5tor c]m Rng 1H phi4n m^L - $hRc hP cA$-!A$ cng th5m gi5 Mo 1H iu hN5 cQ5 hP th0ng 5r5bino1eL uhi cA$-!A$ g(n Mo promoter thX ho#t hI5 ARN-polymer51e cng g(n Mo cqng MW trUL 1C. 5F) h*) n,6:$. - /H <Xm h^m ng23c gTi l 1H Rc ch bKi 1]n phEm cu0i cng l c) ch iu hN5 cI 1H th5m gi5 Rc ch cQ5 ensymeL - /]n phEm cu0i cqng cQ5 gu" trXnh 1inh tVng h3p cI thZ li4n <t Mì ensyme _u ti4n 23c tVng h3p r5 lm bin hXnh di lpp thZ cQ5 ensyme phong b <h] n\ng F[c t"c ng\n c]n 1H 1]n Fut cQ5 1]n phEm cu0i cqngL - CU du8 N{ng d 1]n phEm cu0i cqng i1oleucin trong E.coli c5o 1c li4n <t Mì ensyme _u ti4n l ensyme threonin-de15min51e bin threonin thnh 5cid -<etobutyric M <Xm h^m t"c deng cQ5 ensyme nyL Do I c"c ensyme tuy MOn 23c hXnh thnh nh2ng <hng ho#t dng cho n <hi i1oleucine dqng htL
Bi `. DI TaN 0I 5HN
1. N(= C GJ$ GiK) Mi 2=BTn +i Ph=4n. - Truyn thng tin mdt chiu t t bo cho (donor 15ng t bo nhpn (recipient - T#o h3p t+ tng ph_n (merosygote 8 ThZ cho chh chuyZn mdt o#n MQ5 bd gen 1ng thZ nhpn n4n chh l2bng bdi K mdt ph_n9 c"c ph_n <h"c )n bdiL - ad gen th2Sng chh l mdt phn t+ ADN tr_n9 n4n chh cI mdt nhIm li4n <t gen M t"o tV h3p thHc cht l l5i phn t+ tng o#n t2)ng RngL H. x=\ &hE $U" C Mwn, / b# E.coli 2#n, n,hi(n $Q=. - E.coli a8 t bo chQ cQ5 ph5ge d^y T - E.coli !8 t bo chQ cQ5 ph5ge mdt 13i nh2 j,D7 - E.coli u,8 t bo chQ cQ5 ph5ge C. H"i PiK= !"# $h%& O E.coli. - uiZu 15o chYp thet5 ( 8 G $hn t+ /DN cudn Fo(n cI d#ng hXnh trNn G /5o chYp b(t _u t mdt iZm gTi l iZm Fut ph"t ri M i theo h5i chiu gu5nh MNng trNn G uhi ADN MNng trNn 5ng 15o chYp 1c thy d#ng ADN w con m(tx G !h b5 15o chYp l5n d_n9 cu0i cqng t#o r5 h5i phn t+ ADN l5i - uiZu 15o chYp l\n MNng8 G j]y r5 K gu" trXnh tip h3p cQ5 Mi <huEn G dt m#ch cQ5 ADN 23c c(t bKi ensyme G ADN mì 23c tVng h3p theo chiu A-. W. aV&9i$#n 9 ,Fb Replicon l )n MW 15o chYp dc lppL ad m"y di truyn cQ5 c"c 1inh Mpt nguy4n thQy th2Sng cI , repliconL ^. C. $h/ <# h:& ; Phrn, h#n #n. T#o h3p t+ <hng <hon ton8 Mpt liPu di truyn t t bo cho chh chuyZn mdt ph_n gu5 t bo nhpn8 G :en cQ5 t bo cho gen ngo#i 1inh G :en cQ5 t bo nhpn gen ndi 1inh `. /= 8 G6:$ 2=BTn i !"n, f hF ,Vn NST $X 2=BTn D=" G6:$ Phrn,b Gi-i h3$h. Trong gu" trXnh truyn yu t0 9 hon ton <hng cI mdt o#n ADN no cQ5 N/T 23c truyn gu5 t bo nhpn MX8 G u t0 cI <h] n\ng 15o chYp dc lpp9 <hng li4n gu5n n nhing yu t0 di truyn <h"c G uhi t bo G tip h3p Mì - nI MOn gii nguy4n <iZu gen cQ5 mXnh G /5u <hi tip h3p F]y r59 c] h5i t bo u trK thnh G c. T/ b# H2 9 ,Fb 0F !"# Phi 9"i H2 +7i f hF <# G6:$ Ei s h:& NST $"# h.n Phi 9"i i +7i f. - u t0 cI thZ g(n Mo N/T cQ5 Mi <huEn9 t bo Mi <huEn ny 23c gTi l m6rL - uhi I nI <hng cNn <h] n\ng 15o chYp dc lpp ni5 m 23c 15o chYp bW dng cqng M Mì N/TL
d. i bi/n hnh f Phi n#b 0 Phi n# bi/n hnh H2bgb - $l51mid cI thZ tH nhi4n mt9 <hi I G -. - nh2ng Ut F]y r5 M h_u nh2 <hng cIL - /H 1"t nhpp cQ5 epi1ome Mo N/T thX <hng bn9 epi1ome cI thZ bin Vi ng23c l#i d#ng tH do nh2 l , yu t0 L - uhi gu5y ng23c nh2 th9 epi1ome thhnh tho]ng m5ng theo c"c o#n gen cQ5 N/T theo cqngL - , yu t0 m 1"t nhpp mdt 10 gen N/T 23c gTi l yu t0 L e. Gi7i n<& 9 ,Fb Bi/n n<& 9 ,Fb T?n !8 bi/n n<&b - uhi mdt t bo tip h3p Mì t bo nhpn -9 nI <hng chh chuyZn yu t0 m cNn gen cQ5 <z chQ m nI m5ng theo cqngL - :iì n#p l 1H truyn tUnh tr#ng cQ5 t bo cho 15ng t bo nhpn gu5 L - ain n#p l 1H Fm nhpp cQ5 ADN tr_n h5y tH do 23c phIng thUch bKi , Mi <huEn cho 15ng , Mi <huEn nhpn lm th5y Vi tUnh tr#ng Mi <huEnL - ain n#p l mdt ph2)ng ph"p gu5n trTng trong MiPc t"i tV h3p Mi <huEn tuy r%ng chh F]y r5 K , gu_n thZL 1j. Th3 n,hi) $hQn, )inh b-n $h\ $U" E$ nhn bi/n n<& 9 DNAb - $h"t hiPn cQ5 rederic< :ri66ith8 G ng ph"t hiPn thy 1H bin Vi <huEn l#c d#ng R(rough- nh\n nheo <hng cI n5ng cQ5 Diplococcus pneumoniae thnh d#ng /(1mooth-tr)n cI <h] n\ng t#o n5ngL G D#ng / gy bPnh cI n5ng b%ng poly15cch5ride9 c]n trK b#ch c_u ph" Mb t boL D#ng ny t#o <huEn l#c tr)n l"ng tr4n mJt th#chL G D#ng R <hng gy bPnh9 <hng cI n5ng9 t#o <huEn l#c nh\n nheoL G ThU nghiPm8 Ti4m cho chudt , liu Mi <huEn d#ng / {ng thSi , liu Mi <huEn d#ng / ^ cht MX nhiPt -. chudt MOn cht T m"u chudt cht ng ^ phn lpp 23c d#ng / iZn hXnh -. Biu I cI nghy5 Mi <huEn d#ng / ^ cht MX nhiPt ^ truyn <h] n\ng t#o n5ng cho c"c t bo d#ng R lm cho nI trK thnh d#ng / M tUnh cht ny 23c di truynL - ThU nghiPm cQ5 TLAMery9 cLkeod M cL!5rty8 G Nu t bo / F+ lz b%ng b%ng prote51e (ensyme phn hQy protein hoJc ARN-51e (ensyme phn hQy ARN thX ho#t tUnh bin n#p MOn cNn -. protein M ARN <hng ph]i l t"c nhn gy bin n#p G Nu t bo cht 23c F+ lz b%ng ADN-51e (ensyme phn hQy Jc hiPu ADN thX ho#t tUnh bin n#p <hng cNn ni5 -. ADN l t"c nhn bin n#p 11. CE$ GiT= Pin L-B 2" bi/n n<&b - uUch th2`c o#n ADN8 ,>-,>D d5lton9 chR5 ,>-*> gen (<ho]ng ,?*>>-,?A>> hP gen t bo cho L !"c o#n nh| h)n <hng cI <h] n\ng bin n#pL - T bo nhpn ph]i cI <h] n\ng dung n#p8 G Tr#ng th"i 1inh lz Jc biPt Z nhpn o#n ADN G uh] n\ng thu nhpn ADN t mi tr2Sng - !"c yu t0 t"c dng n <h] n\ng dung n#p8 t"c dng l4n b mJt t bo9 lm th5y Vi <h] n\ng thm gu5 mng hoJc l nhing the thZ b mJtL - CU de8 Tr2Sng h3p E.coli8 Ci <huEn 23c t"c dng Z cI <h] n\ng dung n#p b%ng c"ch F+ lz Mì ion c5nFi hoJc 1hoc< nhiPtL 1H. B" ,i"i G#<n $U" bi/n n<&. f Th) nhk& $U" ADN8 13i ADN m#ch <Yp cQ5 dNng Mi <huEn / 15u <hi chui gu5 mng t bo cQ5 dNng R thX mdt m#ch / 1c bW nucle51e cQ5 t bo nhpn c(t9 cNn l#i mdt m#ch nguy4nL f BN $J&8 ADN cQ5 t bo nhpn R 1c bin tUnh t"ch rSi * m#ch K mdt o#n Z b(t cJp Mì mdt o#n ADN cQ5 t bo cho / M5 chui MoL f S"# $h%&8 15u <hi b(t cJp t#o o#n l5i R-/9 phn t+ ADN 15o chYp t#o r5 * 13i9 mdt 13i <Yp R-R M mdt 13i <Yp <h"c cI m5ng o#n ADN t bo nhpn /-/ 1C. T-i n<& 9 ,Fb T]i n#p l hiPn t23ng chuyZn ADN t t bo cho 15ng t bo nhpn nhS thHc <huEn thZL
