Nguyen Thj H6ng Gam Tap chi KHOA HQC & CONG N G H $ 115(01): 1 7 5 - 179
XAY DlTNG QUY TRINH PCR B A MOI
XAC DINH SU* METHYL HOA PROMOTER CUA G E N E B N A l
Nguyen Thj Hdng Gam*
Tnrdng Dgi hgc Y Dugc - DH Thdi Nguyen TOM T A T
Mi^c dich: Protein EBNAI cua EBV dupe bilu Id d tdt cd thi tilm dn va sir dung cac promoter khdc nhau, bao g6m Wp, Cp va Qp. Su dilu h6a bilu Ip gen EBNAl do nhilu c a chi, trong do c6 su methyl hoa cytosin cua CpG d vfing promoter. Muc tieu nghifin cuu la thilt k l cac cSp m l i d l xdc djnh mirc do methyl hda cdc promoter cua gen EBNAl. P h u o n g phdp: tiln hanh xay dung quy trinh PCR da moi bSng su dyng d i i tugng 1^ t l bao dong Namawa, Raji . K i t q u i : Hai cap mil xac djnh methyl hoa va khong methyl hda cua Wp, Cp va Qp cho kit qua duong tinh tit nhat d nflng dp ADN tucmg duong 1000 t l bdo Namalwa va Raji. D6i vdi Cp, hai cgp m l i xac djnh methyl hba va khdng methyl hoa cho Raji dIu cho kit qua am tinh K i t luan: Dilu kien phan img PCR da m6i va cdc c3p moi tu thiet ke dac hieu vdi Namalwa va Raji, ngogi trir Cp cQa Raji.
Tir khda: PCR da mdi. methyl hda. Wp. Cp vd Qp
DAT V A N D E
EBNAl ( E B V nuclear antigen 1) cd chieu dai 641 acid amin ( B 9 5 . 8 ) d u g c b i l u Id hdu n h u lat cd te bdo nhiem E B V va d d n g mpt vai trd quan trpng trong viec d u y tri episom ( d a n g vong) ciia EBV t r o n g nhan t l bao chu, ciing nhu chirc nang phien m a c u a virut [1].
EBNAl cung cd t h e lien ket vdi cac inlron dau lien xudi c h i l u ciia p r o m o t e r Q p d l t y dilu hda b i l u Id protein E B N A l . S y bieu Id ciia E B N A l d u d i s y dieu hda ciia cac promoter C p , W p hoac Q p , tiiy t h e o cac t h i nhilm tiem an k h a c nhau ciia E B V trong te bao chii [2, 3].
Sy methyl hda A D N d i l n ra d c a c vimg promoter ndy la m g l trong n h d n g yeu Id tac dpng len dieu hda phien mS dan den iirc che hoac tdng c u d n g bieu Id gen [4]. Hien t u g n g nay xuat hien d o mdt gdc - C H 3 lien ket d d n g hda trj vdi carbon sd 5 c u a Cytosin n a m d vj trf hai nucleotid C y t o s i n - G u a n i n t h e o chieu 5', viet tat Id C p G (p la lien k i t phosphodieste).
CpG t h u d n g tgp trung d c a c viing p r o m o t e r va hinh thanh nen c a c d a o C p G . D o dd, viec xdc djnh mirc dp methyl hda cua viing nay cd the gop phan tim h i l u nhitng c o c h e bieu ig hogc lie c h i b i l u Ig ciia gen EBV Irong ung t h u v6m miii hgng.
Tel:
N g d y nay, n h d nhiing tien bd ciia sinh hpc phdn t u ngudi ta cd the xac djnh d u g c mirc dp methyl hda ciia mpt gen dac hieu hay todn bd bd gen. Cdc ky thuat nay d y a vao nhOng nguyen ly khdc nhau, cd nhDng ky thugt rdt phuc lgp, n h u n g cd nhirng ky thuat don gidn vd thdng d u n g [5]. Dd cd n h u n g c d n g trinh xdc djnh m d c dp methyl hda cac promoter cua E B N A l biing phdn u n g PCR don moi [6], nhung c h u a cd c d n g trinh nghien ciru ve PCR da mdi d u g c c d n g bd. Vi v a y c o n g t r i n h n a y n h a m m u c tieu: xdy dimg quy trinh PCR da mdi de xdc dinh mirc dg methyl hda cdc promoter Wp. Cp vd Qp cua EBNA i tren hai dong li bdo Namalwa vd Raji.
