Bi tiu lun: Phng php Northern blot, Southern blot, Western blot Nhng ngi thc hin: 1.

Nguyn Xun Trung 2. L c Thun 3. L Hong Lm 4. Nguyn Hu Phc 5. ng Nguyn Lu Vy Vi

A.Lai trn pha rn 1. Nguyn tc Lai trn pha rn c cng nguyn tc vi lai trn pha lng. im khc bit y l mt trong hai trnh t b sung (thng l trnh t ch , tc trnh t cn tm) c c nh trn mt gi th rn. Thun li ca vic s dng gi th rn l to d dng trong thao tc v trong vic tch cc trnh t khng lai ra cc phn t lai, mt khc cn ngn s ti bt cp gia hai mch ca cng mt phn t. B li, vic phn tch nh lng cc phn t lai sau km chnh xc v hiu qu thp. Tht vy, vn tc lai trn pha rn thp hn 10 ln so vi vn tc lai trn pha lng do mt phn cc nucleic acid c c nh trn gi th b che khut, khng tip xc c vi cc trnh t b sung. 2. Cc yu t k thut quan trng trong cc phng php lai trn pha rn Gi th rn dng c nh nucleic acid l cc mng lai. C hai loi mng lai : mng lai bng nitrocellulose l loi gi th c s dng u tin hin nay khng cn thng dng do hai nhc im : bn c hc km nn kh thao tc, khng th tch ri cc phn t lai trn mng lai tr li vi mt mu d khc; mng lai bng nylon, c bn c hc cao v cho php lai nhiu ln vi nhiu mu d khc nhau (cn loi b mu d c trc khi lai vi mu d mi).

Cc bc lai trn pha rn

3. ng dng ca cc phng php lai trn pha rn Trong rt nhiu ng dng ca cc phng php lai trn pha rn , ni bt ln ba phng php c s dng rng ri nht :phng php Southern blot, Northern blot v Western blot . 3.1. Southern blot Phng php ny c s dng nh v nhng trnh t c bit trn DNA b gen ,hay nhng DNA kch thc nh nh DNA plasmid, phage C s ca phng php l k thut chuyn (tranfer) DNA t gel ln mng lai do Southern m t nm 1975. Phng php Southern blot gm cc bc sau: - DNA b gen c ct thnh nhng on kch thc khc nhau bi 1 hay nhiu enzyme gii hn (RE) ; cc on ny c phn tch da vo kch thc qua in di trn gel agarose .

S phng php lai Southern blot

- DNA c lm bin tnh (bin thnh 2 mch n ) ngay trn gel ri c chuyn ln 1 mng lai. Trong qu trnh chuyn, v tr cc on c gi nguyn . - DNA c nh trn mng c em lai vi mu d c nh du phng x. Sau qu trnh lai, ngi ta ra mng lai loi b cc mu d khng bt cp chuyn bit vi DNA c nh trn mng. - Cui cng, ngi ta dng k thut phng x t ghi (autoradiography) nh v cc phn t lai (DNA b gen ; mu d ) trong k thut ny, ngi ta t 1 phim nhy cm vi tia x p st vo mng lai. Cc phn t lai (ng hn l mu d c nh du phng x ) s tc ng ln phim v kt qu c th hin qua cc chm en trn phim. Cc ng dng ca Southern blot : - Lp bn gii hn ca 1 gen . - Pht hin cc a dng trnh t (frament polymorphism ) ca cng 1 gen cc chng hay cc c th khc nhau qua s so snh bn gii hn ca chng . - Pht hin cc t bin mt on , t bin im hay ti t hp trn 1 gen v chng lm thay i bn gii hn . 3.2 Northern blot

