서지주요정보
Capillary electrophoretic microdevice for variable number tandem repeat genotyping = 전기영동 마이크로 소자를 이용한 다수연쇄반복구간 유전자형 분석
서명 / 저자 Capillary electrophoretic microdevice for variable number tandem repeat genotyping = 전기영동 마이크로 소자를 이용한 다수연쇄반복구간 유전자형 분석 / Yu-Chao Chen.
저자명 Chen, Yu-Chao
발행사항 [대전 : 한국과학기술원, 2011].
Online Access 원문보기 원문인쇄

소장정보

등록번호

8023112

소장위치/청구기호

학술문화관(문화관) 보존서고

MCBE 11026

SMS전송

도서상태

이용가능

대출가능

반납예정일

초록정보

Variable number tandem repeat (VNTR) typing is a powerful tool for forensic identification due to the unique profile for an individual by using the highly polymorphic nature of short tandem repeat (STR) markers. Polymerase chain reaction followed by electrophoretic separation of the amplified products for STR analysis allows simultaneous amplification and analysis of multiple loci, and has been accepted as the gold standard for human identification in the forensic society. The commercial STR kits such as Powerplex 16 and AmpFlSTR Identifiler are designed to genotype 16 autosomal STR loci including the 13 Combined DNA Index System (CODIS) STR loci providing high discrimination power. Likewise, Y chromosome-specific STR typing kits (PowerPlex Y and AmpFlSTR Yfiler) are developed specifically for analyzing male genomic DNA such as sexual assault cases, paternity test, missing person identification, and evolutionary studies. In particular, Y-STR typing is very valuable in sexual assault cases since a vast majority of crimes involve males as the perpetrator. Lab-on-a-chip (LOC) or microfabrication technology has paved its way in a variety of chemical and bio-logical applications as a miniaturized analytical tool due to the benefits such as small sample consumption, high throughput synthesis and screening capability, fast analysis time and portability. Recently, the forensic human identification based on short tandem repeat (STR) genotyping has been performed on a microdevice platform, and the superior performance has been demonstrated. In our study, we focused on the sensitivity issue by designing an improved sample stacking capillary electrophoretic microdevice and used it for highly sensitive Y STR typing as well as PCR-free variable number tandem repeat genotyping. In the first part, we developed a sample stacking capillary electrophoretic (CE) microdevice for mini Y STR genotyping. The mini Y STR includes redesigned primer sequences to generate smaller-sized PCR ampli-cons to enhance the PCR efficiency and the success rate for a low copy number and degraded DNA. The mini Y STR amplicons occupied in the 5 and 10 mm stacking microchannel are preconcentrated efficiently in a defined narrow region through the optimized sample stacking CE scheme, resulting in more than 10-fold improved fluo-rescence peak intensities compared with that of a conventional cross-injection ??CE method. Such signal en-hancement allows us to successfully analyze the Y STR typing with only 25 pg of male genomic DNA, with high background of female genomic DNA, and with highly degraded male genomic DNA. The combination of the mini Y STR system with the novel sample stacking CE microdevice provides the highly sensitive Y STR typing on a chip, making it promising to perform high-performance on-site forensic human identification. In the second part, We demonstrated a proof-of-concept for novel minisatellite tandem repeat typing, called PCR-free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a liga-tion reaction instead of PCR thermal cycling, magnetic bead based solid phase capture for purification, and an elongated sample stacking micro-CE (μCE) for sensitive digital coding of repeat number. We designed a 16-bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin-coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min by using this method. In our study, we first focused on the capillary electrophoretic microdevice for highly sensitive and high-throughput Y STR typing by developing a sample-stacking CE operation with the multiplex mini Y STR typing system. These combinations would enable the superior low copy number (LCN) and degraded DNA genotyping to the conventional Y STR method with high speed and lower undesirable allelic drop-out and drop-in occurrence. Then, a proof-of-concept for novel minisatellite tandem repeat typing was demonstrated, called PCR-free digital VNTR (variable number tandem repeat) typing. In this method, we aimed to realize the high speed and portability of VNTR genotyping by using isothermal ligation reaction instead of PCR thermal cycling.