DOI T U O N G V A P H U O N G P H A P Doi tuong
T I bdo ddng Namalwa, va Raji dugc cung cdp bdi Labo nghien cuu ung thu vdm mui hpng, Khoa Vi sinh, Sinh hpc tl bao va Khdi u (MTC), Vien Karolinska, Stockholm, Thyy Diln. Cdc ddng t l bdo dugc nudi cay trong mdi trudng RPMI 1640, 10%. PCS vd dl tu am 37" C. T I bao dupe thu hogch va chiet tdch ADN theo quy trinh dudi day.
Phu-ong phap
- Thill kl moi: Dk xac djnh muc dg methyl hda, 2 cap mdi cho mdi promoter dugc thiel 175
Nguyen Thi Hdng Gdm Tap chi KHOA HOC & CONG NGHE 115(01): 175- 179 kl. Mdt cap mdi diing de xac djnh ADN bi
methyl hda (M) va mpt cap mdi diing de xac dinh ADN khdng bj methyl hda (K). Sir dung phdn mem chuyen dung Methyl Primer Express Software vl.O. (Applied Biosystem, Hoa ky). Trinh ty mdi dugc sd dung dya vao trinh ty nucleotid cac promoter cda EBV thudc te bao ddng chuan ciia ngan hang gen (V01555).
- Chiet tach ADN
Ky thuat chill tach ADN dugc tiln hanh bdi
"DNA extraction kit" (Qiagen, Dire), theo quy trinh hudng ddn bdi cdng ty. Chdt lugng ADN, dugc kiem tra bang dien di san phdm Iren gel agarose 0,8% trong TBE Ix ( kit qua d hinh 1), do ndng dp san pham bang may NanoDrop.
- Xu Iy ADN b^ng Bisulfite
Cac budc xu ly ADN dugctach tir mau sinh thiet ung thu vdm mui hpng bang Bisulfite dugc tien hdnh theo bp kit Epitecl Bisulfite (Qiagen, Due).
- Phan irng PCR da moi
Phan irng PCR da mdi dac hieu su dung cap mdi gen ngi chudn actio va cac cap mdi ddc hieu xac dinh muc dp methyl hda trong phan ling 25^1 gdm: dung dich dem PCR, Mg2+, dNTPs, TaqGold polymerase va ADN khudn mlu. Chu ky nhiet gdm: bien tinh ADN va hogt hda TaqGold polymerase 95 CxlO', 5 chu ky nhiet: 94" Cx45", 60" Cx30", 72"
Cx45". Va 35 chu ky 94" Cx45", 56" Cx30", 72" Cx45"; 72" Cx7'. Dien di san phdm PCR, nhupm ethidium bromide va chup anh KET QUA
Danh gia chat luang ADN chilt tach bdng dien di (Hinh 1)
Hinh anh dien di cho thdy ADN dugc chilt tach cd chat lugng tdt vdi trpng lugng phdn tir cd kich thudc Idn, ddng thdi khdng cd diit gay va khdng lln cac thanh phan khac nhu protein.
lang 1. Cdc loai cap moi sir dung trong phdn irng MMSP Cac cap m6i xac
C P - U F 2 - C P - U R 2 Wp - UR2.
