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K thut da trn c s PCR

a hnh di cc on khuych i Amplified fragment length polymorphisms (AFLPs)

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences

Ni dung
Nhng nt chnh Cc bc ca k thut AFLP
Phn gii v kt ni DNA Phn ng PCR v s thm d Tm tt ca k thut ny

Phng tin Dch m cc bands AFLP Nhng thun li v bt li ca AFLP ng dng


Biotechnological Section, Department of Biology, Hue University of Sciences

9-2007

Nhng nt chnh ca k thut AFLP


K thut AFLPs c pht trin bi Keygene (Netherlands), mt cng ty cng ngh sinh hc t nhn v thuc quyn s hu cng ngh ca cng ty ny. Nhng nt chnh:
Mt s kt ni gia k thut RFLP v PCR Da vo s khuych i PCR mt cch chn lc cc phn on ct hn ch ca DNA b phn gii Phng php c nhy cao trong vic tm du vt ca DNA ca bt k ngun gc no hay s phc tp no C th thc hin vi DNA ca ton h gen hay vi cDNA (bn sao nguyn bn)
9-2007 Biotechnological Section, Department of Biology, Hue University of Sciences

Bn bc chnh
1. DNA c phn gii bi hai enzyme ct hn ch khc nhau 2. Phn ni (adapter) oligonucleotide c ni vi nhng u tn cng ca cc phn on DNA 3. Cc tp hp con c trng ca cc sn phm phn gii DNA c khuych i, s dng kt hp cc primer chn lc 4. Thm d s a hnh c th thc hin c vi cc ng v phng x, nhum hunh quang hay nhum bc
9-2007 Biotechnological Section, Department of Biology, Hue University of Sciences

Phn gii v kt ni DNA


Enzyme ct hn ch th nht (im nhn bit 4 bases, vd:Msel) Enzyme ct hn ch th 2 (im nhn bit 6 bases, vd: EcoRI) Nhng phn kt ni 2 dy c tng hp c bit i vi mi im ct hn ch c gn vi on DNA tip theo

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences

PCRs v s thm d
Mt phn ng PCR th nht c thc hin (preselective PCR), dng nhng primer oligonucleotide (1721 nucleotides) c trnh t b sung vi adapter v restriction sites. Mt nucleotide c thm vo primers ch chn nhng tp con ca cc phn on Nhng sn phm khuych i tin chn lc ri phi tri qua mt phn ng PCR khc, li mt ln na mt tp hp con ca cc phn on li c chn. Thng th i vi s khuych i chn lc th 2, 2 nucleotides ph s c thm vo primers Nhng phn on c phn tch bng in di gel gy bin tnh polyacrylamide (gel trnh t) hay in di gel thm thu
9-2007 Biotechnological Section, Department of Biology, Hue University of Sciences

S tm tt ca AFLPs

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences

Phng tin
Ngun
Nc ct Ho cht

Dng c

9-2007

T lnh, t ng su Laminar c dng ht kh My li tm My PCR My iu chnh ngun in Microwave pHmeter Cn chun My in di phng thng ng My soi mu cc tm (UV transilluminator)
Biotechnological Section, Department of Biology, Hue University of Sciences

Dch m cc bands AFLPs


C s phn t ca a hnh AFLP thng c to ra mc nucleotide K thut AFLP thm d s a hnh xut hin t cc thay i (hnh dng hay kch c) trong nhng im ct hn ch hoc cnh nhng im ct ny, c gy ra do thiu cc nu. u 3 v lm ngn cn s khuych i
Nhng enzyme ct hn ch khc nhau c th c s dng, nhng s kt ni khc nhau ca cc nucleotide chn lc v tin chn lc s gia tng tm kim cc a hnh c ch Cng nhiu nucleotide chn lc th cng t a hnh c thm d Nhng bands thng c ghi c khi hin din hay khng Nhng bands d hp ngc li vi ng hp c th c thm d da vo dy cc du hiu, mc d iu ny c th i hi s tinh t

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences

Mt bn gel chy vi thuc nhum hunh quang


y l hnh nh mt AFLP gel chy trong my c trnh t t ng. Trc khi load mu vo gel, mu c gn vi 3 loi thuc nhum hunh quang (yellow, blue, green). Mu gn vi mu iu khin cng vi cc mu khc iu khin s trnh din trong in di. Vic bit chc s hin din hay vng mt ca mt band nht nh l khng th lm c bng mt thng m phi nh vo my laser v d liu c thu thp vi s gip ca phn mm my tnh c bit

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences

AFLPs c thm d bng nhum bc

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences

Nhng u im ca k thut AFLPs


AFLPs cho php kim tra nhanh s ng hnh trn ton b gene Bi v mt lng ln cc bands c sinh ra, mi marker s cho ra mt du hiu informative cao Kh nng lp li cao Khng cn bit trc v thng tin ca cc trnh t v vic to ra cc u d l cn thit Rt c ch trong trong vic to ra nhanh bn gene transcript profilling (s chp li nguyn bn-sao bn)

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences

Nhng bt tin ca phng php ny


AFLPs to ra mt lng ln thng tin cn phi c phn tch t ng, v th cn s gip ca k thut my tnh cao AFLP marker biu hin s u th Trong xy dng bn di truyn, AFLPs thng tp trung li vng centromeres v telomeres Chng i hi nhng k thut phng th nghim v c bit l phn tch d liu
9-2007 Biotechnological Section, Department of Biology, Hue University of Sciences

ng dng
nh gi a dng di truyn Phn tch di di truyn Kim tra du hiu di truyn Phn tch b su tp ngun gene (germplasm collections) Xy dng bn gene Qun l cc ch th thm d
9-2007 Biotechnological Section, Department of Biology, Hue University of Sciences

Tm li
K thut AFLP da vo s khuych i chn lc cc phn on hn ch sau khi phn gii DNA N thm d s a hnh gy ra bi nhng thay i trong nhng im hn ch hay nhng vng ln cn K thut ny to nn mt lng ln bands trong mt ln chy, v cc bc tin hnh i hi k thut cao
9-2007 Biotechnological Section, Department of Biology, Hue University of Sciences

Cu hi
Cc bc khc nhau lin quan n qu trnh thc hin k thut AFLPs Nhng quan tm chnh khi dch m cc bands AFLP Nhng thun li v bt li ca AFLP trong phn tch a dng di truyn

9-2007

Biotechnological Section, Department of Biology, Hue University of Sciences