A repository of 14 PCR-loci Italian gene frequencies in the World Wide Web.

A collection of 6830 typing results produced by the Immunohematology Laboratory at the UCSC, pertaining to 11 STRs (FES/FPS, vWA31, HUMTH01, F13A1, MBP, D21S11, D7S460, D18S51, CD4, TPOX, CSF1PO) and 3 AmpFLPs (D1S80, APO-B, COL2A1), is publicly available as an electronic archive at a website.

Fluorescence-based classification of microsatellites using a single-wavelength semiautomatic sequencer: genotype assignment and identity tests by analysis of comigrating peak profiles.

Microsatellite analysis, based on fluorescein labeling and reading through a semiautomatic single wavelength sequencer, is described. Pairs of labeled polymerase chain reaction (PCR) samples, mixed in equimolar proportion, were electrophoresed and the specific peaks read in a single gel lane. Identity was asserted when peaks overlapped in a unique fluorescent signal which, compared with individual sample profiles, had a twofold intensity. Classification was achieved by blending individual PCR products to 'locus specific allelic ladders' (composite samples containing a repertory of fragments allelic to a given locus) and by noticing the specific peak enhancement. The resulting protocol of analysis assigned no size and classified allelic forms by tandem repeat number. Applied to a large repertory of PCR products and compared with manual electrophoresis, this protocol proved to be reliable and reduced times and costs of genotype analysis. Analysis of comigrating peak profiles is highly objective and provides convincing evidence for diagnostics and identity tests.

Simple purification and secondary structure evaluation of Sendai virus neuraminidase water soluble fragment.

We have developed a quick and simple method to purify the water soluble fragment of Sendai virus neuraminidase (cHN). We used the trifluoperazine (TFP) as detergent because of its ability to selectively solubilize the HN (Baiocchi et al., (1988) FEBS Lett. 238, 171-174). Here we describe conditions by which trypsin produces the cHN from the TFP treated virions. The cHN is further purified by size exclusion chromatography and it fully retains the neuraminidase activity and the allosteric inhibitory site as described in (Dallocchio et al., (1991) Biochem. Int. 25, 663-668). Both circular dichroism (CD) spectra, analyzed by deconvolution methods and secondary structure prediction were used to assess the secondary structure of cHN.

Distribution of alleles and genotypes from three hypervariable DNA loci in individuals from central and southern Italy.

Three hypervariable locus-specific DNA probes (D2S44, alpha globin 3'HVR, D12S11) used in the routine typing activities of our laboratory have been studied using a representative sample of individuals from central and southern Italy. We report allele and genotype distribution of these systems, and discuss several problems met when typing quasi-continuous arrays of restriction fragments.

Errors in sizing bands of hypervariable DNA profiles on autoradiograms: are they Gaussian?

The accuracy of procedures for sizing hypervariable restriction fragments by Southern blot analysis (SBA) has been tested under three different experimental conditions: (i) intrablot serial analyses: three heterozygous DNA profiles were tested 14 times each in the same gel electrophoresis; (ii) intralaboratory analyses: we replicated three profiles (six autoradiographic bands) in over 100 SBA experiments; (iii) interlaboratory analyses: 15 serial measurements produced in a recent collaborative study (Forensic Sci. Int. 1991, 49, 1-15) were taken into account. In these three cases, a typical U-shaped correlation curve between molecular size and coefficient of variation was found. We explain decrease of accuracy at both extremities of the gels in terms of: (i) enlarged shapes of bands and mean electrophoretic resolution at the cathode; (ii) diffusion and blurring of bands at the anodal edge. The three populations of data were subjected to a chi 2 test and to the Kolmogorov-Smirnov test in order to verify their compliance with normal distribution. Twenty-three out of 27 tests indicated no significant deviation from the assumption of Gaussian distribution. We recommend the adoption of tests for normality to validate the use of symmetric confidence intervals for calculating gene frequencies and asserting a match between adjacent bands.

Estimating allele frequencies of hypervariable DNA systems.

Several polymorphisms of human DNA have been shown to be hypervariable due to the recurrence of a variable number of tandem repeats (VNTRs) in the lengths of allelic restriction fragments. The recurrence of allelic variants in this novel class of polymorphisms seems to comply well with a model of continuous random variables. Based on this assumption, we have compiled some simple algorithms for classification of continuous data and estimation of classes of relative frequencies and have implemented these routines for the management of databases storing hypervariable single locus DNA genetic systems. The algorithms are compiled in BASIC language and can be incorporated in task-oriented computer programs. Three procedures are discussed, based in turn on: (a) using predetermined, arbitrary classes; (b) point estimations of frequencies for single fragments using error measurements associated with the kilobase value assignment; (c) estimates of phenotype frequencies according to error measurements. Error measurements are obtained from a statistic of values pertaining to several restriction fragments (genomic controls) repeatedly tested in different experiments. Problems related to these approaches are discussed.

Efficient, small scale electroelution of high molecular weight DNA from agarose gels by a miniature vertical electrophoresis cell.

A miniature, vertical electrophoretic system is described that allows quick elution and efficient harvesting of double-stranded, high molecular weight DNA fragments from agarose gels. A gel slice containing the band of interest is excised from the agarose gel after submarine electrophoresis and placed into an electrophoresis cell, assembled by inserting a truncated micropipette tip into a 2.0 mL polypropylene tube. The tip supports the agarose slice and is connected to dialysis tubing. Platinum wire electrodes are placed over the tip and at the bottom of the test tube. DNA molecules are quickly eluted into the dialysis bag (60 s to 10 min for bands ranging from 2 to 23 kb) and then easily recovered. The method is highly efficient and allows considerable time saving. Of special interest is its applicability to small starting amounts of DNA (less than 0.5 micrograms per band). Purity of the DNA is comparable, if higher, to that obtainable by conventional electroelution on dialysis bags and DEAE-cellulose membrane electrophoresis. The system is simple enough to be used as a routine method for eluting multiple bands in short times.

Selective extraction of haemagglutinin and matrix protein from Sendai virions by employing trifluoperazine as a detergent.

Incubation of trifluoperazine, a local anaesthetic, at concentrations higher than the cmc with Sendai virus particles produces the selective solubilization of the haemagglutinin neuraminidase (HN) and matrix (M) proteins. This phenomenon involves aggregation of the Sendai virions and therefore the separation of HN and M from the rest of the particle can be performed by bench centrifugation. The supernatant contains the HN and M proteins and HN, once inserted into liposomes, elicits its own biological activities. Therefore, the method seems suitable for purifying large amounts of HN.