1W. H"i GiT= Pin !inh !-n $U" &h",V. $h5ge cI * c) ch 1inh 1]n trong t bo Mi <huEn8 !hu trXnh ti4u gi]i M chu tXnh ti4u gi]i tim En 5L !hu trXnh ti4u gi]i8 - !"c thHc <huEn thZ lm cht t bo chQ gTi l ph5ge dc M ch[ng 1inh 1]n theo chu trXnh ti4u gi]iL - !hu trXnh ti4u gi]i tim En8 G a(t _u <hi 13i ui cQ5 thHc <huEn thZ g(n Mo iZm nhpn tr4n mJt ngoi t bo E.coliL G ng ui co l#i t#o lV thQng Fuy4n thnh M mng t bo M b)m ADN Mo trong t bo9 c5p1id rvng cQ5 thHc <huEn thZ cNn l#i b4n ngoi t boL G T bo E.coli nh5nh chIng thHc hiPn phi4n m^ M dWch m^ c"c gen cQ5 Miru1L G @nsyme _u ti4n t#o r5 1c c(t ADN cQ5 t bo chQL G ad gen cQ5 Miru1 <iZm 1o"t ton bd ho#t dng cQ5 t bo Z t#o r5 c"c cu ph_n cQ5 nI8 ADN Miru19 c5p1id (g{m8 _u 5 diPn9 0ng ui M 13i ui L !h[ng tH r"p l#i Mì nh5u thnh Mirion conL G ut th[c <hi ensyme ly1osyme 23c t#o r5 ph" Mb M"ch t bo Z ,>>-*>> Mirion tho"t r5 M lJp l#i chu trXnh mìL bL!hu trXnh ti4u gi]i tim En8 - $h5ge 1inh 1]n m <hng lm cht c"c t bo chQ gTi l ph5ge n hN5L - !I * <h] n\ng 1inh 1]n8 chu trXnh ti4u gi]i M ti4u gi]i tim En <hng lm cht t bo chQ - $h5ge g(n Mo b mJt t bo E.coli M b)m ADN MoL - ADN cQ5 ph5ge T7 t#o MNng trNn M th5m gi5 Mo , trong * chu trXnh8 GTrHc tip i Mo chu trXnh ti4u gi]i G:(n Mo N/T cQ5 Mi <huEn nhS t"i tV h3p K iZm chuy4n biPt Z <hKi _u chu trXnh ti4u gi]i tim En9 23c gTi l proph5ge - ADN cQ5 ph5ge cng 23c 15o chYp M chi5 u M c"c t bo con nh2 t bo Mi <huEn - $roph5ge cI thZ ngOu nhi4n t"ch r5 <h|i ADN Mi <huEn h5y do t"c dng cQ5 phIng F# h5y hI5 cht thnh ph5ge M b(t _u chu trXnh ti4u gi]i 1^. 0F !"# +i Ph=4n + &h",V $X !Z Gtn, i/n hX"b - !"c t bo Mi <huEn M ph5ge <z 1inh cI 1H {ng tin hI5L - Ci <huEn bin Vi Z ch0ng l#i ph5ge8 G ain Vi mng t bo Z ph5ge <hng b"m Mo 23c G @nsyme c(t giì h#n c(t c"c ADN cQ5 ph5ge - $h5ge bin Vi Z Fm nhpp Mi <huEn - !I tr2Sng h3p c] h5i cqng t{n t#i nh2 trong chu trXnh ti4u gi]i tim En 1`. T-i n<& $X hK $h=BKn 1jj ,Vn NST $U" / b# $h# !"n, / b# nhkn Phrn,b 0F !"#b - T]i n#p chh chuyZn <hng M23t gu" ,-* hP gen cQ5 Mi <huEn MX8 G mP gen cQ5 Mi <huEn di h)n hP gen cQ5 ph5ge tì ,>> l_n G !hh cI c"c gen n%m c"ch nh5u <hng gu" ,?,>> chiu di hP gen mì cI thZ truyn i cqng nh5u G m5y chh nhing gen li4n <t chJt chc Mì nh5u mì cI <h] n\ng cqng nh5u truyn 15ng t bo nhpn 1c. T-i n<& Phrn, GJ$ hi=. Ch# +3 M]. - Th2Sng do ph5ge <iZu $, thHc hiPn - at <X gen no cQ5 Mi <huEn cng 23c t]i n#p - T]i n#p cI 23c do gIi nh_m ADN cQ5 t bo chQ <hi ph5ge tr2Kng thnh - !"c thZ t"i tV h3p )n bdi 23c t#o r5L - CU de8 G Dqng * chQng dt bin Salmonella typhimurium M <h] n\ng tVng h3p 5rgininL G !hQng tVng h3p 23c <U hiPu l ArgG M <hng tVng h3p 23c l ArgG Tin hnh thU nghiPm trong 0ng chi ;9 K "y 0ng h5i ph_n 23c ng\n c"ch Mì nh5u b%ng mng lTc Mi <huEn nh2ng cho ph5ge lTt gu5 G Nh"nh A chR5 Mi <huEn ArgG9 cNn nh"nh a chR5 Mi <huEn Arg-
G /5u mdt thSi gi5n nui cy9 nh"nh a cI Mi <huEn cI <h] n\ng tVng h3p 5rginin -. ph5ge ^ t]i gen ArgG t nh"nh A 15ng nh"nh aL G T_n 10 Fut hiPn l c"c t bo ArgG <ho]ng ,>-AL 1d. T-i n<& GJ$ hi=. Ch# +3 M]. T?n !=\ -i n<&. - !hh li4n gu5n n mdt cem gen Mi <huEn nht Wnh - CU de8 G $h5ge => b"m Mo cem gen TryG - t]i n#p gen tVng h3p tryptoph5n G $h5ge l5mbd5 b"m Mo cem gen :5lG t#o cu tr[c -g5lG t]i n#p gen g5lG Mo t bo nhpn g5lG uhng gi0ng pl51mid 9 ph5ge l5mbd5 chh g(n Mo , MW trU chuy4n biPt9 , _u l gen :5lG M _u <i5 l gen :5l- M gen bioL G Thhnh tho]ng <hi l5mbd5 t"ch <h|i N/T K cu0i ph5ge ti4u gi]i9 nI g^y 15i MW trU t2)ng tH nh2 1H t#o thnh t , m6r G dt o#n N/T cQ5 E.coli g(n Mo N/T ph5ge l5mbd5L $h5ge m5ng , o#n gen cQ5 E.coli cqng M)i gen l5mbd5 23c gTi l ph5ge t]i n#p Jc hiPuL !h[ng cI thZ m5ng gen g5l h5y gen bio Mo E.coli <h"cL G Nu Mi <huEn g5l- 23c nhifm bKi l5mbd5 g5l 1c t#o t bo l2bng bdi , ph_n8g5l- - g5lG G Ci <huEn nhifm {ng thSi bKi ph5ge ho5ng d#i M ph5ge g5lL $h5ge ho5ng d#i 1c hv tr3 ph5ge g5l chuyZn gen g5lG cho t bo g5l- Mì t_n 1ut c5oL - T_n 1ut t]i n#p8
Bi c. IT BIN G@N
1. N,=B(n nhn ,B 2" [ 9 G> bi/n ,Vn $"# h\& O n,6_i. Th3 M]. - uUch th2`c Uch8 gen cI Mqng m^ hI5 protein rt l`n9 cI nhiu b51e cI thZ bW th5y Vi hoJc lm mt chRc n\ng cQ5 genL - BiZm nIng8 mdt 10 gen n%m trong Mqng N/T nh#y c]m h)n Mì 1H ph" hQy?th5y Vi di truyn9 hoJc gen cI chR5 c"c trXnh tH df bW th5y Vi bKi c"c dt bin tH ph"t h)nL CU de8 gen chRng lo#n 1]n 1en cI chR5 iZm nIng (trXnh tH !p: H. CRn $Q $\& G> n# GK GEnh ,iE ?n !8 G> bi/nb 9?n !"# $h%&' 9?n P h=k $-) Qn,. T_n 10 dt bin 23c "nh gi" c\n cR tr4n8 G mdt l_n 15o chYp G mdt l_n phn bo G mdt gi5o t+ G mdt t bo trong mdt th hPL C. n, M]n, P h=k $-) Qn,. uy thupt c]m Rng dt bin ^ 23c 1+ deng8 G Trong cy tr{ng M Mpt nui Z t#o r5 c"c chQng mì G Trong cng nghP Mi 1inh hTc hiPn #i Z t#o r5 c"c chQng Mi 1inh Mpt lm t\ng n\ng 1ut M t#o r5 1]n phEm mìL W. N,=B(n nhn Hk= D=T3nh hti bi/n ^. Phn bi G> bi/n GiK) + G> bi/n G" GiK) +T n,=B(n nhn + hk= D=-. I> bi/n GiK) - hI5 cht - 15i 1It 15o chYp ADN I> bi/n G" GiK) - gen nh]y - c"c lvi <hi 15o chYp cQ5 c"c yt t0 lJp l#i Th5y Vi , b51e trong chuvi Th5y Vi h)n , b51e (t * ADN n hng ngn b51e9 th2Sng l chh Mi b51e !I thZ h{i bin b%ng c"ch uhng h{i bin 23c9 dt chuyZn l#i d#ng ho5ng d#i bin 23c Vn Wnh
CE$ M<n, G> bi/n $U" hin 6:n, h"B h/ $J& b"!V ni .. G Ch=BKn +18 pyrimidin 23c th5y th bKi pyrimidin <h"c h5y purine bKi purine <h"c (A -.:9 ! .T L G I-# $h=BKn8 pyrimidin 23c th5y th bKi purine h5y purine bKi pyrimidin (!?T -.A?: N>i M=n, $U" G> bi/n $vn, n,h"' 9$h n,h" + +r n,h". f I> bi/n $vn, n,h" (dt bin lJn 8 m^ hI5 cho cqng 5cid 5min9 <hng ]nh h2Kng n protein cu0iL CU de8 codon :!A h5y :!: u nghy5 l 5rgininL
`.
