Development of multiplex genotyping methods of genetic variations based on mass spectrometry = 질량분석을 이용한 유전자 변이의 다중 분석 방법 개발

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We herein describe new mass spectrometry-based methods for multiplex SNP genotyping by utilizing allele-specific ligation and isothermal amplification reaction. In these method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific alleles in the target DNA, and the resulting ligated product serves as a template to promote the isothermal amplification reaction using a universal primer, which isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. In chapter 2, we employed strand displacement amplification (SDA) reaction for isothermal amplification to amplify mass markers and by varying the sizes of the mass markers, the multiple SNP sites are simultaneously identified based on peak positions on the mass spectrum. In chapter 4, we employed graphene oxide (GO)-assisted exponential amplification reaction (EXPAR) for isothermal amplification whose detection capability on mass spectrometry is confirmed in chapter 3. By utilizing these methods, we successfully demonstrated the multiplex SNP genotyping capability of these methods by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry.
Advisors
Park, Hyun Gyuresearcher박현규researcher
Description
한국과학기술원 :생명화학공학과,
Publisher
한국과학기술원
Issue Date
2016
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명화학공학과, 2016.8 ,[ix, 96 p. :]

Keywords

Genetic variation; SNP genotyping; mass spectrometry; isothermal amplification; graphene oxide; 유전자 변이; 단일염기 다형성 분석; 질량 분석; 등온 증폭; 그래핀 산화물

URI
http://hdl.handle.net/10203/222160
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=663120&flag=dissertation
Appears in Collection
CBE-Theses_Ph.D.(박사논문)
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