Chuyn RNA t gel ln mng lai S phng php lai

Northern blot

S phng php lai Northern blot

Phng php ny c s dng xc nh kch thc v hm lng ca 1 mRNA c trng trong 1 hn hp RNA. Phng php ny cng bao gm nhng bc sau: - RNA ( c lm bin tnh )s c phn tch theo kch thc nh in di trn gel agarose c cha cc cht lm bin tnh . Cc cht lm bin tnh c tc dng khin cc lin kt yu quy nh cu trc bc 2 ca RNA khng th hnh thnh tr li sau khi RNA bin tnh. V do khng cn tr s di chuyn cng nh s phn tch cc RNA trong gel. - Sau RNA c chuyn ln mnh lai . - RNA c nh trn mng lai c em lai vi mu d c nh du phng x . - Cc phn t lai c pht hin nh k thut phng x t ghi. 3.3. Western blot Western blot l qu trnh protein-protein kt hp c hiu vi nhau. Thng th ngi ta s s dng antibody protein co gn phng xa hoc gn hunh quang pht hin mt loi protein no Western blot l k thut pht hin protein bng phng php in di trn gel SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), c dng rng ri trong chn on cc bnh min dch nh chn on nhim HIV, vim gan virus... Western blot cn c tn gi khc l Western blotting hay l phng php lai thm protein. Western blot bao gm cc bc c bn sau:

- Protein c phn tch bng in di trn gel SDS-PAGE.

- Cc protein c chuyn sang mng lai nitrocellulose, gi nguyn v tr nh phn tch trn gel. - mng lai c nh protein vi mt khng th s cp (primary antibody). Khng th s cp l mt khng th c hiu, s bm vo protein v to thnh mt phc hp protein-khng th i vi protein quan tm. - Tip theo mng lai vi mt khng th th cp (secondary antibody) c enzyme (alkalin phosphatase hoc horseradish peroxidase) i km. Khng th th cp s bm vo khng th s cp ging nh khng th s cp bm vo protein. - Tip tc mng lai trong mt hn hp phn ng c hiu vi enzyme. Nu mi vic u din ra mt cch chnh xc s pht hin thy cc bng bt k ni no c mt phc hp protein-khng th s cp-khng th th cp-enzyme hay ni cch khc l bt k ni no c mt protein quan tm. - t mt phim nhy cm vi tia X ln mng lai pht hin cc im sng pht ra do enzyme.

3.4. Bng so snh cc phng php lai : Southern Blot 1) Trch DNA t t bo. 2) Ct vi enzyme gii hn. Northern Blot 1) Trch RNA t t bo. 2) Lm bin tnh vi formaldehyde . Western Blot 1) Trch protein t t bo . 2) Lm bin tnh vi SDS .

3) Chy trn gel (thng 3) Chy trn gel (thng 3) Chy trn gel (thng dng agarose). dng agarose). dng polyacrylamide gi l SDSPAGE. 3*) Lm bin tnh DNA vi alkali. 4) Chuyn ln mng 4) Chuyn ln mng 4) Chuyn ln mng nitrocellulose (thng nitrocellulose (thng nitrocellulose (thng do bng hat ng mao dn). bng hat ng mao dn). chy in di). 5) Tc nghn vi DNA 5) Tc nghn vi RNA 5) Tc nghn vi protein d. d. d 6) Lai vi mu d DNA 6) Lai vi mu d DNA 6) Lai vi mu d khng c nh du. c nh du. th c nh du. 7) Ra sch khi mu d 7) Ra sch khi mu d 7) Ra sch khi mu d cha c lin kt (di cha c lin kt (di cha c lin kt

s kim ngt)

sat

nghim s kim sot nghim ngt) ng dng - Bao nhiu mRNA ca gene X hin din? - di mRNA ca gene X? ng dng - Bao nhiu protein X hin din ? - ln ca protein X?

ng dng - Bn gii hn ca gene X trong chromosome. Pht hin cc fragment polymorphysm ca cng 1 gen cc c th khc nhau . - Pht hin t bin mt on, im hay ti t hp trn 1 gen.

Nhng c tnh quan trng ca 3 phng php : Southern Northern Ci g chia khi lng phn t ch? Mu d DNA b ct bi enzyme ct DNA mang gene X c tnh phng x RNA b lm bin tnh vi formaldehyde

Western Protein b lm bin tnh vi SDS

DNA mang gene X c Khng th i khng tnh phng x vi protein X c nh du phng x hay enzyme