f I> bi/n 9$h n,h"8 m^ hI5 cho 5cid 5min <h"c9 cI thZ lm protein <hng cI chRc n\ngL CU de8 Trong chuvi protein -globin gy bPnh thiu m"u h{ng c_u lim9 :A: m^ hI5 cho glut5m5te nu th5y b%ng :;: m^ hI5 cho M5linL f I> bi/n +r n,h"8 m^ hI5 cho codon <t th[c9 cI thZ lm cet proteinL T"c dng cQ5 c"c dt bin M nghy5 tqy thudc protein bW cet b5o nhi4u M mRc d c_n cho ho#t dng cQ5 proteinL c. B-n $h\ $U" G> bi/n 9$h Ph=n, + hk= D=-. - a]n cht8 G Do chn hoJc mt mdt h5y nhiu nu trong Mqng m^ hI5 cQ5 gen lm th5y Vi <hung TcL G Th5y Vi tt c] 5cid 5min phU5 15u dt bin M df t#o 1]n phEm <hng chRc n\ng MX <h"c hln Mì protein bXnh th2Sng h5y c"c <hung Tc 15i th2Sng chR5 c"c codon 1top 1c lm gu" trXnh tVng h3p protein dng 1`mL - mpu gu]8 G Nu 55 mì cI Jc tUnh t2)ng tH 55 bW th5y th (tUnh "i n2`c9 tUnh 5cid -. Ut t"c dng tr4n chRc n\ng protein G Nu 55 bW ]nh h2Kng c"ch <hng F5 MW trU t"c dng cQ5 ensyme h5y t#i cu tr[c c_n thit <h"c -. ]nh h2Kng l`n G Nu 55 dt bin l 55 cI trong cu tr[c bpc *9 (MU de cy1tein -. ]nh h2Kng nghi4m trTng n ho#t tUnh?d Vn Wnh protein d. CE$ M<n, G> bi/n Z nhi(nq h bi/n' Ph; ")in' 9$h Ph=n, + #LB hX" &hE hUB M# $E$ ,8$ Z M# n>i M=n, $U" n, M<n,. ". H bi/nq - !"c b51e cI <h] n\ng t{n t#i ho"n chuyZn gii5 h5i d#ng (<eto -.enol9 5mino -.imino 8 G a51e pyrimidin8 th2Sng cI d#ng <eto (!' chuyZn thnh d#ng enol (!-m 8 T-:9 !-A G a51e purine8 th2Sng cI d#ng 5mino (-Nm* chuyZn thnh imino ('Nm 8 A-!9 :-T - ut gu]8 dOn n b(t cJp nh_m M gy c"c dt bin chuyZn MW trong gu" trXnh 15o chYp b. 5h; ")in8 - cyto1ine -. ur5cil G Nm (chhnh 23c b%ng c"ch lo#i ; MX ; <hng cI tr4n ADN M th5y b%ng ! - A-methylcyto1in-. thymin G Nm (<hng chhnh 23c MX <hng nhpn diPn 23c Thymin lvi - cyto1ine t#i c"c trXnh tH !: (!p: th2Sng bW methyl hI5 -. wiZm nIngx cho dt bin $. I> bi/n 9$h Ph=n,q - !hn hoJc mt nu tr4n mdt 13i - Th2Sng l lvi cQ5 polymer51e <hi 15o chYp c"c o#n lJp l#i cQ5 mdt o#n nuL M. OLB hX" &hE hUB do c"c g0c oFy8 - !"c g0c oFy t\ng trong t bo do chuyZn hI5 oFy hI5 hoJc t"c nhn Mpt lzL - CU de8 gu5nine -. =-hydroFy gu5nine M b(t cJp 15i Mì A t#o ]o chuyZn8 :-! thnh T-A e. H"i E$ nhn ,B G> bi/n $-) Qn,q hX" hS$ + bQ$ L<. ".TE$ nhn hX" hS$8 g{m 7 nhIm - NhIm b51e {ng lng8 !u tr[c hI5 hTc gi0ng c"c prymidin M purine M cI thZ g(n Mo ADN t#i MW trU c"c b51e bXnh th2Sng <hi 15o chYp ADNL aromour5cil (a; 8 l h3p cht nhn t#o dqng rdng rî trong nghi4n cRu9 gi0ng Mì T (cI nguy4n t+ ar th5y cho nhIm methyl 9 b(t cJp Mì A cI <h] n\ng hv bin thnh d#ng enol (a; M b(t cJp Mì : A-T -. A-a; -. a;-: -. :-! :-! -. :-a; -. a;-A -. A-T Aminopurine8 l {ng lng cQ5 A9 cI thZ b(t cJp Mì T hoJc (rt Ut Mì !9 gy chuyZn MW A-T thnh :-! hoJc :-! thnh A-TL !"c {ng lng b51e gy chuyZn MW nh2 gy hv bin tH ph"t - !"c cht hI5 hTc th5y Vi cu tr[c M Jc tUnh b(t cJp cQ5 c"c b51e8 !"c cht <h+ 5min8 MU de
5cid nitr) lm ! -. ;9 methyl!yto1in -. T9 A -. hypoF5nthin <h+ 5min9 hypoF5nthin b(t cJp Mì ! gy chuyZn MWL hydroFyl5min (Nm*m 8 chh <h+ 5min cQ5 !9 cI t"c dng rt giì h#nL !"c cht 5l<yl hI58 gy dt bin b%ng c"ch8 Th4m nhIm methyl h5y ethyl Mo : t#o b51e {ng lng cQ5 A dOn n 1H b(t cJp bV 1ung 15i t purin do : ^ bW 5l<yl hI5 t#o lv hVng tr4n ADN9 <hi 15o chYp cI lm Rt m#ch ki4n <t chYo gii5 c"c m#ch cQ5 mdt hoJc c"c phn t+ ADN <h"c nh5u lm mt nuL - !"c t"c nhn chn Mo ADN ( lm lPch <hung 8 !"c cht pro6l5Min9 c5m 5cridin9 ethidium bromide l c"c phn t+ 5 MNng phlng t2)ng t"c Mì c"c b51e cQ5 ADN M chn Mo gii5 ch[ngL /H chn lm wd^nx 13i i ADN M ADN polymer51e bW w"nh l5x chn b51e Mo 0i diPn nhiu h)n bXnh th2Sng9 lm lPch <hung ADN t#o thnhL - !"c t"c nhn th5y Vi cu tr[c ADN8 - !"c phn t+ l`n g(n Mo b51e trong ADN M lm ch[ng trK thnh <hng m^ hI5L CD8 N-5cetoFy-*5cetyl5mino6luorene (NAAA - !"c t"c nhn gy li4n <t chYo trong M gii5 c"c 13iL CD8 c"c p1or5len cI trong mdt 10 r5u M dqng trong iu trW mdt 10 bPnh M d5L -!"c cht hI5 hTc gy Rt 13i ADNL CD8 c"c peroFid - !"c hydroc5rbon 5 MNngL CD8 c"c bensypyrene cI trong F\ngL b.TE$ nhn bQ$ L<q - aRc F# ion hI58 ti5 ;C9 ti5 1j. B8n E$ nhn hX" hS$ ,B G> bi/nq b"!V Gtn, Gn, h"B Gsi $\= 2{$ + GJ$ 3nh bN $J& $U" $E$ b"!V $hn +# ADN h"B Gsi $\= 2{$ ADN. - NhIm b51e {ng lng8 !u tr[c hI5 hTc gi0ng c"c prymidin M purine M cI thZ g(n Mo ADN t#i MW trU c"c b51e bXnh th2Sng <hi 15o chYp ADNL aromour5cil (a; 8 l h3p cht nhn t#o dqng rdng rî trong nghi4n cRu9 gi0ng Mì T (cI nguy4n t+ ar th5y cho nhIm methyl 9 b(t cJp Mì A cI <h] n\ng hv bin thnh d#ng enol (a; M b(t cJp Mì : A-T -. A-a; -. a;-: -. :-! :-! -. :-a; -. a;-A -. A-T Aminopurine8 l {ng lng cQ5 A9 cI thZ b(t cJp Mì T hoJc (rt Ut Mì !9 gy chuyZn MW A-T thnh :-! hoJc :-! thnh A-TL !"c {ng lng b51e gy chuyZn MW nh2 gy hv bin tH ph"t - !"c cht hI5 hTc th5y Vi cu tr[c M Jc tUnh b(t cJp cQ5 c"c b51e8 !"c cht <h+ 5min8 MU de 5cid nitr) lm ! -. ;9 methyl!yto1in -. T9 A -. hypoF5nthin <h+ 5min9 hypoF5nthin b(t cJp Mì ! gy chuyZn MWL hydroFyl5min (Nm*m 8 chh <h+ 5min cQ5 !9 cI t"c dng rt giì h#nL !"c cht 5l<yl hI58 gy dt bin b%ng c"ch8 Th4m nhIm methyl h5y ethyl Mo : t#o b51e {ng lng cQ5 A dOn n 1H b(t cJp bV 1ung 15i t purin do : ^ bW 5l<yl hI5 t#o lv hVng tr4n ADN9 <hi 15o chYp cI lm Rt m#ch ki4n <t chYo gii5 c"c m#ch cQ5 mdt hoJc c"c phn t+ ADN <h"c nh5u lm mt nuL G !"c t"c nhn chn Mo ADN ( lm lPch <hung 8 !"c cht pro6l5Min9 c5m 5cridin9 ethidium bromide l c"c phn t+ 5 MNng phlng t2)ng t"c Mì c"c b51e cQ5 ADN M chn Mo gii5 ch[ngL /H chn lm wd^nx 13i i ADN M ADN polymer51e bW w"nh l5x chn b51e Mo 0i diPn nhiu h)n bXnh th2Sng9 lm lPch <hung ADN t#o thnhL G !"c t"c nhn th5y Vi cu tr[c ADN8 *=L !"c phn t+ l`n g(n Mo b51e trong ADN M lm ch[ng trK thnh <hng m^ hI5L CD8 N-5cetoFy-*5cetyl5mino6luorene (NAAA
