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vn

Bi tiu lun:

PHN LOI SINH HC PHN T

Ngi hng dn: PGS.TS Chu Hong Mu

NCS: Nguyn Th Thu Ng

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NI DUNG

I. M u

II. CC PHNG PHP C BN CA PHN LOI

SINH HC PHN T

III. Kt lun

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I. M u
Sinh hoc phn t l mn khoa hoc nghin cu gii sinh vt mc phn t. Sinh hoc phn t ch yu nghin cu tng tac gia cac h thng cu truc khac nhau trong t bao, gm quan h qua lai gia qua trinh tng hp DNA, RNA, Protein v cch thc iu hoa cac mi tng tac nay. Ngy nay nhng tin b trong sinh hc phn t m ra kh nng ng dng c hiu qu trong phn loi hc v nghin cu a dng sinh vt.

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Phn loi Sinh hc phn t la phng phap phn loai sinh vt bng

c im gen mc phn t. Tc la phn loai bng cac dn liu so sanh hoa sinh cac phn t ln nh DNA, RNA, protein.

Cc phng php sinh hc phn t tp trung vo cc k thut ch yu sau:

+ Phn tch acid nucleic + Phn tch protein + Phn tch glicolipit + Ha phn loi hc

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II. CC PHNG PHP C BN CA PHN LOI
SINH HC PHN T

1.Phn tch v so snh acid nucleic

1.1. Phn tch DNA plasmid (S dng trong phn loi vi sinh vt 1.2. Phn tch ADN nhim sc th 1.3. Lai ADN/ADN 1.4. Lai DNA/RNA

2. K thut nhn gene PCR

2.1. K thut nhn gene PCR (Polymerase Chain Reaction)

2.2. Cc ci tin ca k thut PCR

3. Gii trnh t ADN 4. Phn tch trnh t r-RNA (ribotyping)

5. So sanh Protein
s

Ti liu chia s ti: wWw.SinhHoc.edu.vn 1. Phn tch v so snh acid nucleic

1.1. Phn tch DNA plasmid (S dng trong phn loi vi sinh vt)

Phng php phn tch y bao gm: + tch plasmid + tinh sch plasmid + x l bng enzym ct hn ch + in di trn gel agarose + so snh s a hnh ca cc phn on phn bit cc chng vi sinh vt vi nhau.

Di chuyn ca plasmid mch thng (L) v siu xon (OC) khi in di

1. Phn tch v so snh acid nucleic

1.2. Phn tch ADN nhim sc th
Phng php phn tch y bao gm:
+ tch ADN ca nhim sc th + ct bng enzym ct hn ch phn bit gia cc sinh vt vi nhau. Cc on ct nn c kch thc nh hn hoc bng 50 kb. Vic phn tch cc on ct c thc hin da vo k thut in di trong

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trng xung in (PFGE = Pulsed Field Gel Electrophoresis)

 K thut chinh ca PFGE la: i chiu dong in chay qua gel

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in di, gy xung in theo chu k. u im: k thut PFGE co kh nng phn tach theo kich thc nhng oan DNA kich thc ti vai megabase.

1. Phn tch v so snh acid nucleic

1.3. Lai ADN/ADN - Nguyn tc c tin hnh nh sau: + ADN tng s c tch ra v x l vi enzym ct hn ch (c

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trng hp khng cn x l vi enzym ct hn ch)

+ in di trn gel agarose + chuyn ln mng lai + Mu d (probe) c chun b v lai vi mng ADN trn. Kt qu php lai cho bit s khc bit gia cc mu ADN khc nhau.

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Phng php chuyn ADN ln mng cho k thut Southern Blot Mt s nhn t tham gia vo kt qu ca php lai: mu d ADN, phng php nh du ADN, mu ADN lai, iu kin tin hnh v phng php nh gi kt qu php lai.

1. Phn tch v so snh acid nucleic

1.4. Lai DNA/RNA

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- Phng php ny cho php so snh v phn bit ngun gene ca nhng sinh vt thuc cc taxon khc nhau c quan h xa.
-Trong php lai DNA/RNA s dng r-RNA hoc t-RNA. - K thut lai cng gm c cc khu c bn tng t nh lai DNA/DNA.

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2. K thut nhn gene PCR

K thut lai ADN c s dng rng ri cho nhiu nghin cu xc nh v phn loi trn i tng sinh vt. Ngun ADN ch i khi ch c hm lng t. Kh khn ny c khc phc bng cch lm tng ngun ADN ch mt cch nhanh chng bng k thut nhn gene PCR.

2.1. K thut nhn gene PCR (Polymerase Chain Reaction)

Nguyn l: da trn kh nng xc tc ca enzyme DNA polymerase

trong phn ng ko di on trnh t b sung bt u t on mi trnh t ngn gn trn 2 si n ca on DNA khun to phin bn ca on DNA cn phn tch.

Ti liu chia s Bc 1: bin tnh ADN, ti: wWw.SinhHoc.edu.vn c tc dng tch hai si

n t si khun xon kp.

Bc 2: bt cp hai mi vo hai si n ca khun.

Bc 3: ko di chui
theo mi, chiu 5-3 vi qu trnh gn cc dNTP

dc theo si khun to
thnh phin bn mi theo nguyn tc b sung.

