I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN Lê thị bích trâm Nghiên cứu số locut đa hình str người việt nam nhằm sử dụng khoa học hình sự, nhận dạng cá thể xác định huyết thống Luận án tiến sĩ sinh học H NI - 2016 I HC QUC GIA H NI TRNG I HC KHOA HC T NHIấN Lê thị bích trâm Nghiên cứu số locut đa hình str người việt nam nhằm sử dụng khoa học hình sự, nhận dạng cá thể xác định huyết thống Chuyên ngành : Di truyền học Mã số : 62 42 70 01 Luận án tiến sĩ sinh học Người hướng dẫn khoa học: PGS.TS Đinh đoàn long pgs.Ts trịnh đình đạt H NI - 2016 LI CAM OAN Tụi xin cam oan tt c cỏc kt qu nghiờn cu lun ỏn l trung thc v cha tng c cụng b bt k cụng trỡnh no khỏc Nu sai tụi xin chu trỏch nhim hon ton Tỏc gi Lờ Th Bớch Trõm LI CM N Lun ỏn ny c hon thnh ti Phũng K thut Sinh hc nghip v thuc Vin K thut Húa hc, Sinh hc v Ti liu nghip v, Tng cc Hu cn - K thut, B Cụng an Vi lũng kớnh trng v bit n sõu sc, tụi xin chõn thnh cm n : PGS.TS inh on Long, Ch nhim B mụn Y dc hc c s, Phú ch nhim khoa Y- Dc, H Quc Gia H Ni ó tn tỡnh hng dn, giỳp tụi quỏ trỡnh hon thnh lun ỏn PGS.TS Trnh ỡnh t, B mụn Di truyn hc - Khoa Sinh hc - H Khoa hc t nhiờn, H Quc Gia H Ni ó tn tỡnh hng dn, giỳp tụi quỏ trỡnh hon thnh lun ỏn TS Nghiờm Xuõn Dng, Thc s Trn Minh ụn, Thc s Lng Th Yn - Tng cc Hu cn - K thut - B Cụng an ó giỳp v úng gúp nhng ý kin quý bỏu cho tụi quỏ trỡnh hon thnh lun ỏn Khoa Sinh hc cựng cỏc phũng chc nng, phũng o to sau H Trng H Khoa hc t nhiờn - H Quc gia H Ni ó to iu kin thun li cho tụi quỏ trỡnh thc hin lun ỏn Tụi cng xin chõn thnh cm n PGS.TS Nguyn Th Hng Võn cựng th cỏn b, giỏo viờn B mụn Di truyn hc - Khoa Sinh hc - Trng H Khoa hc t nhiờn - H Quc Gia H Ni v Lónh o Vin K thut Húa hc, Sinh hc v Ti liu nghip v ó ng h v luụn to iu kin thun li cho tụi quỏ trỡnh hon thnh lun ỏn Cui cựng xin gi li cm n ti ton th cỏn b Phũng K thut Sinh hc nghip v - Vin K thut Húa hc, Sinh hc v Ti liu nghip v cựng gia ỡnh, bn bố luụn bờn tụi, quan tõm, ng viờn v to iu kin thun li nht tụi cú th hon thnh bn lun ỏn ny Tỏc gi MụC LụC Trang Mc lc Cỏc ch v t vit tt Danh mc cỏc bng Danh mc cỏc hỡnh M U 10 CHNG TNG QUAN 15 1.1 Cỏc phng phỏp nhn dng cỏ th ngi 15 1.1.1 Phng phỏp hỡnh thỏi hc 16 1.1.2 Nhn dng cỏ th bng cỏc yu t cú bn cht 17 protein 1.1.3 Nhn dng cỏ th ngi qua phõn tớch ADN nhõn 19 1.1.4 Phng phỏp phõn tớch ADN ty th 20 1.2 ADN v cỏc phng phỏp phõn tớch a hỡnh ADN ng 22 dng nhn dng cỏ th ngi 1.2.1 Cu trỳc a dng trỡnh t ADN cú ý ngha 22 nhn dng cỏ th 1.2.2 Kỹ thuật phõn tớch đa hình chiều dài on ADN 24 bng enzym giới hạn 1.2.3 Phng phỏp nhõn bi ADN (PCR) 24 1.2.4 K thut PCR a mi (multiplex PCR) 25 1.2.5 K thut in di 26 1.2.6 K thut gii trỡnh t 28 1.3 Cỏc locut STR h gen ngi v ng dng ca 29 chỳng nhn dng cỏ th 1.3.1 Khỏi nim cỏc on lp v STR 29 1.3.2 C s khoa hc ca phõn tớch STR nhn 30 dng cỏ th v xỏc nh huyt thng 1.3.3 Cu trỳc ca STR v danh phỏp quc t 31 1.3.4 Vai trũ ca cỏc STR phõn tớch hỡnh s 32 1.3.5 Vai trũ ca vic la chn, phi hp cỏc locut 34 STR khỏc xỏc nh huyt thng 1.4 Tỡnh hỡnh nghiờn cu nhn dng cỏ th ngi bng 35 phng phỏp phõn tớch ADN trờn th gii v ti Vit Nam 1.4.1 Tỡnh hỡnh nghiờn cu trờn th gii 35 1.4.2 Tỡnh hỡnh nghiờn cu ti Vit Nam 41 1.5 Cỏc locut s dng nghiờn cu lun ỏn 43 1.5.1 Cỏc locut STR 43 1.5.2 Locut Amelogenin 45 CHNG VT LIU V PHNG PHP NGHIấN CU 46 2.1 i tng nghiờn cu 46 2.1.1 Mu sinh phm 46 2.1.2 Cỏc locut a hỡnh STR v locut gii tớnh 46 Amelogenin 2.2 Thit b v húa cht 46 2.2.1 Thit b 46 2.2.2 Húa cht 47 2.3 Cỏc phng phỏp nghiờn cu 49 2.3.1 Thit k mi 49 2.3.2 Tỏch chit ADN 51 2.3.3 Phng phỏp PCR a mi (multiplex PCR) 51 2.3.4 Phng phỏp in di v phõn tớch sn phm PCR 53 2.3.5 Phng phỏp ch to thang alen 55 2.3.6 Phng phỏp xỏc nh trỡnh t cỏc nucletotide 57 2.3.7 Kho sỏt tn sut alen v x lý s liu bng cỏc 57 ch s thng kờ CHNG KT QU NGHIấN CU V BN LUN 3.1 Thit k mi v la chn iu kin PCR thớch hp cho 60 60 phc locut F13A01, D8S1179, Amelogenin, HPRTB 3.1.1 Thit k mi cho locut F13A01 60 3.1.2 Thit k mi cho locut D8S1179 61 3.1.3 Thit k mi cho locut HPRTB 61 3.1.4 Thit k mi cho locut Amelogenin 62 3.1.5 Sng lc multiplex 63 3.1.6 La chn iu kin PCR thớch hp cho phc 64 locut F13A01, D8S1179, HPRTB v Amelogenin 3.2 Kho sỏt tn sut alen v ỏnh giỏ mc a hỡnh 74 ca 15 locut STR qun th ngi Vit (Kinh) bng cỏc ch s thng kờ 3.2.1 Kt qu kho sỏt tn sut alen 15 locut STR 74 3.2.2 ỏnh giỏ tớnh c trng qun th v mc a 77 hỡnh ca 15 locut STR 3.3 Ch to thang alen locut F13A01, D8S1179, 99 Amelogenin, HPRTB 3.3.1 Kt qu ch to thang alen locut F13A01 99 3.3.2 Kt qu ch to thang alen locut D8S1179 102 3.3.3 Kt qu ch to thang alen locut Amelogenin 105 3.3.4 Kt qu ch to thang alen locut HPRTB 106 3.4 xut hng ng dng cỏc locut STR nhn 109 dng cỏ th v xỏc nh huyt thng Vit Nam 3.5 Ch to b KIT v th nghim phõn tớch 3.5.1 Ch to b kit nhõn bi ADN phc locut 110 110 F13A01, D8S1179, HPRTB, Amelogenin 3.5.2 Phõn tớch xỏc nh kiu gen cỏ th 111 3.5.3 Phõn tớch xỏc nh huyt thng 115 KT LUN 118 KIN NGH 120 DANH MC CC CễNG TRèNH KHOA HC CA TC GI 121 LIấN QUAN N LUN N 122 TI LIU THAM KHO PH LC CC CH V T VIT TT Ch/t vit tt ADN ARN APS bp CPI Ting Anh Deoxyribonucleic acid Ribonucleic acid Amonium persulphate base pair Combined Parternity Index dNTP EDTA NST PBS PCR