*L !"c t"c nhn gy li4n <t chYo trong M gii5 c"c 13iL CD8 c"c p1or5len cI trong mdt 10 r5u M dqng trong iu trW mdt 10 bPnh M d5L >L !"c cht hI5 hTc gy Rt 13i ADNL CD8 c"c peroFid ,L !"c hydroc5rbon 5 MNngL CD8 c"c bensypyrene cI trong F\ngL 11. NhX) b"!V Grn, Gn,q b2#)#=2"$i9 ^fB= + ")in#&=2inV. N/= $. $h/ + $E$ M<n, $h=BKn +1. aromour5cil (a; 8 l h3p cht nhn t#o dqng rdng rî trong nghi4n cRu9 gi0ng Mì T (cI nguy4n t+ ar th5y cho nhIm methyl 9 b(t cJp Mì A cI <h] n\ng hv bin thnh d#ng enol (a; M b(t cJp Mì : A-T -. A-a; -. a;-: -. :-! :-! -. :-a; -. a;-A -. A-T Aminopurine8 l {ng lng cQ5 A9 cI thZ b(t cJp Mì T hoJc (rt Ut Mì !9 gy chuyZn MW A-T thnh :-! hoJc :-! thnh A-TL 1H. NhX) $E$ $h\ h"B Gsi $\= 2{$ + bN $J& !"iq Ph; ")in + "9PB9 hX". N(= $. $h/ ,B bN $J& !"i h"B 9) GQ )<$h' h#J$ )\ n= !"c cht <h+ 5min8 MU de G 5cid nitr) lm ! -. ;9 methyl!yto1in -. T9 A -. hypoF5nthin <h+ 5min9 hypoF5nthin b(t cJp Mì ! gy chuyZn MWL G hydroFyl5min (Nm*m 8 chh <h+ 5min cQ5 !9 cI t"c dng rt giì h#nL !"c cht 5l<yl hI58 gy dt bin b%ng c"ch8 G Th4m nhIm methyl h5y ethyl Mo : t#o b51e {ng lng cQ5 A dOn n 1H b(t cJp bV 1ung 15i G t purin do : ^ bW 5l<yl hI5 t#o lv hVng tr4n ADN9 <hi 15o chYp cI lm Rt m#ch G ki4n <t chYo gii5 c"c m#ch cQ5 mdt hoJc c"c phn t+ ADN <h"c nh5u lm mt nuL 1C. CE$ $h\ GiKn hFnh ,B "9PB9 hX" + b" $E$h ,B G> bi/n ,Vn $U" $h{n, h() nhX) )V hB9 h"B V hB9 )\ &=2inV 9i(n P/ $h%# ,i" $E$ )<$h ADN 9) )\ n= - !"c cht iZn hXnh gy 5l<yl hI58 nitro1ogu5nidin(NT: 9 methyl meth5n 1ul6on5t (/ 9 ethyl meth5ne 1ul6on5t (@/ - a5 c"ch gy dt bin gen cQ5 ch[ng8 G Th4m nhIm methyl h5y ethyl Mo : t#o b51e {ng lng cQ5 A dOn n 1H b(t cJp bV 1ung 15i G t purin do : ^ bW 5l<yl hI5 t#o lv hVng tr4n ADN9 <hi 15o chYp cI lm Rt m#ch G ki4n <t chYo gii5 c"c m#ch cQ5 mdt hoJc c"c phn t+ ADN <h"c nh5u lm mt nuL 1W. CE$ E$ nhn hX" hS$ $hn +# ADN 9) 9$h Ph=n,. - !"c cht pro6l5Min9 c5m 5cridin9 ethidium bromide l c"c phn t+ 5 MNng phlng t2)ng t"c Mì c"c b51e cQ5 ADN M chn Mo gii5 ch[ngL - /H chn lm wd^nx 13i i ADN M ADN polymer51e bW w"nh l5x chn b51e Mo 0i diPn nhiu h)n bXnh th2Sng9 lm lPch <hung ADN t#o thnhL 1^. CE$ E$ nhn hX" hS$ 9) h"B Gsi $\= 2{$ ADN. - !"c phn t+ l`n g(n Mo b51e trong ADN M lm ch[ng trK thnh <hng m^ hI5L CD8 N-5cetoFy-*5cetyl5mino6luorene (NAAA - !"c t"c nhn gy li4n <t chYo trong M gii5 c"c 13iL CD8 c"c p1or5len cI trong mdt 10 r5u M dqng trong iu trW mdt 10 bPnh M d5L - !"c cht hI5 hTc gy Rt 13i ADNL CD8 c"c peroFid - !"c hydroc5rbon 5 MNngL CD8 c"c bensypyrene cI trong F\ngL 1`. 0"i 2w ,B G> bi/n $U" i" 0. - ADN hp thu ti5 ;C m#nh nht K ' *ADnm - !"c tVn th2)ng gy cht chUnh l c"c dimer pyrimidin trong ADN (t#o bKi ;C-a M ;C-! - Ng\n chJn 1H phi4n m^ M 15o chYp ADN M gy cht nu <hng 1+5 chi5L
- uUch thUch dt bin9 1(p Fp l#i N/T 1c. B" $. $h/ h6_n, ,J& $h8n, 9 bi/nq I-# n,h1$h !"i h}n,' 9#<i b} !"i h}n,' M=n, n<& !"i h}n,. *L B]o nghWch 15i h|ng8 G tu5ng phec h{i8 !I 1H th5m gi5 cQ5 mdt ensyme cI <h] n\ng c(t c"c dimer pyrimidin (lm Rt c"c li4n <t {ng hI5 trW <hi cI "nh 1"ngL @nsyme photoly51e F[c t"c ph]n Rng ny cI nhiu trong Mi <huEn9 t bo nhn thpt bpc thp9 cn trqng M thHc Mpt9 <hng cI trong dng Mpt G /+5 15i b%ng c"ch lm mt nhIm 5l<yl8 1d. B-n $h\ $U" D="n, &h]$ hti. D<n, nB tn <i O nhn, !inh +k n#. f !I 1H th5m gi5 cQ5 mdt ensyme cI <h] n\ng c(t c"c dimer pyrimidin (lm Rt c"c li4n <t {ng hI5 trW <hi cI "nh 1"ngL - @nsyme photoly51e F[c t"c ph]n Rng ny cI nhiu trong Mi <huEn9 t bo nhn thpt bpc thp9 cn trqng M thHc Mpt9 <hng cI trong dng MptL N(= +3 M] !;" !"i G> bi/n bn, $E$h 9) )\ nhX) "9PB9 hX" O E.coli. - :en ada m^ hI5 cho ensyme T51e (<D5 - CW trU !y1 tr4n ensyme nhpn g0c methyl t pho1phodie1ter M th5y Vi cu hXnh cho phYp nI ho#t hI5 promoter cQ5 c"c gen ada, alkA (methyl5denin ADN glyco1yl51e 9 5l<a M 5ida-. t\ng <h] n\ng <h"ng Mì c"c t"c nhn 5l<yl hI5 - CW trU !y1*, nhpn g0c methyl t -e:u5nin M 7-eThymin Z <hi phec b51e bXnh th2SngL B" $. $h/ 9# bi/n bn, Ei s h:& + bn, G> bi/n 2#n, M<n, M=n, n<& ADN !"i h}n,. - /+5 chi5 b%ng t"i tV h3p8 G Th[c Ey t"i tV h3p Z lp <huyt 13i con <hng dimmer9 c"ch chYp 0i phI tVn th2)ng l2u tri <hng m^ hI5 trong ADN G $h]n Rng li4n <t _u <hng t2)ng {ng Z 1+5 c"c _u ADN bW Rt - /+5 chi5 b%ng dt bin8 G Th5y Vi tUnh Jc hiPu cQ5 ADN-polymer51e Z cI thZ chn bt cR nu 0i diPn dimer no M tip tec 15o chYp (c"ch wdt bin h5y l chtx G j]y r5 K Mi <huEn9 cI thZ F]y r5 K t bo nhn thpt nh2ng ch25 r} c) ch G BI l lz do t#i 15o i <hi 1+5 chi5 cI thZ gy dt bin
BI d q 5 TH T SINH HOC PHN T

1 .c im chng JM109,DH 5 Alpha , DH 5Alpha F, ! 1 "l#$ , M1je Dqng Z nhn b]n c"c Mector cQ5 ph5ge , L !I thZ dqng Mì pl51mid p;!9 l c"c pl51mid 1+ deng < thupt bV 1ung 5lph5 Z chTn lTc dNng t"i tV h3pL !hQng ny bW mt gen -g5l5cto1id51e tr4n nhifm 1(c thZ9 nh2ng cI m5ng gen cho o#n omeg5 cQ5 -g5l5cto1id51e tr4n pl51mid 9 cho phYp nI bW nhifm bKi c"c ph5ge d#ng 13i nh2 ,L Nu mt pl51mid 1c lm mt <h] n\ng ny9 do I ph]i duy trX chQng ny tr4n mi tr2Sng t0i thiZuL !I dt bin tr4n gen recA9 lm cho nI mt <h] n\ng t"i tV h3p t2)ng {ngL DH^f !I Jc tUnh bV 1ung 5lph5L !hQng ny m5ng gen recA, dt binL Ngoi r59 nI cNn m5ng dt bin deoR lm df dng cho MiPc thu nhpn c"c m]nh ADN l`nL DH^fg Rt t0t cho MiPc nhn b]n Mector , mpL !ho phYp t#o r5 ADN 13i )n t pl51mid cI origin 6, (nh2 Mector , <hi bW nhifm {ng thSi mdt ph5ge tr3 gi[p (helper ph5ge L uh"c Mì ,>9 epi1ome9 c_n Z nhn b]n ph5ge 13i )n9 Vn Wnh9 <hng c_n 1+ deng mi tr2Sng t0i thiZu Z duy trX nIL xL1fB9=V Rt t0t Z nhn b]n c"c th2 MiPn cADN h5y genome 5ng c"c dt bin lm bt ho#t h_u ht c"c R@ cQ5 @Lcoli9 do I ng\n MiPc c(t c"c ADN cI m5ng ! hoJc A 23c metyl hI5 5ng dt bin h1dR9 ng\n 1H c(t ADN ^ t#o dNng bKi @couL 5ng recA9 M < thupt bV 1ung 5lph5L 5ng gen <h"ng tetr5cyclin n4n <hng dqng Z chTn lTc dNng 15u bin n#pL %&# ' ( )ai *+, ca -$c*o+ *.o /,ng *+ong 0H12 -3 45# c6# ca -$c*o *.o /,ng 0"i 2w
