The specificity and reliability of locked nucleic acid (LNA) substitution at the 3' position of allele-specific PCR (AS-PCR) primers for SNP detection was investigated in direct comparison to DNA primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3' LNA mismatch combinations were tested in triplicate with the plasmid target. LNA primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3' LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3' LNA primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3' LNA primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications.

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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.

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 thermalcycling parameters. Loci analyzed were the apolipoprotein B (APOB) 3' hypervariable region (HVR), phenylalanine hydroxylase 3' HVR (PAH), and D1$80. Coamplification of a monomorphic 13-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.The use of amplified fragment length polymorphism (AFLP) systems for human identification in forensic and parentage testing is expanding rapidly. (1-6) This report describes a systematic approach to human identity testing using nonisotopic PCR technology (7) and modified enzyme immunoassay detection. PCR reaction and thermal cycling parameters were optimized for three AFLP loci: apolipoprotein B 3' hypervariable region (APOB HRV), (11)(12)(13)(14) and phenylalanine hydroxylase 3' HVR (PAH). (Is'j6) Coamplification of a monomorphic 1327-bp Bglobin fragment (]7) with each APOB and PAH reaction assured that amplifiable DNA was present and inhibitors were absent in each AFLP reaction. Parameters were optimized from those published previously by the following differences: use of biotinylated primer, direct detection of blots, reduction of genomic template DNA, [3-globin coamplification, and sample temperature monitoring during thermal cycling. The parameters titrated for each locus were concentrations of MgC12 primers, Taq polymerase, and template DNA, along with annealing temperature, extension time, and number of PCR cycles. Standard conditions were established prior to AFLP data basing.One objective of this study was to simplify and streamline the methodology for AFLP typing. This was done using an inorganic DNA extraction procedure, PCR amplification with biotinylated primers, agarose gel electrophoresis, Southern transfer, and direct detection with either a colorimetric or chemiluminescent end point. AFLP typing was done by comparison to locus-specific allele ladders (AL) containing known variable number tandem repeat (VNTR) numbers. The time required to process and type samples with one AFLP system was -36 hr, with 4 hr of hands-on time.Coamplification of the APOB primers described with an HLA-DQoL kit was attempted on a limited number of known samples to assess whether both loci typed correctly. Specificity testing of APOB and PAH with nonhuman DNA under low-and high-stringency PCR was conducted. Future adjustments in AFLP ...