Dissertation > Excellent graduate degree dissertation topics show

Enrichment of Minority Alleles in Mixture by COLD-PCR

Author: LiXiaoZhu
Tutor: ZhouHaiMei；ZhaoGuiSen
School: Henan University of Science and Technology
Course: Forensic
Keywords: Mixed samples Polymerase chain reaction ( PCR ) Heteroduplex Enrichment
CLC: D919.2
Type: Master's thesis
Year: 2011
Downloads: 35
Quote: 0
Read: Download Dissertation

Abstract

Background from the mixing of two or more individual samples is one of the types of forensic evidence examination often encounter difficult samples. With the popularity of the application of the highly sensitive detection of special technology, high polymorphism and sex-linked genetic markers, the ability to obtain evidence from the mixed samples continues to increase. But even for experienced specialists, the actual case detection and interpretation of the results of some of the mixed samples is still a huge challenge. Differences in the small amount of ingredients in a large mixing samples of component content sub-type, is one of the problems of the current forensic DNA analysis. The purpose of the establishment of the human Y chromosome M175 insertion / deletion loci COLD-PCR (co-ampLification at lower denaturation temperature PCR) technology, comparing COLD-PCR and conventional PCR amplified sample typing results, mixed in different proportions to explore COLD- PCR allele for forensic mixed samples of low-level enrichment amplified typing feasibility. First, establish the human Y-M175 locus conventional PCR-polyacrylamide gel electrophoresis (PCR-PAGE) the silver staining typing technology, survey 103 Henan Han male volunteers genotype. Proteinase K digestion - phenol / chloroform extraction insertions and deletions of individual genomic DNA mixed samples were prepared by mixing different proportions. Gradient insulation heteroduplex abatement method for the determination of the large M175 amplicon (171/166bp) is critical denaturation temperature (critical denaturation temperature, Tc), high-resolution melting curve (high resolution melting analysis, HRM) determination M175 amplicon (91/86bp), Tc values. Tc based on the value increase between the denaturation and annealing in conventional PCR amplification product of hybridization and heteroduplex denaturation two steps were established and optimized based on PAGE-silver staining (amplicon) and real-time quantitative COLD-PCR technology Act (amplicon), compared with conventional PCR and COLD-PCR amplification genotyping effect of sample mixed in different proportions. Results Henan Han Y-M175 locus type of frequency of insertions and deletions were 0.1650,0.8350. M175 amplicon Tc is 76.4 ℃, small amplicon Tc is 78.4 ℃, COLD-PCR using optimized heteroduplex denaturation temperature of 76.4 and 78.2 ° C, respectively. Amplicon PAGE-silver staining, conventional PCR can be effective genotyping mixed sample component ratio of 5:1, COLD-PCR can reach at least 20:1. The small amplicon real-time quantitative conventional PCR can be effective genotyping mixed sample component ratio of 16:1, COLD-PCR can reach at least 64:1. Conclusions using conventional PCR with the same equipment and reagents, COLD-PCR can be achieved Trace enrichment amplified alleles in the mixed samples, and expand the scope of the subject material of the separable type mixing, to improve genotyping results, in the mixed samples in forensic potential application of DNA typing.

Related Dissertations

CLC: > Political, legal > Legal > UNIVERSITY > Forensic > Forensic Laboratory Science
© 2012 www.DissertationTopic.Net Mobile