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JOURNAL OF SCIENCE OF HNUE DOI: 10 18173/2354-1059.2017-0014 Natural Sci. 2017, Vol 62, No. 3, pp. i 14-120

This paper is available online at http://stdb.hnue.edu.vn

X A C DJNH D A HINH rsl0811661 GEN CDKN2A TREN Q U A N T H E NGU"6l VIET NAM SU' DUNG PHU'CNG PHAP AS-PCR VA RFLP-PCR

N g u y e n T h i T r u n g T h u ' , Bui Thj N h u n g ^ v a T r a n Q u a n g Binh^

'Khoa Smh hoc. Truang Dai hoc Sir phgm Hd Ndi

^Khoa Dinh du&ng hgc duang vd Ngdnh nghe, Vien Dinh duang Qudc gia

^Khoa Mien Dich vd Sinh hf>e phdn tu. Vien sinh dich te Trung uang

Tom tit. Gen CDKN2A cd lien quan den benh tien dai thao dudng va dai thao dudng typ 2 thong qua giam kh6i luang te bao P va suy giiim tiet insulin. Phirong phap AS-PCR va RFLP- PCR dugc su dung de xac djnh kieu gen cua da hinh rsl0811661 gen CDKN2A tren lugng mau Idn cfla quan the ngudi Viet Nam. Phuang phap AS-PCR sfl dung mdi nguoc phat hien alen C (Rc): 5'-GGTAATAGACTTACTGTCATCG-3' va alen T (Rt).5--GGTAATAGAC TTACTGTCATCA-3' va m6i xuoi (F): 5'- TCAGTTAAGCAGATGAAATTC-3', nhiet do bat mdi la 52 "C vdi su co mat ciia alen C hoac T thi san pham c6 kich thuuc 208 bp. Phirong phap RFLP-PCR su dung mdi xuoi (F): 5'-ACCTTCAGCCACCTCTCTGTCTTTC-3' va mdi ngugc (R): 5"-CCCATCCTGGGTAGGAGGAGCC- 3 ' , nhiet dp bSt mdi la 62 "C, san pham co kich thudc 350 bp. Sau khi sfl dung enzyme cat gidi han PagI thi kieu genCC cd 1 san pham (350 bp), kieu gen TT c6 2 san pham (280 bp va 70 bp), kieu gen CT 3 san phjm (350 bp, 280 bp va 70 bp).

Tirkhda: AS-PCR, RFLP-PCR, relOSl 1661, gen CDKN2A, xac dinh kieu gen

1. ivitfd^u

Gen CDKN2A nim tren canh ngSn cua NST so 9 tai vi tri 21 tfl vi tri 21967751 bp d^n vi iri 21994490 bp, vdi kich thude: 26739 kb [1]. Clu true cfla CDKN2A gdm 3 exon (exon la, exon ip, exon 2, exon 3) va 3 introa Exon 2 va exon 3 dugc ghep vao vdi mdt trong 2 exon dau tien thay the la, lp t^o thanh 2 khung dgc khac nhau. Dieu nay dan den tao ra2 san phim protein P16 vii P14'^'*''- chdl uc che khdi u anh hudng den tuyen tyy tang sinh te bao p [2].

Gen CDKN2A lang tinh nhay cam cfla tien dai thao dudng va benh dai thao dudng typ 2 thdng qua giam Ididi lugng te bao p va sau do suy giam tiet insulin can thiet trong co the vdi nhu cau insulin tang len [3]. Mdt trong nhflng chflc nang quan trgng anh hudng tryc tiep ldi benh dai thao dudng lyp 2 dugc nghien CUTI la gen CDKN2A cd vai trd trong ung thu luyen luy [4].

xac dinh dot bien CDKN2A d 6/28 ngudi (chiem 21%) trong cac gia dinh dugc xac dinh chac chan mac benh ung thu tuyen tuy [5]. Vi vay, vi?c xac dinh dac diem cfla da hinh CDKN2A cd vai tro quan trgng trong nghi6n cunj anh hudng cfla gen den cac benh lien quan.

