Human amplified fragment length polymorphism and animal genomic identification systems

• Main Author: Latorra, David
• Format: Dissertation
• Language: English
• Subjects: biology, genetics; biology, molecular
• ISBN: 9780599093799; 059909379X

This research project was an investigation of new genetic assays in human and animal species identity applications. The two main areas of research focused on analysis of human amplified fragment length polymorphism (AFLP) systems and animal genetic profiling using defined repetitive oligonucleotides (DROs). These two concepts were linked by the common goal of identification and PCR-based genomic hybridization specificity in both systems. Data supporting this research consisted of 16 tables and 54 print figures.
The characterization of human AFLP systems apolipoprotein B (APOB), DIS80 and phenylalanine hydroxylase (PAH) was accomplished using a non-isotopic, polymerase chain reaction (PCR)-based protocol. Assays were optimized for amplification reaction, thermalcycling and detection parameters along with allele ladder construction. A monomorphic beta-globin amplification control for PCR inhibition and DNA degradation was incorporated. Over 270 alleles from each of three population groups were genotyped to establish allele frequency databases with these three systems. This analysis allowed the utilization of specific AFLP assays in forensic and paternity testing casework at AGTC, Inc. of Denver, CO since 1992 as described in two published journal articles. Four other AFLP loci were also analyzed for identification applications.
Human specificity of AFLP systems APOB, PAH and D1S80 with beta-globin was assessed under high and low stringency PCR parameters as part of forensic validation testing. Species-specificity of resulting amplicon band patterns was examined. Multiplex PCR capability and uracil N-glycosylase (UNG) sensitivity and specificity were also examined in these AFLP systems. Seven AFLP systems were used to test genomic DNA samples from one nuclear family for parentage verification and individualization. The PCR assay for HLA DRB discriminated each family member except two brothers.
The hypothesis that defined repetitive oligonucleotides (DROs) used as primers would produce informative aniplicon patterns for identity applications in humans, primates and animal species was investigated. Evidence for species genetic identification and polymorphism within species was shown for a non-human genomic DNA panel tested with specific individual and combination DROs. In total, this body of research helped to define and establish these AFLP and DRO systems for analysis of genetic relationships and identification testing in human and animal species.