W P - U F 2 OP - UF2 QP - UR2 Cac cap moi xac Cp - MF2
Cp - MR2 Wp - MF2 Wp - MR2 O P - M F 2
Q P - M R 2 Cap mot gen noi
ACTB-R4
dinh ADN khong methyi hoa (5'-3') GGGTAAAGGGGTTTTATGGGT CTACATTTTTCAAATCATAAACAAA ACCTCTAACCCAATACCAAA TTTGGGGTTATTTTTATTATTGTT GTAGGTTTTGTTTTGGGGT CAAAAAAATCAACCTCCAAC dinh ADN methyl hoa (5'-3')
TAAAGGGGTTTTACGGGC CATTTTTCAAATCGTAAACGAA CGGGGTTATTTTTATTATCGTC ACCTCTAACCCGATACCG GTAGGTTTTGTTTTCGGGGC CAAAAAAATCGACCTCCGAC chuan actin dijng cho ADN da durcrc xu*
CTTTACACCAACCTCATAACCT
Kich thu'o'C 83bp
l l l b p
105bp
77bp
lOElbp
104bp iy bisuifite (5'.3')
92bp
Nguyen Tlij Hong Gim Tap clii KHOA HOC & CONG NGHE 115(01): 175- I-')
•^v^
H i n h i Hinh dnh dien dl ADN sau khi chiet tach
da mdi ddi vdi ddng t l bdo Raji, thuc hien ddng thdi vdi ddng tl bao Namalwa. Cap moi dimg cho ADN khdng methyl hda cua Wp cho ket qua dm tinh, Irong khi do cap moi diing cho ADN methyl hda cd kit qua duong linh mgnh d hai ddng t l bdo. Qp thi hoan loan ngiroc lai, ddi vdi moi dimg cho ADN khdng methyl hda thi duong tinh manh va mdi dimg cho ADN Op methyl hda Igi am tinh d ca hai ddng le bdo. Cap moi dimg cho ADN khdng methyl hda cua Cp duong tinh yeu a Namalwa. cdn lai deu am tinh (Hinh 3).
Xdc dinh lu-png ADN khudn m5u ciia phan ung PCR da mdi, sir dung te bao ddng Namalwa (Hinh 2A va 2B).
Te bdo ddng Namalwa la logi tl bdo cd EBV vdi sd lugng tir 1-2 ban sao trdng 01 tl bao.
Do dd, ngudi la thudng diing loai te bdo nay de xac djnh dp nhay ciia phan ung PCR, vdi cac nong dp le bao pha loang khac nhau.
Nghien ciru sir dung ndng dp ADN tuong duang 1000 te bdo Namalwa va sau dd pha lodng bgc 10 thanh 100. 10 va I te bdo (tuong ling vdi mdu 2, 3, 4. 5, Hinh 2A vd 2B). Kit qua cho Ihay cap mdi ciia gen ndi chuan actin hoat dpng tot d phdn irng PCR \ac djnh methyl hda va khdng methyl lida. giam dan theo dp pha loang vd d ndng dp 01 te bao van duong tinh rd ret. Moi Qp dimg cho ADN khong methyl hda dirong tinh mgnh va giam ddn d tdi ca ndng do, irong khi mdi Op cho ADN methyl hda cd mpt bang yeu d ndng do ADN tuong duang 1000 tl bao. cdc ndng do khac khdng thdy bang. Ngirgc lai. Wp khdng methyl hda Ihl cho bang rat yeu. ngay ca d ndng dp 1000 tl bdo, va Wp methyl hda cho bang rat ddm d cac ndng dp vd ket qud gidm dan.
Miie do khdng methyl hoa va methyl hda cua cac promoter d hai ddng te bao Namalwa va Raji
Tren eo sd dp nhgy cua phan irng d ddng te bao Namalwa. ndng dp ADN luang duang 1000 tl bao dugc sii diing cho phan dng PCR
isobfj .
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M
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'T-
M
-Wp Op
r^arker H,0 Namalwa Raji
Hinh 3: K: Khong methyl hoa M: Methyl hoa BAN LUAN
Ky thugt PCR da mdi ciing dya tren nguven ly ciia PCR don mdi. nhung nd cd it nhdt tir 02 cap mdi trd len dugc khucch dai trong mpt dng nghiem PCR [7]. Tuy nhien. PCR da mdi Cling cd nhung dac diem rieng cdn phai chii \ 177
Nguyen Thj Hong Gam Tap chi KHOA HQC & CONG NGHE 115(01): 175- 179 tir khau Ihiet ke mdi nhu nhiet dp gan mdi
luong ty nhau. kich thudc san pham khac nhau. va cac thanh phdn khdc cua hdn hgp PCR cung phdi tang len [8]. Viec xdc djnh mirc dp methyl hda ciia viing promoter bang ky thual PCR dan mdi hay da mdi deu tuan thii nhirng nguyen tae tren, nhung cung cd them nhiing khac biet dgc trung do trinh ty ADN da dugc chuyen ddi bdi bisulfite [5].