Variable number tandem repeat (VNTR) typing is a powerful tool for forensic identification due to the unique profile for an individual by using the highly polymorphic nature of short tandem repeat (STR) markers. Polymerase chain reaction followed by electrophoretic separation of the amplified products for STR analysis allows simultaneous amplification and analysis of multiple loci, and has been accepted as the gold standard for human identification in the forensic society. The commercial STR kits such as Powerplex 16 and AmpFlSTR Identifiler are designed to genotype 16 autosomal STR loci including the 13 Combined DNA Index System (CODIS) STR loci providing high discrimination power. Likewise, Y chromosome-specific STR typing kits (PowerPlex Y and AmpFlSTR Yfiler) are developed specifically for analyzing male genomic DNA such as sexual assault cases, paternity test, missing person identification, and evolutionary studies. In particular, Y-STR typing is very valuable in sexual assault cases since a vast majority of crimes involve males as the perpetrator. Lab-on-a-chip (LOC) or microfabrication technology has paved its way in a variety of chemical and bio-logical applications as a miniaturized analytical tool due to the benefits such as small sample consumption, high throughput synthesis and screening capability, fast analysis time and portability. Recently, the forensic human identification based on short tandem repeat (STR) genotyping has been performed on a microdevice platform, and the superior performance has been demonstrated. In our study, we focused on the sensitivity issue by designing an improved sample stacking capillary electrophoretic microdevice and used it for highly sensitive Y STR typing as well as PCR-free variable number tandem repeat genotyping. In the first part, we developed a sample stacking capillary electrophoretic (CE) microdevice for mini Y STR genotyping. The mini Y STR includes redesigned primer sequences to generate smaller-sized PCR ampli-cons to enhance the PCR efficiency and the success rate for a low copy number and degraded DNA. The mini Y STR amplicons occupied in the 5 and 10 mm stacking microchannel are preconcentrated efficiently in a defined narrow region through the optimized sample stacking CE scheme, resulting in more than 10-fold improved fluo-rescence peak intensities compared with that of a conventional cross-injection ??CE method. Such signal en-hancement allows us to successfully analyze the Y STR typing with only 25 pg of male genomic DNA, with high background of female genomic DNA, and with highly degraded male genomic DNA. The combination of the mini Y STR system with the novel sample stacking CE microdevice provides the highly sensitive Y STR typing on a chip, making it promising to perform high-performance on-site forensic human identification. In the second part, We demonstrated a proof-of-concept for novel minisatellite tandem repeat typing, called PCR-free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a liga-tion reaction instead of PCR thermal cycling, magnetic bead based solid phase capture for purification, and an elongated sample stacking micro-CE (μCE) for sensitive digital coding of repeat number. We designed a 16-bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin-coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min by using this method. In our study, we first focused on the capillary electrophoretic microdevice for highly sensitive and high-throughput Y STR typing by developing a sample-stacking CE operation with the multiplex mini Y STR typing system. These combinations would enable the superior low copy number (LCN) and degraded DNA genotyping to the conventional Y STR method with high speed and lower undesirable allelic drop-out and drop-in occurrence. Then, a proof-of-concept for novel minisatellite tandem repeat typing was demonstrated, called PCR-free digital VNTR (variable number tandem repeat) typing. In this method, we aimed to realize the high speed and portability of VNTR genotyping by using isothermal ligation reaction instead of PCR thermal cycling.

서지기타정보

서지기타정보
청구기호 {MCBE 11026
형태사항 vi, 50 p. : 삽도 ; 30 cm
언어 영어
일반주기 지도교수의 영문표기 : Tae-Seok Seo
지도교수의 한글표기 : 서태석
수록잡지명 : "Sample Stacking Capillary Electrophoretic Microdevice for Highly Sensitive Mini Y Short Tandem Repeat Genotyping". Electrophoresis, 31, 2974-2980(2010)
수록잡지명 : "PCR-free digital minisatellite tandem repeat genotyping". Electrophoresis, 32, 1456-1464(2011)
학위논문 학위논문(석사) - 한국과학기술원 : 생명화학공학과,
서지주기 References : p.34-37
주제 Capillary electrophoresis
Sample stacking
Mini Y STR
PCR-free
QR CODE qr code