Cector t#o dNng l Mpt liPu di truyn trung gi5n Z chuyZn gen gii5 c"c t bo do ch[ng cI <h] n\ng 23c 15o chYp M phn ph0i Mo c"c t bo mì trong gu" trXnh phn bo9 nhS I gen 5ng nghi4n cRu cng 23c 15o chYp M hiPn diPn K tt c] c"c t bo Fut ph"t t t bo nhpn gen b5n _uL V= $"= 8 !I thZ 23c 15o chYp bKi t bo nhpn nIL !I trXnh tH nhpn diPn cQ5 ensym giì h#n Z c(t M 25 gen ngo#i l5i MoL 5ng mdt h5y nhiu yu t0 chTn lTc no I Z phn biPt hoJc chTn lTc t bo nhpn 23c Mector Mì t bo <hng nhpn 23cL %&# 7 ( 8c im -3 45# c6# ca -$c*o 1la9mi/ $l51mid l mdt phn t+ ADN 13i i9 d#ng MNng <Un9 nh| cI thZ nhn b]n dc lpp Mì nhifm 1(c thZ trong t boL (= $?=q !I yu t0 "nh du Z chTn lTc dNng t"i tV h3p9 th2Sng l mdt h5y nhiu gen <h"ng <h"ng 1inhL !I iZm <hKi _u 15o chYp (ri cho phYp nhn b]n pl51mid trong t bo cht <hng phe thudc N/TL !"c pl51mid 23c 1+ deng hiPn n5y th2Sng cI iZm ri cI thZ <iZm 1o"t 23c LTrXnh tH cQ5 ri cng guyt Wnh 10 b]n 15o cQ5 pl51mid trong mdt t boL !I Mqng t#o dNng (ultiple !loning /ite - !/ 8 l mdt trXnh tH DNA ng(n9 m5ng nhiu trXnh tH nhpn diPn duy nht cQ5 c"c R@ <h"c nh5u n%m li4n tip (polylin<er 9 cho phYp c(t Z mK MNng pl51mid M n0i o#n gen mong mu0n MoL Ngoi r59 mdt 10 pl51mid cI thZ m5ng yu t0 "nh du chn Z nhpn bit pl51mid 15u <hi 25 Mo t bo ^ 23c n0i Mì gen ngo#i l5i trong Mqng t#o dNng h5y ch25L $l51mid 23c 25 Mo t bo chQ b%ng c"ch bin n#p (tr5n16orm5tion h5y thEm iPn (electropor5tion dt 10 pl51mid thng deng p;! paR** p:@ M palue1cript rr : .8c im ca -$c*o phag$ !am"/a k , ph5ge ti4u gi]i9 nghy5 l 15u <hi Fm nhpp t bo chQ9 ph5ge 1c nhn b]n ADN cQ5 mXnh M t#o c"c Mirion9 ly gi]i t bo chQ r{i phIng thUch ph5geL Cector8 uhi 23c dung lm Mecto ng2Si t5 c(t b| mdt ph_n bd gen cQ5 ph5ge <hng li4n gu5n n gu" trXnh ti4u gi]i M th5y th b%ng o#n ADN c_n t#o dNngL uUch th2`c 15u t"i tV h3p ph]i l`n h)n D= (= * (A, <b thX mì Ing gIi df dng thnh ph5ge 23cL $h]i ch[ z n <Uch th2`c o#n gen mu0n chn9 nu o#n gen mong mu0n <hng Q <Uch th2`c thX ng2Si t5 1c n0i th4m ADN dn <h"c Z Q <Uch th2`c cI thZ Ing gIi 23cL u iZm trong Fy dHng c"c th2 MiPn t"i tV h3pL BIng gIi in MitroL Biu ny rt cI z nghy5 MX <hi I chh c_n mdt l23ng Ut Mector M ADN c_n chnL BIng gIi in Mitro l c"ch )n gi]n nht Z 25 ADN t"i tV h3p Mo t bo @L coli chQL !"c th2 MiPn ph5ge cI thZ df dng ph"t hiPn Mì mOu dN9 <huch #i M b]o gu]n trong mdt thSi gi5n diL %&# 5 ( ;<ch *h=>c *?i a ca o.n g$n$ c@ *h *.o /,ng "Ai cBc -$c*o+ ChBc nha# $l51mid M ph5ge l5mbd58 ,A <b !o1mid8 7A <b
%&# D ( Eg#45n *Fc ho.* Gng ca -$c*o+ co9mi/ !(t Mector b%ng R@ t#i MW trU mu0n t#o dNng M trdn Mì o#n gene mu0n (cI chiu di t A - 7A <b 9 Cector M o#n chn 1c 23c n0i l#i b%ng lig51e9 t#o thnh mdt 13i ADN thlng Mì o#n chn bW <&p gii5 * ph_n cQ5 Mector M tpn cqng Mì c"c MW trU co1 K * _uL BIng gIi in Mitro nhS nhpn diPn * MW trU co18 /3i DNA ny <hi Q Mì c"c protein M| 1c t#o thnh ph5ge M 23c gy nhifm Mo @L coliL /5u <hi Mo t bo chQ9 ADN t"i tV h3p 1c t#o thnh MNng nhS MW trU co1 M ho#t dng nh2 mdt pl51midL IJ$ GiK) $U" $#!)iM !o1mid l mdt d#ng l5i gii5 ph5ge M pl51mid 5ng yu t0 chTn lTc9 iZm ri cQ5 pl51mid9 CW trU t#o dNng 8 !"c trXnh tH cQ5 ph5ge l5mbd5 m^ hI5 cho MW trU co1L ;u iZm cQ5 co1mid 8 T#o dNng c"c o#n gen cI <Uch th2`c l`n n A> <bL !I thZ Ing gIi in Mitro M 25 gen Mo t bo b%ng c"ch gy nhifm9 do I )n gi]n9 hiPu gu] M tit <iPm ADN Trong t bo ho#t dng nh2 pl51mid8 df th5o t"c9 duy trX %&# H ( %I# *.o chJc nng -3 ph&n lo.i KE C\= <# @nsym c(t h#n ch (re1triction ensym - R@ l c"c endonucle51e cI <h] n\ng c(t ADN t#i h5y g_n mdt trXnh tH nhpn diPn Jc hiPuL ChQ$ nRn, !(t M tho"i hI5 c"c ADN ngo#i l5i (MU de ADN ph5ge gi[p cho bd gen cQ5 Mi <huEn 23c Vn WnhL Phn 9#<i a@8 cI lo#i ko#i r M rrr m5ng mdt phRc hP g{m c] * ho#t tUnh c(t giì h#n M methyl hI59 lo#i ny c_n AT$ Z cI n\ng l23ng M nI c(t ADN t#i MW trU <h" F5 iZm nhpn diPnL m5i lo#i ny Ut 23c dqng trong c"c thHc hnh 1inh hTc phn t+L ko#i rr chh cI ho#t tUnh endonucle51e9 <hng c_n AT$ Z ho#t dng M c(t ADN t#i hoJc rt g_n MW trU nhpn diPnL ko#i rr 23c 1+ deng nhiu trong thHc tifn 1inh hTc phn t+ M hiPn ng2Si t5 ^ bit <ho]ng ,>>> LR@ lo#i ny M cI Mi tr\m lo#i ^ 23c th2)ng m#i hI5L %&# L ( )M *+< nhNn /iOn cF* ca KE CW trU nhpn diPn cQ5 5 10 R@ lo#i rr th2Sng chR5 7 - nucleotid theo <iZu p5lindromeL !I 7 <iZu 1(p Fp cQ5 trXnh tH nhpn diPn 8 $5lindrome (MU de a5mmr9 @coRr $5lindrome bW ng(t (min6r9 jmnr TrXnh tH bW tho"i bin9 th2Sng cI ph_n cNn l#i cQ5 p5lindrome (@coRrr9 mincrr uhng ph]i p5lindrome (borr L R@ chh c(t ADN 13i i M t#o r5 * _u tpn A pho1ph5te M hydroFylL B_u c(t cI thZ t (tq hoJc 1o le (cNn gTi l _u dUnh L Nu trXnh tH nhpn diPn cI ph_n no tUnh 0i FRng thX iZm c(t th2Sng n%m gii5 nI M K MW trU gi0ng nh5u cho mvi 13iL Ng23c l#i thX MW trU c(t c"ch <ho]ng 1o Mì nI %&# 9 ( 8c im ca KE lo.i PP -3 cBc Ci# cF* ca n@ IJ$ GiK) !hh cI ho#t tUnh endonucle51e9 <hng c_n AT$ Z ho#t dng M c(t ADN t#i hoJc rt g_n MW trU nhpn diPn TrXnh tH nhpn diPn c(t g{m 7-= nucleotid9th2Sng cI <iZu p5lindrome9 CD8 :AATT! 5iK= $N B_u tq8 c(t h5i m#ch t#i cqng mdt iZm B_u tq8 c(t h5i m#ch t#i cqng mdt iZm
B_u 1o le?dUnh8 c(t mvi m#ch t#i mdt iZm <h"c nh5u %&# 10 ( %Bc 8c *<nh ca 1ol4m$+a9$ H#< 3nh sn, h:& ^gf Cg8 mo#t tUnh <Yo diL Dqng dNT$ Z g(n th4m , deoFynucleotid-Amonopho1ph5te Mo _u hydroFyl cQ5 m{i M phIng thUch pyropho1ph5teL Tqy theo ensym9 ph]n Rng c_n cI <hun mOu ADN9 ARN9 h5y <hngL H#< 3nh VL#n=$9V"!V Cf ^8 chRc n\ng w1+5 lvixL uh] n\ng ny 0i Mì m#ch )n m#nh h)nL Tr4n m#ch <Yp nu cI Q dNT$9 ho#t tUnh tVng h3p m#nh h)nL mo#t tUnh ny cng m#nh l4n <hi t\ng nhiPt dL H#< 3nh VL#n=$9V"!V ^gf Cg8 phn hQy o#n m{i h5y <hun mOuL H#< 3nh 2ib#n=$9V"!V H8 !hh cI K mdt 10 polymer51e9 nI phn hQy Jc hiPu ARN <hi cI 1H hiPn diPn cQ5 phRc l5i ARN?ADNL D1$h $h=BKn !:i 8 dt 10 polymer51e cI <h] n\ng chuyZn 13i c t M5i trN <hun mOu thnh 13i mì 5ng 23c tVng h3pL 5h- nRn, L; 9| q k <h] n\ng cQ5 , phn t+ polymer51e cI thZ tip tec tVng h3p tr4n 13i <hun tr2`c <hi t"ch r5 M bW th5y th bKi mdt polymer51e <h"cL T?n !