Ngaynhan bai: 22/3/2016, Ngay nh^n dang: 20/5/2016 Tac gia hen h?: Tr^n Quang Blnh, email: binhnihe@yalioo.com

Xdc dinh da hinh rsl08!I66I gen CDKN2A tren qudn the nguoi vi^t nam sir dung phuong phdp...

Trong cac nghien cuu trudc deu sfl dyng phuang phap RFLP-PCR de xac dinh da hinh rsl0811661 gen CDKN2A [6. 7], Tai VietNam, chira cd nghien cuu nao ve xac dinh kieu gen da hinh rslOSl 1661 gen CDKN2A. Dd dap flng viec nghien cuu da hinh rsl0811661 gen CpKN2A tren quin the ngudi Viet Nam vdi cac thiel bi hien cd va nhu cau mau ldn, chung tdi lien hanh phuang phap AS-PCR de xac dinh kieu gen rslOS 11661 gen CDKN2A tren lugng mau Idn va su dung phuang phap RFLP-PCR de kiem tra do cliinh xac efla phucmg phap.

2. Noi dung nghien ciru

2.1. Doi tuwng va phuoTtig phap nghien ciiu

* Ddi tuang nghien cuu

Mlu mau su dung trong nghien cflu lay tfl 141 ddi tugng tai thanh phd Phfl Ly, linh Ha Nam.

Mau ADN duge tach chiet tu 300 pL mau loan phin lay tfl tinh mach, chdng ddng bang ETDA vdi bd kit Wizard Genomic DNA purification Kit (Promega Cat #A1125, USA) theo hudng din cua nha san xuat. Dp tinh sach va ndng do ADN dugc do bang may NanoDrop. Npi dung nghien cuu da dugc Hdi ddng dao due trong nghien euu Y sinh hpc - Vien Ve sinh dich te Tmng uang thdng qua. Cae ddi tugng deu dugc giai thich ve nghien cflu va ki vao giay ddng y tham gia nghien cflu.

* Thiit ki quy trinh AS-PCR cho xdc dinh kiiu gen rsl08lI661 gen CDKN2A - Thiet ke mdi

+ Budc 1. Xac djnh trinh lu tni'de va sau rsl0811661 gen CDKN2A tren genebank tfl NCBI [I].

+ Budc 2. Nhan bidt trinh tu nucleotide mismatch va thidt kg mdi xudi (ngugc) d6 xac dinh alen C hoac T cfla rsI0811661 gen CDKN2A dya theo Wangkuhang va cdng sy [8].

+ Budc 3. ThiSt k^ mdi ngugc sfl dyng phan mem Oligo 7 Primer Analyse [9] va UCSC In-Silico PCR tryc tuygn [10] dk chgn cap mdi thich hgp ddng nhat ve nhiel do bat mdi (T„) va hai mdi khdng bat cap nhau.

- Thiit ki quy Irinh PCR

De thuc hien phan ung AS-PCR phat hien kieu gen cfla da hinh rsl0811661 gen CDKN2A, moi ki^u gen duge xae dinh nhd hai phan flng dgc lap phat hien alen C va alen T.

Mdi phan ung gdm cac thanh phin: 0,8 ^iL nudc (khdng nucleotide); 2,5 yL PCR master mix, 0,35 pL mdl xudi, 0,35 ^L moi ngugc va 1,5 pL ADN mau Cin xac dinh chu trinh nhiel eho phan UTig PCR, dac biet la phan flng bat mdi dya tren cap mdi sfl dung.

San phim PCR dugc xac dinh bing nhudm vdi Redsafe, dien di trong gel agarose 30 phfli d 100 V, 0,5 X dem TIBE, SO sanh vdi marker *X174 DNA Haelll Digest. Bang ADN duqz phat hien su dung may anh Geldoe-Il™ gel. Batig cua ADN dugc kiem tra lieu cd phfl hgp vdi ngan hang gen.