Chung tdi ty thiet ke mdi bang phan mem chuyen dung vd tim dieu kien tdi uu cho phan iing dua tren cac nghien ciru cua chiing tdi vdi cac gen ire che ung thu va gen EBV. Cap mdi gen npi chuan la actin dimg cho ADN sau khi da xu ly vdi bisulfite cung dugc thiet ke va cho kel qua rat lot d cac lan phan tich the hien trong cdc Hinh 2 va 3. Theo nhieu lac gia va kinh nghiem ciia chiing tdi. khi sir dung mdt cap mdi indi cho phan irng PCR. cdc ndng dp Mg2+ khac nhau can phai thir de tdi uu hda phan ung. Ddi vdi phan ung PCR da mdi dac liieu methyl hda, cac thanh phan ciia phan irng PCR dupe tang len, tuy nhien se khdng lang theo ty le sd cap mdi su dung [7].
Ndng dp IV1g2+ dugc sir dung la 4 mM vd enzym dugc hogt hda d nhiet dp cao la AmpliTaq Gold cho cac phan irng PCR da mdi dac hieu. Mac dau cac bang dac hieu the hien rat ro, tuy nhien van cdn nhirng bang klidng dac hieu trong san pham PCR. Cd the do dieu kien phan irng chua tdi iru hoan loan, can phai dieu chinh cac yeu td tham gia phan irng PCR da mdi nhu ndng dp cac mdi dua vao, nhiet dp gan nidi, ndng dp Mg2+ theo mpl ty le thich hop han.
Trong nghien ciru nay ADN ciia hai ddng le bao Namalwa va Raji dugc xir ly va khuech dai bdi cac cap mdi tu thiet ke vi chimg da duoc \ac djnh mirc dp methyl hda d cdc vimg promoter Iheo phuong phap giai trinh ly gen, day coi nhu la chudn vdng cua ky thuat PCR (hitp://gbrowse.bioinfo.cnio.es/ cgi- bin/VIRUS/HS4/). Tai vj tri gan mdi ciia cac cap mdi Wp va Cp ciia EBV d hai ddng te bao 178
dIu bi methyl hda, trong khi dd, Qp hau nhu khdng methyl hda. Kel qua nghien ciiu thu dugc ve eac cap mdi Wp va Qp phii hgp vdi chuan vang cua Namalwa va Raji va ket qua nghien ciru b^ng PCR don mdi (6, 9]. Ddi vdi Op ciing phii hgp vdi Tao. Q vd cpng sy, Qp hdu nhu khdng bj methyl hda d Namalwa, Raji va cho thay Op chua bao gid im lang [3].
Vdi cap mdi xac dinh methyl hda va khdng methyl hda ciia Cp cho bang ylu hogc dm tinh d Raji cd the do nhieu ly do bao gdm, dp nhgy ciia cap mdi, ndng dp mdi dua vao phdn iing hoac chdt lupng moi chua dii tdt hoac cd the do cd dot bien trong trinh ty ADN gan mdi hoac nhiing ly do khac chua biet rd [7, 8], Cdc promoter Wp. Cp va Qp cd khac nhau ve cau true va mat dp CpG. Rieng Wp. trong cdu triic cd den 12 promoter gidng nhau lap Igi nen ndng dp mdi sii dung cho nd bj giam di so vdi cdc ndng dp mdi Cp va Qp.
KET LUAN
Cac cap mdi de xac dinh su methyl hoa, khong methyl hda cac promoter Wp, Qp ciia EBNAl va cap mdi npi chuan actin boat ddng rdt tdl.Ddi vdi Cp mdi xdc djnh methyl hda vd khdng methyl hda cho ket qua chua ro rang.
