A new allele at the short tandem repeat locus HumF13A01.

We investigated the (AAAG)n short tandem repeat (STR) polymorphism HumF13A01 an Austrian Caucasoid population sample (n = 674). PCR amplified fragments were detected on an automatic A.L.F. DNA sequencer using laser-induced fluorescence. A total of 14 alleles could be identified including a new 179 bp allele which was designated allele 3. Sequence determination of allele 3 confirmed the typing results by revealing three continuous copies of the core repeat, whereas in sequencing of 54 additional alleles no further variants or microheterogeneities could be observed. The population data showed no significant deviation from Hardy-Weinberg equilibrium.

Allelic ladder characterization of the short tandem repeat polymorphism in intron 6 of the lipoprotein lipase gene and its application in an Austrian Caucasian population study.

The short tandem repeat (STR) polymorphism HumLPL (TTTA)n, which is located in intron 6 of the lipoprotein lipase gene, was investigated by AMPFLP (amplification fragment length polymorphism)-technique using an allelic ladder consisting of amplified alleles of this locus as a standard size marker. The allelic ladder was prepared by pooling equal concentrations of six separate alleles, which were identified by their different electrophoretic mobility in native polyacrylamide gel, eluted and subsequently amplified. Sequence analysis of the ladder alleles and allele 7, which is not included in the ladder, showed a regular repeat structure with 7 and 9 to 14 repetitions of the core repeat. The allelic ladder was employed in the analysis of the genotypes of 550 unrelated Caucasoids of Austria. No new alleles were found. The population investigated showed no deviation from Hardy-Weinberg equilibrium (P = 0.195).

Sequence determination of an allele ladder for the STR polymorphism at the CD4 locus and application of the ladder in testing an Austrian Caucasian population sample.

The short tandem repeat (STR) polymorphism at the CD4 locus, designated HUMCD4, was examined by PCR, native polyacrylamide electrophoresis and subsequent silver staining using an allelic ladder of eight distinguishable alleles occurring in an Austrian Caucasian population sample as a standard size marker. The ladder was produced by pooling equal concentrations of eluted, separately amplified and sequenced alleles, which were previously identified by their different electrophoretical migration. Components of the ladder are in regular intervals of five basepairs. Alleles 4 to 8 were designated according to the number of AAAAG repeat units. The four longer alleles 8' to 11 showed a stable A to G transition in one of the repeat units and were designated counting the AAAGG unit for a AAAAG. Allele 8' was not included in the ladder because it showed the same electrophoretic mobility as allele 8. This ladder proved to be a precise and reliable tool in the analysis of 600 chromosomes of the Austrian population. The population investigated showed no deviation from Hardy-Weinberg equilibrium (P = 0.23).

One year's experiences with two different image analysis systems for automated reading of the contrast fluorescence test.

We have tested two different personal-computer based color image analysis systems for automated reading of the microlymphocytotoxicity test (LCT) for HLA-A,B,C-typing and screening. Over 17,000 single LCT-reactions were prepared using the simultaneous double fluorescent variant of the LCT (contrast fluorescence test, CFT). All tests were read visually by experienced laboratory staff members. For image analysis, an automated scanning system was used. In a first step, reactions were recorded on a videotape recorder using a color(CCD)-video camera. In a second step, the recorded reactions were analyzed with the two different image analysis systems by specifically developed programs. Good correlation (r = 0.89) of the score values assigned by digital image analysis with the visual tray reading was obtained. Since also the other main performance characteristics of the prototype system (throughput, reliability, compatibility) were acceptable for routine application, we may conclude that digital image analysis is a feasible and very interesting new technique for automated evaluation of the LCT.

One year's experience with two different image analysis systems for automated reading of the contrast fluorescence test.

We have tested two different personal-computer-based color image analysis systems for automated reading of the microlymphocytotoxicity test (LCT) for HLA-A,B,C typing and screening. Over 17,000 single LCT reactions were prepared using the simultaneous double fluorescent variant of the LCT (contrast fluorescence test, CFT). All tests were read visually by experienced laboratory staff members. For digital image analysis, an automated scanning system was used. The reactions were first recorded on a videotape recorder using a color (CCD) videocamera und subsequently analyzed with the two different image analysis systems by specifically developed programs. Good correlation (r = 0.89) of the score values assigned by digital image analysis with the visual tray reading was obtained. Since also the other main performance characteristics of the prototype system were acceptable for routine application, we may conclude that digital image analysis is a feasible and very interesting new technique for automated evaluation of the LCT.