* Thiet kiquy trinh RFLP-PCR cho xdc dinh kieu gen rs 10811661 gen CDKN2A Phuang phap RFLP-PCR dugc thill kS de kiem ira do chinh xac eua phuang phap AS-PCR.

- Thiel ke mdi.

+ Budc 1. Xac dinh trinh ty tnrdc va sau rsl08II661 gen CDKN2A tren genebank lu NCBI [I].-

+ Budc 2. Xac dinh enzyme cit gidi han phfl hgp de nhan biet alen C va alen T sfl dung phin mgm: restrictionmapper.org [11].

+ Budc 3. Thidl ke mdi xudi va moi nguac cho phan ung PCR.

- Thiil ki quy trinh PCR

115

Nguyen Thi Tmng Thu, Bui Thi Nhung va Trin Quang Binh

Moi phan ung RFLP-PCR phat hien ki6u gen cfla da hinh rsI0811661 gen CDKN2A chua 15 jiL gdm cac thanh phan: 4 \iL nudc (khdng nucleotide); 7,5 jiL PCR master mix; 1 pL m6i xudi; 1 p.L mdi ngugc va 1,5 yL ADN mau. Can xac dinh nhiet do thich hgp cho phan ung gin mdi va chu trinh nhiet cho phan flng. San pham PCR dugc xac dinh bang nhupm v6i Redsafe, dien di trong gel agarose 30 phut d 100 V, 0,5 X dem TBE, so sanh kit qua vdi marker ^X174 DNA Haelll Digest. Bang ADN dugc phat hien su dung may anh Geldoc-Il™ gel.

Bang cua ADN dugc kiem tra li^u cd phfl hgp vdi ngan hang gen.

- U enzyme cdt gi&i hgn vd xdc dinh kit qua

Mdt phan flng fl enzyme gdm: 5 ^L san pham PCR chat lugng tdt, 0,7 )iL lOX dem, 0,15 |xL enzyme cit gidi han dugc lya chpn, 6,0 pL nudc linh sach. U san phim PCR vdi enzyme cat gidi han d nhi?t dp va thdi gian thich hgp theo khuyen cao cua nha san xuat,

Dien di 11,85 ^L san phim sau khi u tren thach agarose 2,5% dem TBE 0,5 X trong 30 phiit d 100 V nhudm RedSafe, cd marker PhiX174//aeIIL Va chyp hlnh san pham dien di sau khi u enzyme cit gidi han bing may GelDoc. Bang san pham dugc kiem ira lieu cd phu hgp vdi ngan hang gen.

2.2. K i t qua nghien cuv va thao lu^n 2.1.1. Xac dinh quy trinh AS-PCR

* Thiit ki mdi

- Buac 1. Xdc dinh trinh tumoi truac vd sau rsl081I661 gen CDKN2A

Tfl dfl Heu NCBI, trinh tu doan gen chua SNP rslOSI I66I gen CDKN2A nhan dugc la [1]:

AATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACAT TAGAACACCATAACCTTrCCGGCCCATTTTCTTTGTCAATAAGCGTTCrrGCCCTGTCA GCAGCTCACCTCCAGCTTTAGTTTTC(C/T)CATGACAGTAAGTCTATTACCCTCCTGAT CTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTC AGAACAAATTTTGGGAAGTAAATATAAGGTTT

Dya vao trinh ly doEin gen chfla SNP, tien hanh chpn 20 - 25 nucleotide trudc va sau SNP de Ihiet ke mdi phu hgp cho phuang phap AS-PCR.

- Budc 2. Chgn trinh tu nucleotide de thiet ke mdi

Dua theo nguyen tic chieu dai cua mdi, ty le GC cua mdi, sy khdng bat cap cfla cac nucleotide cua mdi, nghien cflu chpn trinh ty sau SNP la 5'-AGGGTAATAGACTTACT(3TCATG-3' de thiet ke mdi. Nhiet do bat moi efla mdi xudi la 52,3 "C va mdi ngugc la STC theo khuyen cao cfla phan mem Oligo 7 [9].