Deoxynucleoside triphosphate Ethylendiamin tetraacetic acid PD PE PI Power of Discrimination Power of Exclusion Paternity Index PM FDAH Matching Probability F13A01, D8S1179, Amelogenin, HPRTB Restriction fragment length polymorphism RFLP Ting Vit Axớt Deoxyribonucleic Axớt Ribonucleic Cp baz Ch s quan h huyt thng kt hp Nhim sc th Phosphate buffered saline Polymerase chain reaction STR Short tandem repeat TBE TEMED Tm VNTR Tris Borat EDTA N,N,N,N- tetramethylethylene diamine Melting Temperature Variable number of tandem repeats CODIS Combined DNA Index System CSDL KHHS Phn ng chui trựng hp Kh nng phõn bit Kh nng loi tr Ch s quan h huyt thng Xỏc sut trựng lp a hỡnh chiu di on c ct bng enzym gii hn on ngn lp li liờn tip Nhit núng chy on lp li liờn tip vi s lng thay i H thng ch s ADN kt hp C s d liu Khoa hc hỡnh s DANH MC CC BNG Bng Tờn bng 1.1 Tn sut phõn b alen 15 locut STR qun th ngi Trung Quc 1.2 Tn sut phõn b alen 15 locut STR qun th ngi Ba Lan Trang 38 39 1.3 Tn sut phõn b alen 15 locut STR qun th ngi M (gc Phi) 40 1.4 c im ca 15 locut STR s dng nghiờn cu 45 2.1 Cỏc thụng s th nghim thnh phn PCR phc locut F13A01, D8S1179, Amelogenin v HPTRB 52 3.1 Trỡnh t v thụng s cỏc cp mi ca locut F13A01, D8S1179, HPRTB v Amelogenin 64 3.2 Kt qu la chn thnh phn PCR thớch hp cho phc locut FDAH 72 3.3 Kt qu la chn chu trỡnh nhit thớch hp cho phn ng PCR phc locut FDAH 74 3.4 Tn sut phõn b alen ca 15 locut a hỡnh STR trờn 250 cỏ th ngi Vit (Kinh) 76 3.5 S lng alen kho sỏt c ca 15 locut STR 250 cỏ th ngi Vit (Kinh) 94 3.6 Kt qu la chn alen cho locut F13A01 99 3.7 Kt qu la chn alen cho locut D8S1179 102 3.8 Kt qu la chn alen cho locut HPRTB 106 3.9 Kt qu phõn tớch kiu gen 16 locut cho 10 phm nhõn 115 3.10 Kt qu phõn tớch kiu gen 16 locut mu (B) v (C) 117 KIN NGH Trong khuụn kh ca lun ỏn ny, chỳng tụi ó nghiờn cu xõy dng c t hp locut a hỡnh mi ng thi kho sỏt tn sut alen 15 locut a hỡnh STR qun th ngi Vit (Kinh) Trong thi gian ti, chỳng tụi cú kin ngh cn nghiờn cu v kho sỏt b sung cỏc locut ADN a hỡnh cỏc nhúm ngi thuc cỏc dõn tc khỏc trờn lónh th Vit Nam xõy dng c bng phõn b tn sut cỏc alen phc v giỏm nh, nhn dng cỏ th 120 DANH MC CC CễNG TRèNH KHOA HC CA TC GI LIấN QUAN N LUN N Lờ Th Bớch Trõm, Trn Trng Hi, Bựi Nguyờn Hi, Nguyn Vn H, inh on Long, Trnh ỡnh t (2011), Kt qu nghiờn cu ch to thang alen ch th cỏc locut a hỡnh F13A01, D8S1179 v HPRTB ngi Vit Tp phõn tớch Húa-Lý v Sinh hc, T-16, S 1/2011, trang 10-14 Lờ Th Bớch Trõm, Lng Th Yn, Trn Minh ụn, Nguyn Thanh H, inh on Long, Trnh ỡnh t (2011), Nghiờn cu thit k mi, ti u iu kin phn ng PCR phc ba locut : F13A01 - D8S1179 - HPRTB