CHT PH GIA THC PHM BIN I GEN Tn ging Bp Tc gi / c Gen hu hin quan Mosanto cry3Bb1 / Bacillus thuringiensis subsp. kumamotoensis Dekalb Genetics Corporation cry 1Ac / Bacillus thuringiensis subsp. kurstaki (Btk), bar/ Phng php xt nghim Southern Nm 2001 Hin trng Lm thc phm, thc n gia sc M,Canada,Phil ipines,Nht, Chu u lm thc n gia sc. Dng lm tc phm v thc n gia sc Canada, M, Philippines; ng dng Bo v bp i vi su hi r bp

Phn tch Southern, PCR

1997

Khng su c tri Chu u v thuc c glufosinate

Streptomyces hygrocopicus Mosanto mepsps / Zea mays Phn tch Northern v Western 1997

thc phm Nht, c v i loan Dng lm thc phm v thc n gia sc Canada, Argentina, Nht, M, Philippines; thc phm i loan, c v Hn quc; thc n gia sc Chu u Dng lm thc phm v thc n gia sc Canada, Argentina, Nht, M, Philippines, Nam Phi; thc phm i loan, c; thc n gia sc Chu u c s dng lm thc phm v thc n gia sc Nht v Canada; thc phm M c s dng lm thc phm v thc n gia sc Nht Khng thuc c glyphosate

Pioneer HiBred Internationa l, Inc., Mycoyen Seeds/Dow AgrSciences LLC

cry1Fa2 / Bacillus thuringiensis var. aizawai pat / Streptomyces viridochromogen es

Phn tch Southern, Northern, Direct DNA sequencing, ELISA

2001

Khng su c tri bp Chu u (Ostrinia nubilalis), Ty nam (Diatraea gradiosella), su keo Spodoptera sp., black cutworm (Agrotis ipsilon); khng thuc c glufosinate Khng thuc tr c Glufosinate

Ci du

AgrEvo

pat / Streptomyces viridochromogen es

Phn tch Southern

1995

Plant Genetic System

bar/ Streptomyces hygrocopicus, barstar / Bacillus amyloliquefacien s

PCR, Phn tch Southern, Northern; pat assay, npttII assay Phn tch Southern

1996

Khng thuc tr c Glufosinate, tnh phc hi hu th

Khoai ty

Mosanto

cry 3A / Bacillus thuringiensis

1996

Chp nhn dng Khng b cnh lm thc phm cng Colorado

subsp. Tenebrionis

v thc n gia hi khoai ty sc Canada v M; thc phm c Phn tch Southern,West ern hoc ELISA 1998 Dng lm thc phm v thc n gia sc c, Philippines v M Dng lm thc phm v thc n gia sc M, Canada, Argentina, Brasil, Cng ho Sc, Nh, Mexico Dng lm thc phm v thc n gia sc M; thc phm Canada Dng lm thc phm v thc n gia sc M Dng lm thc phm v thc n gia sc Canada, V M Khng thuc c glyphosate

C ci ng

Monsanto, Syngenta Seeds AG

cp4 epsps /Agrobacterium sp. strain CP4

u nnh

Mosanto

cp4 epsps /Agrobacterium sp.strain CP4

Phn tch Southern, Western; PCR, ELISA

1994

Khng thuc c glyphosate

C chua Mosanto

cry1Ac / Bacillus thuringiensis subsp. kurstaki (Btk) cp4 epsps /Agrobacterium sp.strain CP4 bar /Streptomyces hygroscopius

Phn tch Southern

1998

Khng su thuc h Lepidoptera

La m

Mosanto

Phn tch Southern

2004

Khng thuc tr c glyphosate

Bng vi

Bayer CropSci

Phn tch Southern, PCR

2003

Khng thuc c glufosinate

CC CY TRNG BIN I GEN Loi Lipase Sn phm Tc gi / c quan Gen hu dng / ngun gc Phng php xt nghim Hin trng ng dng

IUB3.1.1.3

Novo Nordisk

Acid bo cao cho cng ngh sn xut b sa Hiu sut cao lipase / Rhizomuco r meihie
Phn tch Southern NHT ph duyt

a-Amylase

TS-25 Maxiren

Novozymes A/S GISTBROCADES N.V. Genencor International, Inc. (USA)

Rennet

Hiu sut cao

Pullulanase

Optimax

Hiu sut cao

Riboflavin

Ribiflavin F. Hoffman(vitamin B2) La AMG-E Novozymes A/S

Hiu sut cao

Glucoamylase

Hiu sut cao