Muc nhiel dp nay kha phu hgp cho phan fltig bit c?p cfla mdi vdi Irinh ty nucleotide tren m^ch, - Buac 3. Xdc dinh nucleotide mismatch vd thiet ke mdi nhdn biet alen C/T

Nucleotide thu 2 tinh tfl dau 3' cua moi dugc su dung de thiet ke I mismatch Iheo Wangkumhang va cgng su [8]. KSt qua 2 mdi ngugc de phat hien SNP la:

Mdi phat Men alen C (Rc): 5 - GGTAATAGACTTACTGTCATCG - 3'.

Mdi phat Men alenT (Rt): 5 -GGTAATAGACTTACTGTCATCA - 3',

Dtem misyiatch la nucleotide khac vdi nucleotide tren mach gdc, cd tac dung lam lang bat cap mdi va tinh dac hieu cfla enzyme trong phan ung [12].

- Budc 4. Thiit ke moi xuoi

Sfl dyng phan mim Oligo 7 [9] va UCSC In-Silico PCR (true tuydn) [10] de thiet kl mii xudi sao cho moi xudi va mdi ngugc cd su luang ddng ve nhi?t do bit mdi va khdng bat clip va kich thudc cfla san phim PCR trong khoang 200 bp. Ket qua mdi xudi chflng ldi chpn la:

Moi xudi la: F: 5'-TCAGTTAAGCAGATGAAATTC-3' 116

Xdc dinh da hinh rslOSl 1661 gen CDKN2A tren qudn thengie&i vift nam su dung phuang phdp..

San pham PCR su dung cap mdi xudi va mdi ngugc d iren cd kich thudc la 208 bp theo dfl lieu NCBI [I]:

TCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCT GTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCAT AACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCT CCAGCTTTAGTTTTC(C/T)GATGACAGTAAGTCTATTAC

* Thih ki chu trinh PCR

Vdi cap mdi sfl dung, chdng tdi sfl dyng phan mem Oligo 7 [9] va UCSC In-Silicon PCR (true tuyen) [10] de xac djnh rdiiet do cfla mdi xudi la 52,3 "C va mdi ngugc la 51,0 °C va. Vi vay, de chgn nhiet do gan mdi (Ta) tMch hgp cho phan flng PCR, chflng tdi thyc Men kiem tra tiMet do bit mdi d 4 nhiet dg- 48 °C, 50 "C, 52''C va 54 "C trong 31 chu ki (kit qua khdng hiln thi). Trong dd, nhiet do 52 "C cho kit qua ro net nhat. Ket qua dien di cac san phim theo phuang phap AS-PCR d nhi^l dp gan moi 52 "C dugc the Men d Hinh 1. Cac mau ed alen C hoac T cho bang 208 bp phu hgp vdi du li?u tfl NCBI.

Hinh L Ket qud xac dinh kteugen cuaSNP rsl08I1661 tren gen CDKN2A bdng phuang phap AS-PCR

M: Marker <l>Xl74HAEm. I (TT). 2 (CC). 3 (CT). 4 (TT), 5 (CT). 6 (TT). 7 (CC) Vi v|y, chu trinh phan ung gdm bien tinh ADN d 94 "C trong 3 phut, tiep theo 32 chu ki gom bien linh d 94 "C irong 30 giay, gan mdi d 52 "C trong 30 giay, keo dai mdi d 72 "C trong 30 giay va keo dai d 72 "C trong 8 phfli, cudi cung la gifl hon hgp d 15 "C sfl dung may may PCR mastercycle epgradient (hang Eppendorf). Sfl dyng phuang phap AS-PCR de xac djnh kieu gen cua 100 miu nghien cuu cho thiy ty le dgc rat eao 99% (99/100).

