Practical applications of genotypic surveys for forensic STR testing.

Legitimate genotype frequency estimation for multiallelic loci relies on component allele frequencies, as population surveys represent only a fraction of possible DNA profiles. Multilocus genotypes from two ethnic human populations, African American (n=195) and U.S. Caucasian (n=200), were compiled at 13 STR loci that are used worldwide in forensic investigation (D3S1358, vWA, FGA, D16S539, TH01, TPOX, CSF1PO, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820). Sex-specific AmpFlSTR multiplexes provided stringent PCR-based STR typing specifically optimized for multicolor fluorescence detection. Heterozygosity at each STR locus ranged from 0.57 to 0.89 and encompassed from seven (TH01) to twenty-one (D21S11) alleles. Homozygosity tests, tests based on the distinct numbers of observed homozygous and heterozygous classes, log likelihood ratio tests, and exact tests assessed that the degree of divergence from theoretical Hardy-Weinberg proportions for all 13 STRs does not have practical consequence in genotype frequency estimation. Departures from linkage equilibrium, between loci, that imposed significance to forensic calculations were not indicated by observed variance of the number of heterozygous loci or Karlin interclass correlation tests. For forensic casework, reliable multilocus profile estimates may be obtained from the product of component genotype frequencies, each calculated through application of the Hardy-Weinberg equation to population database allele frequency estimates reported here. The average probability that two randomly selected, unrelated individuals possess an identical thirteen-locus DNA profile was one in 1.8x10(15) African Americans and one in 3.8x10(14) U.S. Caucasians.

General method for HPLC purification and sequencing of selected dsDNA gene fragments from complex PCRs generated during gene expression profiling.

Gene expression profiling using an AFLP-based technique generates a large number of gene fragments that require identification by sequencing. The DNA fragments vary in length from about 50-500 bp. Ion-pair reversed-phase HPLC can be used to purify selected double-stranded DNA fragments that represent differentially expressed genes. The gene fragments are sequenced directly after vacuum drying of the collected HPLC fractions.

Human 18 kDa phosphotyrosine protein phosphatase (ACP1) polymorphism: studies of rare variants provide evidence that substitutions within or near alternatively spliced exons affect splicing result.

The mammalian low molecular weight phosphotyrosine protein phosphatase is expressed as two distinct isoforms. The human 'fast' and 'slow' isoforms differ only in the sequence of an internal segment of 34 residues, and the ACP1 gene contains two adjacent exons (E3F and E3S) which encode these segments. We have previously suggested that the fast and slow isoforms are generated by mutually exclusive pre-mRNA splicing of E3F and E3S. The common alleles ACP1*A, *B and *C express the fast and slow isoforms in different ratios. The *A and *C alleles differ from *B by C --> T transitions in E3S and E3F respectively. To test the idea that the fast : slow ratio is determined by nucleotide substitutions in the E3F-I3F-E3S region, four groups of rare ACP1 variants with unusual fast : slow ratios and the rare *E and *R alleles, expressing fast∶slow ratios similar to *C and *B, respectively, were analysed. Gene segments of the I2-I3S region were amplified by PCR and analysed by SSCP and variant bands were excised and sequenced. For each of the rare isozymic variants one of six different nucleotide substitutions in E3F (nts+42, +85, +109, +110), I3F (nt+1) and I3S (nt+8) was observed. The *E and *R alleles showed C and B sequence, respectively, in accordance with the fast : slow ratio. The results support the hypothesis that the fast : slow ratio is constitutive.

TWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis.

Studies were performed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) committee to validate the AmpFISTR Blue PCR Amplification Kit for forensic casework applications. The kit coamplifies the tetranucleotide short tandem repeat (STR) loci D3S1358, vWA, and FGA. The dye-labeled amplification products were electrophoresed and detected directly using the ABI PRISM 377 DNA Sequencer or the 310 Genetic Analyzer. CEPH family studies demonstrated Mendelian inheritance of these loci and probability of identity values from population studies were 1/4,830 (African-American), 1/5,479 (U.S. Caucasian), and 1/3,443 (U.S. West Coast Hispanic). In all studies examining different body tissues and fluids, the expected genotypes were observed. Studies to determine and test the PCR reagent components and thermal cycling parameters demonstrated specificity, sensitivity, and balance over a wide range of conditions. Reliable results were obtained from DNA quantities as low as 0.25 ng. A variety of environmental studies were performed, as forensic samples are often exposed to different environmental conditions and substances which may degrade DNA or inhibit the amplification process. Highly degraded samples demonstrated that FGA was the first locus to become undetectable, followed by vWA, and then D3S1358; this is the expected pattern according to locus size. In studies of PCR inhibition, the pattern in which the loci became undetectable was different; FGA was the first locus to become undetectable, followed by D3S1358, and then vWA. Single versus multiple locus amplifications revealed no benefit to single locus analysis, even in cases of degradation or inhibition. The occurrence of preferential amplification was very rare, particularly in noncompromised, unmixed samples. Artifact peaks were not observed in any instance. Mixture studies confirmed the ability to detect mixed DNA samples and included the characterization of stutter and peak height ratios; the limit of detection was 1:10 for 1 ng total genomic DNA and 1:30 for 5 ng. DNA extracted from nonprobative case evidence was successfully amplified and genotyped. All such studies indicate that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results.

Exon structure at the human ACP1 locus supports alternative splicing model for f and s isozyme generation.

The human ACP1 locus encodes a genetically polymorphic cytoplasmic low-molecular-weight acid phosphatase. Each of the common alleles encodes two isoforms, f and s. Both isozymes are of equal length (157 residues) but differ in sequence over an internal 34 residue segment. Substantial portions of the ACP1*A, *B and *C alleles common to Europeans have been sequenced. Six linearly positioned exons containing codons 14 to 157 were identified. Two exons of equal length (114bp) interspaced by a short (41bp), probably nonfunctional, intron encode the specific f and s segments, respectively. These findings strongly support an alternative RNA splicing hypothesis. In addition, three allele-specific base substitutions were encountered.

MPP+ is transported by the TEA(+)-H+ exchanger of renal brush-border membrane vesicles.

Rabbit renal brush-border membrane vesicles (BBMV) were used to study the transport of the cationic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). An outwardly directed H(+)-gradient stimulated MPP+ uptake and led to the development of an active accumulation of MPP+ within the vesicles. H(+)-gradient driven MPP+ transport was saturable, with a maximal transport rate of 3 nmol.mg-1.min-1 and an apparent Michaelis constant (Kt) of 8 microM. MPP+ and tetraethylammonium (TEA) behaved as competitive inhibitors of one another's transport in renal BBMV, suggesting the presence of a common transport pathway for these organic cations. At an ambient pH of 7.5, preloading BBMV with MPP+ failed to stimulate TEA uptake, although trans TEA did stimulate MPP+ uptake. Increasing ambient pH to 8.5 (i.e., reducing competition between H+ and these organic cations for a common transport pathway) led to a clear reciprocal trans stimulation of TEA and MPP+ fluxes. With an equilibrium-shift protocol, a trans concentration of MPP+ energized uphill transport of TEA. We conclude that MPP+ and TEA share a common organic cation-H+ exchange pathway in the renal brush border, although turnover of an MPP(+)-loaded exchanger is slow compared with that for a TEA or H(+)-loaded exchanger.