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2. K thut nhn gene PCR

2.2. Cc ci tin ca k thut PCR

-Nested PCR (PCR lng)

-Revert transtriptase PCR

-Asymmetric PCR (PCR mt chiu) -Phn tch RFLPs vi sn phm PCR khi dng cp mi c hiu

-K thut PCR cho ribotyping (phn loi vi sinh vt)

-K thut rep-PCR -K thut RAPD (Random amplified of polymorphic DNA)

Ti liu chia s ti: wWw.SinhHoc.edu.vn Nested PCR Reaction Diagram

Ti liu chia s ti: wWw.SinhHoc.edu.vn Revert transtriptase PCR

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Asymmetric PCR (PCR mt chiu)

Ti liu chia s ti: wWw.SinhHoc.edu.vn Phn tch RFLPs vi sn phm PCR khi dng cp mi c hiu

Ti liu chia s ti: wWw.SinhHoc.edu.vn K thut PCR cho ribotyping

Kt qu ribotyping vi Helicobacter pylori.

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Ti liu chia s ti: wWw.SinhHoc.edu.vn K thut RAPD (Random amplified of polymorphic DNA)

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3. Gii trnh t ADN

Phng php gii trnh t ADN theo nguyn tc to

ra cc on ADN c nh du ti u 5 hoc 3. Cc
on c base nh u c tch ra trn gel polyacrylamid. Cc on c phn bit v v tr trn gel

vi s sai khc ch vi mt nucleotid. Vic to ra cc

on ADN sai khc nhau v 1 nucleotid c trnh by theo 2 phng php:

+ Phng php ha hc (Maxam & Gilbert, 1997) + Phng php enzym kt thc phn ng chui (Sanger, 1997)

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3. Gii trnh t ADN

+ Phng php enzym kt thc phn ng chui ca Sanger: Phng php kt thc phn ng chui c thc hin da trn vic nhn ADN t si khun khi c: + on ADN mi vi xc tc ca Klenow hay T7-ADN polymerase + 4 loi deoxyribonucleotid (dNTPs). Nh vy, c 4 phn ng c tin hnh ng thi v trong mi phn ng c mt loi dNTP b thay i bi mt dn xut lm dng phn ng (ddNTP).

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Phn tch trnh t DNA bng my t ng

4. Phn tch trnh t r-RNA (ribotyping)

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Cu to r-RNA bin i chm trong qu trnh tin ha, trnh t nucleotide ca r-RNA a dng, n nh. V vy phn tch v so snh trnh t nucleotide ca r-RNA cung cp nhng bng chng khch quan tin cy lm c s xc nh quan h tin ha gia cc loi, gia cc taxon bc trn loi.

4. Phn tch trnh t r-RNA (ribotyping)

- Phn lp v tinh sch r-RNA. - Dng enzyme phin m ngc v mi oligonucleotide bt cp b sung vi trnh t ca r-RNA to cDNA. - Nhn cDNA bng k thut PCR (Polymerase Chain Reaction) - Phn tch trnh t sn phm PCR thep phng Phng php phn tch mt on php Sanger bng my phn trnh t r-RNA ngn, c trng tch t ng nh phng php phn tch trnh t DNA.

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5. So sanh Protein

1. K thut nghin cu nh
lng phn ng khng th. 2. phng php xc nh v so

snh trnh t acid amin

3. Phng php in di 4. K thut min dch hc so snh khng th 5. K thut in di enzyme a locut

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5. So sanh Protein

Phng php xc nh v so snh trnh t acid amin ca cc protein c cng chc nng l phng php trc tip v c s dng rng ri nht. S liu so snh trnh t cytochrome, cc protein vn chuyn in t (electron transport protein), trnh t cc histone, cc protein sc nhit, protein phin m, dch m c dng rt ph bin trong phn loi hc.
10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.... |.... RAV2-Solanum --------SRYKGVVPQP-NGRWGAQIYEKHQR----------VWLGTFNEENEAARAYDIAAQRFR-----GRDAVTNFK-----------RAV1-Arabidopsis --------SKYKGVVPQP-NGRWGAQIYEKHQR----------VWLGTFNEEDEAARAYDVAVHRFR-----RRDAVTNFKDV---------RAV2-Arabidopsis --------SKYKGVVPQP-NGRWGAQIYEKHQR----------VWLGTFNEQEEAARSYDIAACRFR-----GRDAVVNFKNV---------RAV1-Solanum --------SKFKGVVPQP-NGRWGAQIYEKHQR----------IWLGTFNGEEEAARAYDIAAQRFR-----GRDAVTNFK-----------GmDREBc --------CNYRGVRQRT-WGKWVAEIREP-NRG-------NRLWLGTFPTAIGAALAYDEAAQAMY-----GSCARL--------------GmDREBa --------CNYRGVRQRT-WGKWVAEIREP-NRG-------SRLWLGTFPTAISAALAYDEAAMAMY-----GFCARLNFPNV---------TaDREB1 --------CAYRGVRQRT-WGKWVAEIREP-NRG-------NRLWLGSFPTAVEAARAYDDAARAMY-----GAKARVNFSEQ---------OsDREB2A --------CAYRGVRQRT-WGKWVAEIREP-NRG-------RRLWLGSFPTALEAAHAYDEAARAMY-----GPTARVNFADN---------GhDBP2 --------KLYRGVRQRH-WGKWVAEIRLP-RNR-------TRLWLGTFDTAEEAALAYDKAAYKLR-----GDFARLNFPNLRHHGSHVGDY GmDREB3-DQ055133.1 ---------LYRGVRQRH-WGKWVAEIRLP-KNR-------TRLWLGTFDTAEEAALAYDKAAYKLR-----GDFARLNFPNL---------GmDERBb --------KLYRGVRQRH-WGKWVAEIRLP-KNR-------TRLWLGTFDTAEEAALAYDNAAFKLR------

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III. Kt lun

Nh nhng nghin cu sinh hc phn t, nhiu chng trinh khoa hoc c xy dng phuc vu cho cng tac phn loai sinh vt. Ni bt la y tng ng h phn t v m hinh cy ngun gc chung loai.