v locut gii tớnh Amelogenin, Tp phõn tớch Húa-Lý v Sinh hc, T-16, S 2/2011, trang 39-43 Lờ Th Bớch Trõm, Lng Th Yn, Trn Minh ụn, Nguyn Th Thu Hoi, inh on Long, Trnh ỡnh t (2011), Nghiờn cu kho sỏt tn sut phõn 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Application of the new insertion - deletion polymorphism kit for forensic identification and parentage testing on the Czech population, Int J Legal Med., 127 (1), pp 7-10 106 Zitkiewicz E., Witt M., Daca P., Gala J Z., Goniewicz M., Jarzb B., Witt M (2012), Current genetic methodologies in the identification of disaster victims and in forensic analysis, J Appl Genet., 53 (1), pp 41 -60 107 Ziwen H., Xinnian L., Shaoping L., Yun-Xin F., Eric H., Suhua S., Chung-I W (2013), Estimating DNA polymorphism from next generation sequencing data with high error rate by dual sequencing applications, BMC Genomics, 14, pp 535- 544 NGUN CSDL TRC TUYN 108 CSDL trc tuyn ca Trung tõm thụng tin Cụng ngh sinh hc quc gia (M), truy cp ti a ch http://www.ncbi.nlm.nih.gov/nucleotide 109 CSDL trc tuyn ca Vin Tiờu chun v Cụng ngh quc gia (M), truy cp ti a ch http://www.cstl.nist.gov/strbase 134 [...]... hng ng dng ca cỏc locut ny trong nhn dng cỏ th phự hp vi qun th ngi Vit Do vy, chỳng tụi tin hnh nghiờn cu ti lun ỏn Nghiờn cu mt s locut a hỡnh STR ngi Vit Nam nhm s dng trong khoa hc hỡnh s, nhn dng cỏ th v xỏc nh huyt thng vi cỏc mc tiờu sau : 1 Thit k c cỏc cp mi c hiu nhõn bi v phõn tớch c tớnh a hỡnh 03 locut STR (F13A01, D8S1179 v HPRTB) nhm b sung vo b cỏc locut s dng trong nhn dng cỏ th... HPRTB 5 Nghiờn cu xõy dng thang alen ch th cho 4 locut F13A01, D8S1179, HPRTB v Amelogenin 6 xut hng ng dng 15 locut STR trong nhn dng cỏ th v xỏc nh huyt thng ti Vit Nam * í NGHA KHOA HC V THC TIN 1 Nghiờn cu b sung bn locut F13A01, D8S1179, HPRTB v Amelogenin kt hp vi cỏc locut ó c nghiờn cu cú ý ngha vụ cựng quan trng trong vic: - Tng chớnh xỏc trong phõn tớch nhn dng cỏ th v xỏc nh huyt thng... HPRTB, 06 alen cu locut F13B, 08 alen ca locut FES/FPS v 05 alen ca locut LPL; phỏt hin thờm c 02 alen mi qun th ngi Vit (Kinh) l alen s 8 ca locut CSF1PO, alen s 13 ca locut vWA 3 Xõy dng c 04 b thang alen ch th cho cỏc locut F13A01, D8S1179, HPRTB v Amelogenin s dng trong phõn tớch kiu gen cỏ th m bo chớnh xỏc cao 4 ỏnh giỏ c kh nng ng dng ca 15 locut STR i vi qun th ngi Vit (Kinh) trong nhn dng cỏ... tớch t hp 4 locut a hỡnh mi F13A01, D8S1179, HPRTB v Amelogenin cú th nhõn bi ng thi trong cựng mt phn ng B sung cho cỏc t hp locut ó nghiờn cu, gúp phn lm tng chớnh xỏc trong phõn tớch nhn dng cỏ th ngi v giỏm nh huyt thng phự hp