Phuang phap AS-PCR dugc tien hanh dya tren hai phan ung khulch d?i PCR song song rieng Met, mdi phan img sfl dung mdt cap mdi dac hieu tai dau 3' de nhan biel mpt ADN [8]. Di£u nay dya tren su keo dai cfla mdi cM kM dau 3' eua mdi bat e^p dugc vdi alen cua mau. Nhu vSy, neu cd da hinh dan nucleotide xay ra, kit qua cd the xac dinh bing each nhan biet cMeu dai cfla cac san pham PCR. Day la phap don gian, nhanh chdng va dang lin cay, ma khdng ddi hdi nhilu thill bi may mdc dit liln nen kha nang ap dung lai cae phong thi nghiem d Viet Nam la cao.

D|e biet, nghien cuu cd the ung dyng khi xac dinh kieu gen Iren mgt sd lugng ldn mau.

2.1.2. Xac dinh quy trinh RFLP-PCR

De kiem tra dp chinh xac cua phuang phap AS-PCR, chung tdi liln hanh sfl dyng phuang phap RFLP-PCR thdng qua enzyme cat gidi han.

* Thiet ke moi

-Buac I Xdc dinh trinh lu mdi trudc vd sau rsiOSl 1661 gen CDKN2A Kit qua da dugc irinh bay d mye 2.1.1.

- Buac 2. Xdc dinh enzyme cdi gidi hgn ddc hieu

Nguyen Thi Trung Thu, Biii Thi Nhung va Trin Quang Binh

Sfl dung phan mem reslrictionmapper.org [11] nhan thay, enzyme BspHI va EamII05I co kha nang cat tai vi tri alen T. cdn enzyme EamliOSl ed kha nang cat tai vi tri C. Vi vay, chflng tdi lya chgn enzyme cat gidi han PagI de phan biet alen C va alen T.

- Budc 3. Chon trinh tu nucleotide di thiit ki mdi

Thilt ke mdi xudi va mdi nguoc sfl dyng phan mem Oligo 7 [9] va UCSC In-Silico PCR (true tuyin) [10] de chpn cap mdi thich hop ddng nhat ve nhiet do ndng chay (T^), Ididng bit cap, chieu dai mdi khoang 20 nucleotide, ti le GC khdng qua 60%. Chflng tdi lya chpn cap mdi la:

Mdi xudi: 5'-ACCTTCAGCCACCTCTCTGTCTTTC-3' Moi ngugc: 5'-CCCATCCTGGGTAGGAGGAGCC-3'

San phim PCR tfl dfl lieu UCSC In-Silicon PCR (true Uiyln) [10] gdm 350 bp:

ACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTT TTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCA GATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGA A AGC ACTTATC AGTTCTGTCTA ATG A AG AC ATT AG A AC ACC AT AACCTTTCCGGCCC A TTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTC(

C/T)CATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGA TGGG

* Thiit kiquy trinh PCR

NMet dp ndng chay cfla mdi xudi la 65,4 "C va mdi ngugc la 69,2 "C theo UCSC In-Silicon PCR (true tuyin) [10], Vi nhiet do bat mdi kha eao, de dam bao dp nhay va dp dac hieu ciia nhiet dp bat mdi, chung ldi kiem ira d cac nhiet do bat mdi: 58 "C, 60 "C, 62 "C va 64 "C (kit qua khdng hieu thi). Ket qua cho thay, nhiet do bat mdi Ihieh hop la 62 "C (Hinh 2A). Vi vay, ehu Irinh nhiet cfla phan flng PCR gdm biln tinh ADN d 94 "C trong 3 phiit; tilp theo 32 chu ki gdm bien ti'nh d 94 "C trong 30 giay, gan mdi d 62 "C trong 30 giay, keo dai mdi d 72 "C trong 30 giay; va keo dai d 72 "C Irong 10 phut; cudi cflng la giu hdn hgp d 15 "C sfl dung may may PCR mastercycle epgradient (hang Eppendorf).

