Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel.

Using a 52 SNP marker set previously developed for forensic analysis, a novel 49plex assay has been developed based on the Genplex typing system, a modification of SNPlex chemistry (both Applied Biosystems) using oligo-ligation of pre-amplified DNA and dye-labeled, mobility modified detection probes. This gives highly predictable electrophoretic mobility of the allelic products generated from the assay to allow detection with standard capillary electrophoresis analyzers. The loci chosen comprise the 48 most informative autosomal SNPs from the SNPforID core discrimination set supplemented with the amelogenin gender marker. These SNPs are evenly distributed across all 22 autosomes, exhibit balanced polymorphisms in three major population groups and have been previously shown to be effective markers for forensic analysis. We tested the accuracy and reproducibility of the Genplex system in three SNPforID laboratories, each using a different Applied Biosystems Genetic Analyzer. Genotyping concordance was measured using replicates of 44 standardized DNA controls and by comparing genotypes for the same samples generated by the TaqMan, SNaPshot and Sequenom iPLEX SNP typing systems. The degree of informativeness of the 48 SNPs for forensic analysis was measured using previously estimated allele frequencies to derive the cumulative match probability and in paternity analysis using 24 trios previously typed with 18 STRs together with three CEPH families with extensive sibships typed with the 15 STRs in the Identifiler kit.

Functional asymmetry of the two nucleotide binding domains in the ABC transporter Ste6.

The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1. We constructed a set of 26 Ste6 mutants using a random mutagenesis approach. Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6. Trafficking, therefore, does not seem to be affected in most of the mutants. To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N). Suppressor mutations were identified in TM12 and upstream of TM6. Surprisingly, these mutations also suppressed the Walker A mutation G397D, which should be defective in ATP-binding and hydrolysis at NBD1. Photoaffinity labeling experiments with 8-azido-[alpha-32P]ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12. The experiments further suggest that the two NBDs of Ste6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.