=\ 9i8 uhi 1+ deng in Mitro t_n 1ut ny th2Sng c5o h)n in MiMo M phe thudc Mo iu <iPn tin hnhL a4n c#nh I nI cng tqy Mo <h] n\ng w1+5 lvix M F+ lz cQ5 polymer51eL %&# 11 ( 1h&n lo.i 1ol4m$+a9$ $hn lo#i dH5 tr4n 1]n phEm M <hun mOu ADN polymer51e phe thudc ADN8 @nsym 15o chYp ARN polymer51e phe thudc ADN8 @nsym phi4n m^ DNA $olymer51e phe thudc ARN8 ReMer1e Tr5n1cript51e $olymer51e <hng phe thudc <hun mOu8 $rim51e %&# 1' ( QR# *? S#4R* Mnh *6n 9#I* lTi ca 1ol4m$+a9$ @nsym cI ho#t tUnh 1+5 lvi h5y <hng8 mo#t tUnh eFonucle51e - A cI chRc n\ng w1+5 lvixL uh] n\ng ny 0i Mì m#ch )n m#nh h)nL Tr4n m#ch <Yp nu cI Q dNT$9 ho#t tUnh tVng h3p m#nh h)nL mo#t tUnh ny cng m#nh l4n <hi t\ng nhiPt dL T_n 1ut lvi8 uhi 1+ deng in Mitro t_n 1ut ny th2Sng c5o h)n in MiMo M phe thudc Mo iu <iPn tin hnhL a4n c#nh I nI cng tqy Mo <h] n\ng w1+5 lvix M F+ lz cQ5 polymer51eL %&# 17 8c im -3 Jng /Ung ca DEA pol4m$+a9$ P ca E.coli -3 o.n ;l$noV IJ$ GiK) k ADN polymer51e phe thudc ADN B23c phn lpp t @L coli9 nh2ng ngy n5y ^ 23c 1]n Fut t chQng Mi <huEn cI <h] n\ng 1i4u tVng h3p ensym nyL n, M]n, !huyZn <hU5 (nic< tr5n1l5tion 8 Rng deng trong "nh du 5cid nucleic TVng h3p 13i thR * cQ5 cADN I#<n 59Vn# Thu 23c nhS t"c dng cQ5 ensym thQy phn protein tr4n polymer51e r cQ5 @L coli LBo#n uleno <hng cI ho#t tUnh eFonucle51e A-L :_n y Mì cng nghP di truyn ^ t#o 23c dNng 1]n Fut o#n uleno <hng cI ho#t tUnh eFonucle51e (eFo- uleno ensym /tr5t5gene L /+ deng km t _u bW het (_u dUnh ko#i b| _u nh r5L B"nh du _u cQ5 ADN 13i i9 Jc biPt 0i Mì _u bW hetL TVng h3p 13i thR * cQ5 cADN9 cI 2u iZm h)n polymer51e r MX <hng cI ho#t tUnh eFonucle51e AL B"nh du b%ng < thupt m{i ngOu nhi4nL
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pm dung dWch 23c nng l4n ,* - ,*9A b%ng N5m lm cho c"c ADN thlng bW t"ch thnh 13i )n9 ADN pl51mid K d#ng 1upercoil (1i4u Fo(n n4n <hng bW t"c dngL uhi th4m 5cid Z lm h# pm9 ADN 13i )n l#i t"i h3p9 nh2ng MX 13i gu" di n4n <t h3p <hng chUnh F"c t#o n4n bI r0i dy Jc9 <hng t5n M cI thZ df dng lo#i b| b%ng ly tmL $h2)ng ph"p ny cNn thupn l3i h)n MX trong iu <iPn ly gi]i nh2 Mpy9 h_u ht protein M ARN cng trK thnh <hng t5n M bW lo#i b|L %&# 1L ( %Bc ph=Wng phBp *inh chR Aci/ E#cl$ic LB ) &hn G#<n8 dung dWch 5cid nucleic 23c 1i4u ly tm trong th5ng t~ trTng 2Sng 15cc5ro1e h5y !1!l* <hi I c"c phn t+ <h"c nh5u 1c t"ch theo t~ trTng (t~ lP Mì <Uch th2`c phn t+ L !5ch ny 23c "p deng <hi l23ng 5cid nucleic c_n tinh ch l`nL Iin Mi ,V9 + !N$ P| 9S$ ,V9 8 t"ch 5cid nucleic theo <Uch th2`c phn t+ K gui m nh| (iPn di h5y l`n (lTc gel L BiPn di Mì gel poly5cryl5mid ($A:@ cI thZ tinh ch c"c 5cid nucleic Mì <h"c b iPt , nucleotidL HPLC8 23c "p deng chQ yu 0i Mì c"c oligonucleotid do d phn gi]i l4n n , nucleotidL SN$ P| h\& &h]q ARNm th2Sng tpn cqng b%ng polyA9 do I 23c hp phe b%ng re1in cI g(n oligo dT h5y d;L %&# 19 ( EGi /#ng lai ph&n *Z . %Bc 45# *? [nh h=Ang Rn lai ph&n *Z m5i phn t+ 5cid nucleic 13i )n trong iu <iPn thUch h3p cI thZ b(t cJp bV 1ung Mì nh5u b%ng c"c li4n <t hydro Z t#o thnh phn t+ 13i i9 gu" trXnh ny gTi l l5i hI5 5cid nucleic (hybridis5tion L $hn t+ 13i i thu 23c gTi l phn t+ l5i (hybrid h5y dupleFL tu" trXnh ng23c l#i <hi dupleF t"ch r5 thnh c"c 13i )n 23c gTi l 1H bin tUnh (den5tur5tion L $h]n Rng l5i 23c Fem l Jc hiPu <hi 1H b(t cJp bV 1ung hon ton cQ5 mdt phn t+ Mì phn t+ cNn l#i difn r5 tr4n mdt Mqng li4n tec gii5 h5i m#ch9 ng23c l#i <hi mdt phn t+ chh b(t cJp mdt ph_n Mì phn t+ <i5 thX 1H l5i hI5 23c Fem l <hng Jc hiPu h5y <hng hon tonL CE$ B/= 8 -nh h6On, Do b]n cht cQ5 ph]n Rng l5i l 1H hXnh thnh c"c lien <t hydro n4n cn b%ng ph]n Rng9 d Jc hiPu M d bn cQ5 dupleF phe thudc Mo c"c yu t0 15u8 NhiPt d8 l yu t0 guyt Wnh d bn cQ5 li4n <t hydro Bd di c"c c"c trXnh tH l5i8 d di cng l`n dupleF cng bn M thSi gi5n ph]n Rng cng luL T~ lP :! trong dupleF8 t~ lP :! cng c5o dupleF cng bn N{ng d ion8 ]nh h2Kng n d bn cQ5 li4n <t hydro N{ng d c"c phn t+ M thSi gi5n ph]n Rng NhiPt d ch]y (melting temper5ture9 Tm 8 l nhiPt d K I cI A> 10 dupleF 23c hXnh thnhL NhiPt d ny phe thudc chiu di M t~ lP :! cQ5 dupleF9 ngoi r5 n{ng d ion cng cI ]nh h2Kng l`n9 do I c"ch tUnh Tm <h" phRc t#pL $h]n Rng l5i 1c difn r5 K nhiPt d nh| h)n Tm cQ5 dupleF9 ng23c l#i K nhiPt d l`n h)n Tm9 1H bin tUnh cQ5 dupleF 1c chim 2u thL %&# '0 ( %Bc C\ *h#N* lai aci/ n#cl$ic -3 Jng /Ung ( Trong Rng deng9 ph]n Rng l5i difn r5 gii5 mdt phn t+ Uch (phn t+ c_n 23c ph"t hiPn M mdt o#n dN (probe L Bo#n dN l phn t+ oligonucleotid 23c "nh du b%ng phIng F#9hu-nh gu5ng9 ensym h5y c"c < thupt "nh du <h"c CE$ P h=k 9"iq L"i 2(n )n, 2Nn8 k ph2)ng ph"p ph"t hiPn 5cid nucleic trong < thupt blot9 trong I mng nitrocellulo1e th2Sng 23c 1+ dengL !"c bin thZ cQ5 l5i tr4n mng8 /outhern blot Northern blot Dot blot
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%&# '1 ( 1h&n "iO* 0o#*h$+n ]lo* , Eo+*h$+n ]lo* -a Do* ]lo* /outhern blot8 l5i 23c thHc hiPn Mì ADN 15u <hi ^ 23c iPn di trong gel r{i 23c chuyZn M c0 Wnh tr4n mngL Northern blot8 l5i 23c thHc hiPn Mì ARN 15u <hi ^ 23c iPn di trong gel r{i 23c chuyZn M c0 Wnh tr4n mngL Dot blot8 l5i 23c thHc hiPn Mì hvn h3p 5cid nucleic Uch 23c chm trHc tip l4n mngL %&# '' ( o.n /, l3 g^ , )ai *+, ca n@ *+ong lai aci/ n#cl$ic Bo#n dN l oligonucleotid 23c "nh du8 phIng F#9 hu-nh gu5ng9 ensym h5y c"c < thupt "nh du <h"c 0"i 2w 8 NhS I t5 ph"t hiPn 23c 1H l5i cI difn r5 h5y <hng h5y nIi c"ch <h"c phn t+ Uch cI t{n t#i h5y <hng L %&# '7 ( Eg#45n !_ 1%K u thupt $!R dH5 tr4n Jc iZm cQ5 gu" trXnh 15o chYp gene trong t bo L uhun ADN 13i )n 23c g(n m{i nhS l5i hI5 Jc hiPu L Trong iu <iPn cI 1n nucleotid9 ADN polymer51e 1c <Yo di m{i theo nguy4n t(c bV 1ung Mì <hun ADN 13i )n nu _u -m cQ5 m{i cNn tr0ng t#o thnh 1]n phEm 13i i $h]n Rng 23c gu5y MNng nhS m{i th5 M 1+ deng nhiPt d c5o Z t"i t#o <hun ADN 13i )n t 1]n phEm 13i i %&# ': ( %Bc "=>c *iRn h3nh 1 ph[n Jng 1%K . %h# C` nhiO* in h^nh ca 1%K CE$ b67$ i/n hnh q ain tUnh ADN 13i i thnh c"c 13i )n nhS nhiPt d L c"c o#n m{i (chuvi oligonucleotid g(n Jc hiPu Mì DNA <hun mOu Mì c"c 13i )n ADN @nsym polymer51e <Yo di c"c o#n m{i theo nguy4n t(c bV 1ung Mì <hun mOu t#o thnh * 13i i mì L Ch= P nhi 8 dt chu <- nhiPt $!R iZn hXnh nh2 15u 8 Bi/n 3nh PhOi G?= 8 A d trong A ,> ph[t ( K nhiPt d ny tt c] DNA 13i i bt <Z chiu di u bW bin tUnh Ch= 5 Nhi 8 :{m b2`c lpp l#i *>- 7> l_n o ain tUnh 8 7-A d trong > giy n Mi ph[t o :(n m{i 8 7>-D> d trong >-> giy o uYo di 8 A D7 d thSi gi5n tqy theo di <hun 5%# Mi P/ h{$ 8 D* D7 d M thSi gi5n gp Mi l_n thoi gi5n <Yo di LL %&# '5 ( )ai *+, -3 *h3nh ph6n *+ong cBc ph[n Jng 1%K Thnh ph_n ph]n Rng8 *>-,>> l uhun mOu 8 , pg - , g9 n{ng d c5o 1c Rc ch ph]n RngL {i8 >9, - , 9 n{ng d gu" c5o 1c dOn n b(t cJp 15i M lm gi]m tUnh Jc hiPuL
