RESUMO
We have previously characterized and databased three human amplified fragment length polymorphism (AFLP) loci: the hypervariable regions 3' to apolipoprotein B (APOB), phenylalanine hydroxylase (PAH) and at locus D1S80. The analysis utilized polymerase chain reaction (PCR) technology for human identification in forensic and paternity testing. This study extended that work by assessment of specificity of amplicons produced with non-human and human control DNAs for APOB, PAH and D1S80 under high and low stringency PCR conditions. It was seen that primate and other animal templates (with the exception of chimpanzee) yielded products below the human allele range under high stringency PCR parameters. Under reduced stringency PCR with animal and primate samples, reproducible genetic fingerprints were generated spanning the human allele range. The patterns were produced with defined human AFLP primer pairs under specifically relaxed PCR reaction and thermalcycling parameters. They showed genetic relationships between species at the DNA level. Amplicon patterns were compared for band size and intensity matches within the PCR synthesis range defined by the conditions used. This technique could become a useful tool in species identification and molecular evolutionary studies.
Assuntos
Apolipoproteínas B/isolamento & purificação , Fenilalanina Hidroxilase/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Alelos , Animais , Humanos , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
Methodology is presented for amplified fragment length polymorphism (AFLP) typing using a nonisotopic, PCR protocol. Human variable number tandem repeat (VNTR) loci used for identification in forensic and paternity testing were optimized for reaction and thermal-cycling parameters. Loci analyzed were the apolipoprotein B (APOB) 3' hypervariable region (HVR), phenylalanine hydroxylase 3' HVR (PAH), and D1S80. Coamplification of a monomorphic beta-globin fragment serves as an amplification control. Biotin is integrated into PCR amplicon through primer incorporation. AFLP products undergo agarose gel electrophoresis and Southern transfer to a nylon membrane. Amplicons were detected using a streptavidin-enzyme conjugate. Either colorimetric- or chemiluminescent-developed bands are genotyped using locus-specific allele ladders with known VNTR repeat numbers. Using this methodology, we have successfully typed > 500 individuals from three population groups for each locus during data basing and casework.
Assuntos
Alelos , Apolipoproteínas B/genética , Fenilalanina Hidroxilase/genética , Polimorfismo Genético , Sequência de Bases , Temperatura Alta , Humanos , Repetições Minissatélites , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Twenty-three BK virus and JC virus DNA samples obtained from urine of pregnant women had almost exclusively archetypal transcriptional control regions. Rearrangements characteristic of laboratory strains are apparently not required for reactivation in humans. Unexpectedly, alignment shows that many elements identified previously in the BK virus enhancer are conserved in the JC virus archetype.
Assuntos
Vírus BK/genética , Vírus JC/genética , Complicações Infecciosas na Gravidez/microbiologia , Sequências Reguladoras de Ácido Nucleico , Infecções Tumorais por Vírus/microbiologia , Sequência de Bases , DNA Viral/genética , Elementos Facilitadores Genéticos , Feminino , Rearranjo Gênico , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Gravidez , Complicações Infecciosas na Gravidez/urina , Regiões Promotoras Genéticas , Transcrição Gênica , Infecções Tumorais por Vírus/urina , Urina/microbiologiaRESUMO
Restriction fragment length polymorphism analysis of human deoxyribonucleic acid (DNA) using two probes, pYNH24 and CMM101, was performed on the BIOS Timeframe system following the Federal Bureau of Investigation (FBI) Laboratory protocol and some variations of it. Comparable results were obtained by the different methods used.