KIEN NGHI
Cdn phai dieu chinh cdc thdng sd cua PCR hoac thiet ke mdi mdi khac. Ket qua nay cd the irng dung trong viec xac dinh mirc dp methyl hda cac promoter ciia EBNAl d cdc benh ly hoac te bao khac nhau nhiem EBV.
T A I L I E U THAM K H A O 1. Ceccareili, D.F. and Frapuier, L., (2000).
Functional analyses of the EBNAl origin DNA binding protein of Epstein-Barr virus. J Virol, 74 (I I): 4939-4948.
2. Yoshioka. M.. Kikuta, H., Ishiguro, N., Ma, X..
et a!., (2003). Unique Epstein-Barr virus (EBV) latent gene expression, EBNA promoter usage and EBNA promoter methylation status in chronic active EBV infection J Gen Virol, 84 (5): 1133 - 1140.
3. Tao, Q., Robertson, K.D., Manns, A., Hildesheim, A., et al., (1998). The Epstein-Bau virus major latent promoter Qp is constitutively
Nguyen Thj Hong Gam Tgp chi KHOA HOC & CONG NGHE ! 15(01): 175- 179
active, hypomethylaled, and methylation sensitive, J Virol. 72 (9): 7075 - 7083.
4. Salamon. D., Takacs. M., Ujvari. D.. Uhlig. J..
el al.. (2001). Protein-DNA binding and CpG methylation at nucleotide resolution of latency- associated promoters Qp, Cp, and L M P l p of Epstein-Barr virus. J Virol, 75 (6): 2584 - 2596.
5. Dahl, C. and Guldberg, P.. (2003). DNA methylation analysis techniques. Biouerontolouy, 4 (4): 2 3 3 - 2 5 0 .
6. Paulson, E.J. and Speck. S.H.. (1999).
DifTerenlial methylation of Epstein- Barr virus latency promoters facilitates viral persistence in
healthy seropositive individuals. J Virol. 7 (12):
9959 - 9968.
7. Henegariu, O.. Heerema, N.A.. DIouhv. S.R..
Vance. " G . H . . el al., (1997). Multiplex PCR critical parameters and stcp-by-step protocol.
Biotechniques. 23 (3)- 504 - 511,
8. Markoulatos. P.. Siafakas. N . and Moncan>. M..
(2002). Multiplex polymerase chain reaction' practical approach. J Clin Lab Anal. 16(1). 4 7 - 5 L 9 Park. J H.. Jeon, J.P.. Shim. S.M.. Nam, H.Y.. et al.. (2007). Wp specific methylation of highl>
proliferated LCLs Biochem Bioph\s Res Commun. 358 (2)- 513 - 520
SUMMARY
M U L T I P L E X P C R F O R P R O M O T E R iVIETHYLATION O F E B N A l G E N E Nguyen Thi Hong Gam College- oJ.Medicaland Huirmncy - IM Object: EBNAl protein is expressed in all latency program of infecled cells using different promoters including the Wp, Cp and Qp. The EBNAl expression is regulated by some ditTerent mechanisms and methylation of cytosine in CpG positions of promoter region is shown. The aim of the study is to design primer pairs to detect Ihe methylation level of EBNAl. Method:
promoters by the multiplex PCR using cell lines Namalwa Raji. Results, All unmethylation and methylation primer pairs of Wp, Cp and Qp work well with DNA of 1000 Namalwa and Raji cells, excepi the Cp is negative for both unmethyiaied and methylated primer pairs. Conclusion: Wp and Op primer pairs and conditions for the multiplex PCR are specific with Namalwa and Raji lines, except Cp for Raji.
Key words.* multiplex PCR. meihylaiion, Wp. Cp and Qp
Ngdynhgn bdt:3l/l2/2013: Ngdvphan hi('n:20/01/2014. Sgdv duyet ddng 07/02 2014 Phdn biin khoa hoc: TS Xguvin Thi \'goc Hd - Trirdng Dat hgc ) Dirt/c DHTN