BPm8 cung cp pm thUch h3p cho ph]n Rng M cI n{ng d g*G th5y Vi (c_n t0i 2u hI5 9 th2Sng trong <ho]ng >9A-A mL mvn h3p c"c dNT$8 A9 T9 : M !9 n{ng d *>>-*A> ADN polymer51e chWu nhiPt8 >9A - *9A r; cho mvi ph]n Rng N2`c M5 QL %&# 'D ( Eg#Xn g?c -3 8c im ca mG* 9? DEA pol4m$+a9$ chM# nhiO* *h=ang g8p ( a]ng L* ( <hi r5 A c"i thi %&# 'H ( Q5# c6# ca mXi *+ong 1%K . EXng G mXi , cbng *hJc *<nh 2m . o !hiu di ,=-> nucleotid9 cI trXnh tH bV 1ung Jc hiPu Mì <hun mOu L o CW trU b(t cJp tr4n <hun mOu cQ5 h5i m{i 1c guyt Wnh Mqng ADN 23c <huch #i trong $!RL uho]ng c"ch c"c MW trU b(t cJp tr4n <hun mOu gii5 h5i m{i <hng gu" ,> ->L !"c nucleotid AT M :! 23c phn b0 u9 tr"nh d{n Mo mdt chvL B_u cQ5 m{i n4n cI c"c nucleotid : hoJc !L TrXnh tH cQ5 m{i 23c lH5 chTn 15o cho <hng t#o cu tr[c <&p tIc M h5i m{i <hng b(t cJp Mì nh5u o NhiPt d ch]y cQ5 h5i m{i ph]i g_n nh5u M trong <ho]ng 7>-Ao!9 nh2ng t0t nht l A*-A=o! N{ng d {i 8 >9, - , !ng thRc tUnh Tm ' *(AGT G 7(:G! NhiPt d b(t cJp ' Tm - Ao! (c_n 23c t0i 2u %&# 'L ( %Bc 4R# *? c6n =cc *?i =# h@a *+ong 1%K uhun mOu cQ5 polymer51e9 c_n t0i 2u hI5 cho tng <hun mOu <h"c nh5uL Th2Sng trong <ho]ng >9A-A m NhiPt d g(n m{i ' Tm - Ao! (c_n 23c t0i 2u o guyt Wnh tUnh Jc hiPu cQ5 ph]n RngL o gu" c5o ph]n Rng <hI difn r59 gu" thp <hng Jc hiPuL %&# '9 ( QR# *? S#4R* Mnh C<ch *h=>c o.n Ch#Rch .i o uho]ng c"ch gii5 m{i Fui M m{i ng23c o T0i 5 tqy theo <h] n\ng F+ lz cQ5 ensym o Th2Sng <b 0i Mì mdt 10 ensym Jc biPtL %&# 70 ( d# nh=cc im 1%K , ;im 9oB* ngo.i nhiem 1%K . = q Nh5nh 9 )n gi]n 9 nh# 9 Jc hiPu Nh6:$ 8 $h]i bit trXnh tH * _u Z thit < m{i Ngo#i nhifm mt tUnh Jc hiPu 5iK) !#E n,#<i nhi) PCa 8 $h]n Rng df bW ]nh h2Kng bKi 1H nhifm t#p cQ5 c"c o#n ADN ngo#i l5i M ch[ng cI thZ 23c <huch #i cqng Mì mOuL !"c ADN l# ny cI thZ 23c m5ng 15ng t c"c 1H <huch #i tr2`c I (5mplicon hoJc t c"c ngu{n <h"cL o uiZm 1o"t8 tui trXnh thHc hiPn ph]i 23c tun thQ chJt ch uiZm tr5 cht l23ng ensym mdt c"ch nghi4m ngJt /+ deng bd pipet ri4ng !"c thnh ph_n ph]n Rng n4n 23c phn liu tr2`c uhu MHc chit t"ch ADN9 thHc hiPn ph]n Rng M ph"t hiPn 1]n phEm n4n t"ch biPt M th2Sng Fuy4n 23c chiu ;C Z lm gi]m 5cid nucleic trong <hng <hU %&# 71 ( Eg#45n *Fc phB* hiOn 9[n phfm *+ong K$al 2im$ 1%K (
Nh=>) LVn ,i"8 $h]n Rng $!R 23c bV 1ung cht nhudm Fen gii5 nh2 /aR :reen r9 cht ny nu tH do trong dung dWch thX <hng ph"t hu-nh gu5ng9 nh2ng nu 23c chn Mo 13i i ADN9 MU de 1]n phEm $!R9 thX 1c ph"t hu-nh gu5ngL TUn hiPu 23c Tc <hi chu <- $!R <t th[cL Phn hUB G#<n Mwq , o#n dN Jc hiPu Mì trXnh tH 1]n phEm 23c thit < 15o cho , _u 23c g(n phn t+ ph"t hu-nh gu5ng9, _u g(n phn t+ dpp9 do I <hi cNn nguy4n M&n hoJc g(n tr4n trXnh tH Uch thX <hng thZ ph"t hu-nh gu5ng 23cL Nh2ng trong ph5 <Yo di cQ5 $!R9 T5g polymer51e 1c 1+ deng ho#t tUnh A eFonucle51e Z phn hQy o#n dN9 dOn n phn t+ ph"t hu-nh gu5ng M phn t+ dpp <hng cNn K g_n nh5u ni5 n4n hu-nh gu5ng 1c 23c gi]i phIngL TUn hiPu 23c Tc <hi chu <- $!R <t th[cL L"i hX" G#<n Mw8 h5i o#n dN cI g(n phn t+ ph"t hu-nh gu5ng 23c thit < 15o cho u g(n Jc hiPu Mì trXnh tH Uch (1]n phEm $!R theo <iZu ui-_uL Do I <hi cqng l5i hI5 tr4n trXnh tH Uch hu-nh gu5ng cQ5 phn t+ thR nht 1c <Uch thUch 1H ph"t gu5ng cQ5 phn t+ thR * t#o r5 tUn hiPu9 nh2ng trong ph5 <Yo di9 T5g polymer51e 1c t"ch * o#n dN r5 thnh d#ng tH do n4n tUn hiPu 1c mt iL TUn hiPu 23c Tc <hi b(t _u chu <- $!R 15u b2`c g(n m{iL %&# 7' ( 2.i 9ao c6n *.o /,ng g$n$ ? BZ c lpp o#n gen K d#ng tinh <hit9 Mì 10 l23ng l`n M nghi4n cRu cu tr[c9 chRc n\ng cQ5 nI t5 c_n 25 o#n gen I Mo mdt t bo chQ 15o cho nI cI thZ nhn b]n 23c - nghy5 l t#o dNng genL a%ng < thupt t#o dNng gen9 hiPn n5y ng2Si t5 ^ Fy dHng c"c th2 MiPn h5y ngn hng gen cQ5 nhiu lo#i 1inh Mpt - Mì thnh tHu nVi bpt nht l gi]i m^ bd gen ng2Si trong dH "n :i]i m^ ad gen ng2Si (mum5n :enome $roect -trong I mvi gen n%m trong mdt dNng t bo chQL %&# 77 ( %Bc "=>c *.o /,ng g$n$ ( T5o o#n ADN9 N0i o#n ADN c_n t#o dNng Mo Mector Z t#o phn t+ "i tV h3p925 ADN t"i tV h3p Mo t bo chQ !hTn lTc dNng t"i tV h3pL %&# 7: ( %Bc *.o o.n g$n$ mong m#?n *.o /,ng ( Dvn, a@ !(t phn t+ ADN l`n (Md8 ADN nhifm 1(c thZ thnh c"c o#n nh| b%ng R@L Biu ny th2Sng 23c 1+ deng <hi Fy dHng c"c th2 MiPn gen (genomic libr5ry L /+ deng * lo#i R@ <h"c nh5u Z c"c o#n t#o r5 cI <Uch th2`c M5 ph]i cho MiPc t#o dNngL B{ng thSi Mì MiPc c(t b%ng * R@ <h"c nh5u t5 cI thZ phn tUch c"c o#n thu 23c M lpp b]n { c"c MW trU c(t cQ5 bd genL R@ 23c chTn Z c(t c_n l2u z MW trU c(t 1c t#o r5 _u tq (blunt end h5y _u dUnh (1tic<y end 5 h=k PCa Nu cu tr[c o#n gen ^ 23c bit Thng th2Sng o#n ADN thu 23c b%ng $!R cI c"c _u bW 1o le (lPch <hng F"c Wnh M trXnh tHL km t _u 1]n phEm $!R9 g(n lin<er hoJc 5d5ptor Thit < m{i cI chR5 trXnh tH nhpn diPn c(t cQ5 R@ %&# 75 ( 1h[n Jng n?i DEA , cBc gi[i phBp hg *+c -iOc n?i DEA 6# *h ( Dqng li5g1e cQ5 T7 (23c tinh ch t @L coli bW nhifm ph5ge T7 L Mector ^ duvi thlng M o#n ADN Mì lig51e trong iu <iPn thUch h3p9 ph]n Rng difn r5 t#o li4n <t pho1phodie1ter M n0i c"c ADN l#iL Trong tr2Sng h3p t#o dNng9 ADN t"i tV h3p ph]i 23c n0i hon chhnh Mì c"c yu t0 cQ5 Mector Z 1n 1ng 23c 25 Mo M ho#t dng trong t boL CE$ && hs 2: q kin<er ( o#n m{i ligonucleotid 13i i tVng h3p ng(n cI _u t M chR5 trXnh tH c(t (_u dUnh cQ5 mdt R@L kig51e 1c n0i lin<er Mì o#n ADN cI _u tq9 /5u I dqng R@ c(t lin<er Z t#o r5 _u dUnhL AM"& #2