* U enzyme cdt giai hgn vd dien di

Sau dd, 5 - 10 |iL mau cd kel qua PCR tdt se dugc sfl dyng de lien hanh u vdi enzyme cat gidi ban/'ag/d 37 °C trong 16 gid theo khuyen cao cfla nha san xuat. Vj iri cat cfla enzyme-

s'.. T^CATGA. .3' 3'...AGTACtT...5'

Hinh 2. Ket qud xdc dinh kieu gen ciia SNP rsl081I661 trin gen CDKN2A bdng phuang phdp RFLP-PCR tren mptsd mau

A. Kit qud diin di lan I sau phdn ung PCR. B: Kit qud dien di ldn 2 sau ii enzyme BspHI.

M: Marker 0X174 HAEIII. I (TT). 2 (CC). 3 (CT). 4 (TT). 5 (CT). 6 (TT). 7 (CC)

Xdc dinh da hinh rsl 08} 1661 gen CDKN2A tren qudn thi ngu&i vi^t nam su dung phuong phdp...

Do dd, enzyme cd kha nang cit t^i vi Iri SNP cd alen T. Nh^n dinh kieu gen tfl san pham:

kiiu gen CC (350 bp). kieu gen TT (280 bp va 70 bp) va kiiu gen CT (350 bp, 280 bp vk 70 bp).

Bang san phim 70 bp khdng xuit Men do cd kich thudc nhd da chay ra khdi ban Uiach Uong qua trinh chay dien di. Kit qua dien di hinh 2B cho diiy cac bang san phim hoan loan phu hap va cd Ihl xac dinh duac kieu gen cfla cac mau nghien edu.

Nhu vay, ket qua dien di theo phirang phap RFLP hoan loan trung l^p vdi phuong phap AS- PCR. Su dyng phuang phap AS-PCR de xae dinh kieu gen cfla 100 mau nghien cflu cho thay ti le dgc rit cao 99% (99/100). Ket qua dien di tren sd lugng ldn mau chung td ket qua phuong phap RFLP-PCR ehinh xae irong viee xac dinh kieu gen.

Phuang phap RFLP-PCR cuu linh da hinh chieu dai cfla cae phan do^n ADN dya irSn diem cat cua cac enzyme gidi han. Nguyen tac eua ki thuat nay dya trSn dp dac hieu cfla cae enzyme cit gidi han ddi vdi vi tri nhan biel cua chflng tren ADN bd gen. Khi fl vdi enzyme gidi h^n d dung dich dem. pH, nhiel do va thdi gian thich hgp, doan ADN se hi enzyme gidi han eat d vi tri dac hieu de tao ra nhflng phan doan ADN vdi kich thudc khae nhau. Dya vao kich thudc cac doan cat de xac djnh alen va kieu gen. Trong cac ngMen cflu trudc deu sfl dung enzyme PagI de nhan biel alen C va T. Tuy nMen, cap mdi sfl dung cho phan flng PCR khac nhau. Theo ngMen cuu cfla Hubacek va es nam 2013 thi cap mdi su dung cho RFLP-PCR la 5'-GAAGACATTAGAACACC ATAACCTTTCC-3' va 5'-AGGAGGAGCCAGAAGACAGATCAGG-3', san phim l^o ra cd kich thudc 143 bp, tao ra cae doan cit enzyme la 94 bp va 49 bp [6]. Va nghien cflu cfla Singh va cs nam 2012 sfl dyng e|p mSi. 5'-ATAAGCGTTCTTGCCCTGTC-3' va mdi ngugc: 5'-GTCA AAAACCTTCCCCATCC-3', san phim tao ra cd kich thudc 121 bp, tao ra eac doan cit enzyme la 85 bp va 36 bp [7]. San phim PCR trong nghien cuu efla chung tdi cd kich thudc 350 bp (kha ldn). Khi sfl dung enzyme cit gidi han PagI tao ra cac doan cit 280 bp va 70 bp, dl dang nhan biet bang sfl dung gel agarose.