vi iu kin phũng thớ nghim ti Vit Nam 2 B sung s liu kho sỏt tn sut alen 15 locut a hỡnh ngi Vit (Kinh), trong ú ó kho sỏt mi 07 alen ca locut F13A01, 06 alen ca locut HPRTB,... qua trng Cỏc locut STR nm trờn nhim sc th cng theo ú m c di truyn qua cỏc th h (hỡnh 1.1) 30 B M (8-11) (9-12) Con 1 Con 2 Con 3 Con 4 (8-9) (8-12) (9-11) (11-12) Hỡnh 1.1 S minh ho cỏc kh nng di truyn mt s alen thuc locut STR t b, m cho con theo nh lut Mendel (8 - 12: mt s alen thuc locut STR [23]) 1.3.3 Cu trỳc ca STR v danh phỏp quc t Tờn cỏc locut ch th ADN c t theo tờn ca gen nu locut ny nm ... xỏc nh bi v trớ ca chỳng trờn nhim sc th Vớ d, STR locut D5S818 v DYS19 ú l nhng locut khụng nm trong vựng gen Trong trng hp ny ch "D" cú ngha l ADN Con s tip theo l s th t ca nhim sc th Ch "S" thc cht l trỡnh t n l ca ADN Con s cui cựng l v trớ locut nm trờn mi nhim sc th riờng bit Con s ny l duy nht i vi mi locut ADN s dng trong nhn dng cỏ th Vớ d, locut D16S539 cú ngha l D: ADN; 16: Nhim sc th s... tn sut phõn b cỏc alen thuc 15 locut STR ngi Vit (Kinh), gm D5S818, D7S820, D13S317, CSF1PO, TH01, TPOX, D16S539, D3S1358, vWA, F13B, FES/FPS, LPL, F13A01, D8S1179 v HPRTB 3 Xõy dng c thang alen chun cho 03 locut F13A01, D8S1179, HPRTB v locut gii tớnh Amelogenin 4 ỏnh giỏ c mc a hỡnh v xut c hng ng dng ca 15 locut STR trong truy nguyờn cỏ th v xỏc nh huyt thng ti Vit Nam * NI DUNG CHNH CA LUN N Cỏc... nhiờn, mt locut STR ch cú th s dng c cho mc ớch nhn dng cỏ th khi tha món nhng iu kin nht nh Th nht, STR phi cú tớnh a hỡnh v mc d hp t cao iu ny giỳp cỏc nh phõn tớch ch cn s dng ớt nht s lng STR m vn ỏp ng c yờu cu v tin cy Th 19 hai, cỏc locut STR phi ớt b bin i di tỏc ng ca mụi trng xung quanh nh nhit , m, vi sinh vt m bo s nguyờn vn ca phõn on ADN trong quỏ trỡnh phõn tớch Th ba, cỏc STR phi... ATAT ATAT ATAT ATAT - 4 on lp ATAT Cỏc locut h VNTR v STR l nhng locut cha cỏc gc nucleotit lp li nhiu ln, th hin tớnh a hỡnh chiu di on Vớ d locut D5S818 l mt locut thuc h STR, gm 4 nucleotit (AGAT) lp i lp li t 6 n 18 ln 23 cỏc cỏ th khỏc nhau [23] Theo nh lut di truyn t locut cú s lng 13 alen ny cú th to ra (13 x 14)/2 = 91 t hp kiu gen khỏc nhau T hp nhiu locut khỏc nhau s cho kh nng phõn bit cỏ... gi l VNTR, vớ d locut D1S80 cú on lp l bi s ca 16 nucleotit, locut ny cha cỏc alen t s n v lp li 14 ln n 41 ln Trỡnh t lp li l GAGGA CCACCAGGAAG Cỏc on ADN cú cu trỳc lp li t 2 - 6bp c gi l cỏc on lp li ngn (STR) Cỏc cu trỳc VNTR hay STR mang tớnh bo th cao, c di truyn qua cỏc th h v tớnh c trng cỏ th Cỏc VNTR v STR cú th c nhõn bi bng phn ng PCR 1.3.2 C s khoa hc ca phõn tớch STR trong nhn dng cỏ