Nh23c iZm lin<er Ad5ptor - y l mdt oligonucleotid 13i i tVng h3p cI 1n mdt _u dUnh kig51e 1c n0i 5d5ptor Mì o#n ADN cI _u tq M t#o thnh o#n mì cI _u dUnhL $hU5 A cQ5 _u dUnh tr4n 5d5ptor bW th5y nhIm pho1ph5te b%ng nhIm hydroFyl Z tr"nh c"c 5dp5tor tH n0i Mì nh5uL Do I 15u <hi n0i 5dp5tor Fong t5 ph]i dqng polynucleotid <in51e Z tr] l#i nhIm pho1ph5te T<# G=ri h#)#&#9B)V2 /+ deng ensym doFynucleotidyl tr5n16er51e Z th4m mdt lo#t c"c nucleotid (cqng lo#i Mo _u -m cQ5 13i ADN t#o thnh ui homopolymerL Tr4n Mector ^ duvi thlng cng thHc hiPn nh2 Mpy nh2ng Mì nucleotid bV 1ung Mì c"c nucleotid tr4n 13i ADNL ut gu] cng 1c t#o r5 * _u dUnh df dng n0i Mì nh5u b%ng lig51eL %&# 7D ( Eg#45n *Fc cBc ph=Wng phBp =a g$n$ -3o *R "3o 8 ain n#p8 dH5 Mo <h] n\ng "nh b(t ADN cQ5 mdt 10 Mi <huEn T]i n#p8 1+ deng thHc <huEn Z gy nhifm t bo Uch ThEm iPn8 dqng iPn th c5o lm thQng mng t bo Z ADN lTt gu5 !huyZn nhifm8 dH5 tr4n <h] n\ng (t b(t phRc cQ5 ADN Mì mdt 10 cht trong chuyZn nhifm9 "p deng chQ yu cho t bo dng Mpt B#n 1inh hTc8 g(n ADN Mo h#t m5ng M b(n Fuy4n gu5 mng t boL %&# 7H ( ]iRn n.p =a g$n$ -3o *R "3o ( o !huEn bW t bo <h] n#p o j+ lz Mì !5!l* (th2Sng 1+ deng n{ng d A> ,>> m L T#i thSi iZm 15u <hi F+ lz9 ADN chh dUnh K mJt ngoi t bo chR ch25 Mo trongL o T bo <h] n#p ph]i 23c b]o gu]n l#nh M <hi 1+ deng Z K nhiPt d thp (7o! L %&# 7L ( 1h=Wng phBp ch#4en nhiem =a g$n$ -3 *R "3o ( Ch=BKn nhi) nh_ &h#!&h" V $"9$i 8 By l mdt ph2)ng ph"p t2)ng 0i )n gi]n Z 25 pl51mid t"i tV h3p Mo t bo dng Mpt cI M[L a%ng ph2)ng ph"p ny cI thZ 25 cqng l[c * pl51mid (cotr5n16ection Mo t boL ADN 23c trdn trong <t tQ5 cQ5 pho1ph5te c5lci M 23c t bo "nh b(t b%ng c) ch <hng r}L uhi thHc hiPn ph2)ng ph"p ny c_n l2u z dc tUnh Mì t bo cQ5 <t tQ5L !I thZ 1+ deng 10c glycerol (,A Z gi5 t\ng <h] n\ng "nh b(t ADN nu t bo chWu Hng 23cL Ch=BKn nhi) D=" 2#n, ,i"n D@A@ MVL 2"n 8 $h2)ng ph"p ny thUch h3p cho MiPc t#o dNng <hng bn (tr5n1ient tr5n16ection h)nL T bo 23c treo trong Dietyl5minoetyl (D@A@ deFtr5n (phn t+ l23ng A>>L>>> L ThSi gi5n 23c F"c Wnh thHc nghiPm tqy theo lo#i t bo M cI ]nh h2Kng l`n n hiPu gu]L u=" 2=n, ,i"n 9i&#!#)8 $h2)ng ph"p ny nh5nh9 )n gi]n M hiPu gu]9 cI thZ chuyZn {ng thSi * pl51mid Mo t bo Mì t_n 1ut c5oL kipo1om 23c iu ch t c"c cht bYo c5tion nh28 N-(,*9-dioleyloFypropyl-N9N9N-trimetyl5monium cholrid (DTA M dioleoyl pho1photidyl et5nol5min (D$@ (t~ lP ,8, L !"c lipid ny 23c cho tip F[c Mì ADN Z t#o thnh mdt phRc h3p cI thZ 23c "nh b(t bKi t boL !_n l2u z ADN dqng cho ph2)ng ph"p ny ph]i thpt tinh <hit thX hiPu gu] mì c5oL C ph]i 1+ deng Mpt chR5 b%ng poly1tyren th5y MX polypropylen do lipo1om 1c t#o phRc <hng Vn Wnh Mì cht 15uL S; M]n, Li&#V$ ")inV8 T"c nhn ny l mdt d#ng c5tionic lipid 23c iu ch thUch h3p9 23c 1+ deng nhiu trong thSi gi5n g_n y MX cho hiPu gu] chuyZn nhifm c5o Mì nhiu lo#i t bo M c"ch 1+ deng rt )n gi]n9 chh c_n trdn Mì ADN Z t#o phRc M cho Mo mi tr2Sng nui cy t boL T"c nhn ny Jc biPt hiPu gu] <hi c_n chuyZn nhifm ARNi Mo t bo
%&# 79 ( ;\ *h#N* "g 9#ng alpha ( DH5 tr4n 1H hiPn diPn cQ5 ho#t tUnh b-g5l5cto1id51e (chWu tr"ch nhiPm thQy phn l5cto1e thnh g5l5cto1e L @nsym ny l mdt tetr5mer9 trong I mvi monomer cI * m]nh (5lph5 M omeg5 L ad gen cQ5 t bo chQ bW dt bin mt hoJc bt ho#t ph_n m^ hI5 cho m]nh 5lph59 do I chh 1]n Fut 23c m]nh omeg5 cQ5 ensym9 <hng Q Z cI ho#t tUnh (<iZu hXnh l5c-9<hng thQy phn 23c l5cto1e L Trong <hi I9 Mector l#i m5ng
trXnh tH m^ hI5 cho o#n 5lph5 M MW trU chn gen ngo#i l5i 1c n%m trong trXnh tH nyL Do I nu t bo nhpn Mector m5ng ch25 t"i tV h3p thX 1c 23c bV 1ung m]nh 5lph5 M cI <iZu hXnh l5cG9 ng23c l#i nu Mector bW t"i tV h3p Mì gen l# trong trXnh tH m^ hI5 m]nh 5lph5 thX m]nh 5lph5 1c bW bt ho#t M t bo 1c cI <iZu hXnh l5c L %&# :0 ( %Bc ph=Wng phBp chin lic /,ng *Bi *g hcp ( ChSn 9S$ 2Z$ i/&8 y l c"ch t0t nht nh2ng <hI thHc hiPn tr4n thHc tL Trong ph2)ng ph"p ny9 o#n chn 1c m5ng n cho t bo nhpn 23c nI c"c Jc iZm <iZu hXnh m c"c dNng <h"c <hng cI M t5 df dng nhpn r5 <hi phn lppL CU de o#n chn m5ng gen m^ hI5 1(c t0 h5y ensymL L"i PhX) $#9#nB hBb2iMiA" i#nq "p deng cho tr2Sng h3p o#n chn ^ bit trXnh tHL !"c <hIm 15u <hi phn lpp 23c in du l4n mng nitrocellulo1e h5y nylon9 t bo tr4n mng bW ph" hu~ Z bd ld Mpt liPu di truynL ng lTc l[c ny 23c cho l5i Mì o#n dN 23c "nh du t2)ng Rng cQ5 o#n chnL Nu <hIm no m5ng o#n chn 1c cI tUn hiPu9 t MW trU tUn hiPu tr4n mng lTc 1uy r5 <hIm t2)ng Rng tr4n hdp th#ch b5n _uL %&# :1 ( 1h=Wng phBp 0ang$+ -3 %9 jBc Mnh* +^nh *k DEA $h2)ng ph"p ensym?dideoFy8 dH5 Mo 1H ngng tVng h3p ADN <hi gJp ph]i dideoFy nucleotid (rederic< /5nger tui trXnh8 ThHc hiPn 7 ph]n Rng $!R ri4ng biPt Mì , m{i8 , cI dNT$ G ddAT$ *L T8 cI dNT$ G ddTT$ L :8 cI dNT$ G dd:T$ 7L !8 cI dNT$ G dd!T$ vi 0ng 1]n phEm 23c n#p Mo mdt ging iPn di ut gu] iPn di 23c TctrXnh tH %&# :' ( 1h=Wng phBp gi[i *+^nh *k *k Gng ( !"c ddNT$ 23c "nh du hu-nh gu5ng Mì c"c mu <h"c nh5u ThHc hiPn mdt ph]n Rng $!R duy nht Mì 1H hiPn diPn cQ5 c"c dNT$ M ddNT$ /+ deng iPn di m5o gu]n Z t"ch 1]n phEm BTc <t gu] b%ng _u dN l51er %&# :7 ( 1h=Wng phBp Majam -3 lil"$+* $h2)ng ph"p hI5 hTc8 dH5 Mo 1H thQy gi]i Jc tr2ng cQ5 ADN (All5n 5F5m M 5lter :ilbert tui trXnh8 T#o ADN 13i )n t o#n ADN c_n gi]i trXnh tH B"nh du c"c phn t+ ADN K mdt _u ThHc hiPn 7 (hoJc A 0ng ph]n Rng ri4ng biPt8 :8 G dimethyl 1ulph5te:u5nine bW methyl hI5 A:8 G 6ormic 5cidAdenin M :u5nine bW methyl hI5 T!8 G mydr5sinebin Vi Thymin M !yto1in !8 G mydr5sine G ,9A N5!lbin Vi !yto1in A.!8 N5m ,9*N?>o! c(t ADN K Adenin . !yto1in j+ lz 7 0ng tr4n Mì piperidine ,?>o! Z c(t ADN K li4n <t pho1ph5t < nucleotid bW bin Vi vi 0ng 23c n#p Mo mdt ging iPn di ut gu] iPn di 23c Tc M biPn lupntrXnh tH %&# :: ( 0o 9Bnh cBc ph=Wng phBp gi[i *+^nh *k $h2)ng ph"p /5nger u iZm Nh5nh 5F5m M :ilbert :i5i trXnh tH <hng c_n m{i
t dc h#i N\ng 1ut c5o !I thZ tH dng hI5 Nh23c iZm !_n bit trXnh tH <ho]ng *>- !hpm > nucleotid Z lm m{i Bdc h#i N\ng 1ut thp uhng tH dng hI5

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