3. Ket luan

Phuong phap AS-PCR su dyng moi nguge phat hien alen C (Rc): 5'-GGTAATAGACTT ACTGTCATCG-3' va alen T (Rt): J-GGTAATAGACTTACTGTCATCA-S' va mdi xuoi (F): 5'-TCAGTTAAGCAGATGAAATTC-3', nMet do bit moi la 52 "C vdi su cd m^t cfla mdi alen C hoac T thi san phim cd kich thudc 208 bp. Phuang phap RFLP-PCR su dyng mdi xudi (F): 5'-ACCTTCAGCCACCTCTCTGTCTTTC-3' va mdi ngugc (R): 5'-CCCATCC TG GGTAGGAGGAGCC-3', nhiel do bit mdi la 62 °C, san phim cd kich thudc 350 bp. Sfl dung enzyme cit gidi han PagI a 37 "C trong 16 gid dl cat mau cd kieu gen TT hoac CT. Ket qua san phim PCR cua kiiu genCC (350 bp). kiiu gen TT (280 bp va 70 bp), kiiu gen CT (350 bp, 280 bp va 70 bp). Cac kll qua nghien euu xac nhan, phuang phap AS-PCR va RFLP-PCR cd the sfl dung dl xac djnh kiiu gen cua da Mnh rs 10811661 gen CDKN2A tren quin Ihl ngudi Viel Nam.

L&i cam an. D I lai dugc sy tai trg eua Quy phat triin khoa hpc va cdng nghe Qudc gia (NAFOSTED) cho Nghien cuu thu&n tgp 5 ndm ve benh ddi thdo dudng typ 2 vd hpi chimg chuyin hod a nguai VietNam. vai tro yeu to di truyen vd ldi sdng, ma so 106-YS.01-2015.10.

TAI LIEU THAM KHAO

[1] http://www.ncbi.nlm.mh.gov/projects/SNP/snp_ref cgi?rs=10811661 tra cuu ngay 1/4/2014.

[2] http://atlasgeneticsoncology.org/. Tra cuu ngay 1/4/2014.

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sensitivity in 5,327 nondiabetic Finnish men Diabetes, 58, No 9, pp. 2129-2136.

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ABTRACT

Determination of rsl0811661 geneCDKN2A polymorphism in Vietnamese population using AS-PCR and RFLP-PCR methods

Nguyen Thi Trung Thu', Bui Thi Nhung^ and Tran Quang Binh^

'Faculty of Biology. Hanoi National University oJ Education 'Department of School and Occupational Nutrition. National Institute of Nutrition

^Department of Immune and Molecular Biology. National Institute of Hygiene and Epidemiology CDKN2A gene is associated with prediabetes and type 2 diabetes by reducing p cell mass and insulin secretion. In the current stutly, AS-PCR and RFLP-PCR methods were adopted lo determine the genotype of rsl081166I gene CDKN2A potymorpMsm in the samples in Vietnamese population. AS-PCR used reverse primers to detect C allele (Rc): 5'-GGTAA TAGACTTACTGTCATCG-3' and T allele (Rl): 5'-GGTAATAGACTTACTGTCATCA-3' and forward primer (F): 5'-TCAGTTAAGCAGATGAAATTC-3', the annealing temperature is 52''C, vnth presence of allele C or T, the PCR product size is 208 bp. RFLP method - PCR used forward primer (F): 5'-ACCTTCAGCCACCTCTCTGTCTTTC-3' and reverse primer (R); 5'-CCCATCC TGGGTAGGAGGAGCC-3', the annealing temperature is 62 "C and PCR product size is 350 bp.

After using PagI restriction enzymes, CC genot>T3e has one product (350 bp), TT genotype has two products (280 bp and 70 bp) and CT genotype has three products (350 bp, 280 bp and 70 bp).

Keywords: AS-PCR, RFLP-PCR, genotype, rsl0811661. gen CDKN2A.