Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

Cloning of a gene that encodes a new member of the human cytotoxic cell protease family.

A family of unusual serine proteases that are believed to be involved in the effector mechanism of cell-mediated cytotoxicity have previously been described in the mouse. However, in the human only one gene encoding a member has been isolated. By use of a mixture of murine cDNAs as probes, a second human gene has now been isolated. The primary structures of the gene and the predicted protein are very similar to those of the mouse. In addition, in keeping with the postulated involvement in cytolysis, transcripts were detected only in cytotoxic cells. The organization of the coding and noncoding regions of the gene, the clustering of family members, and the chromosomal location, close to the alpha chain of the T cell antigen receptor, are all conserved between human and mouse.

Rescue of BKV from BKV-transformed hamster, rat, and mouse cells: correlation with levels of nonintegrated viral DNA.

Infectious BKV was rescued from 39 of 40 lines of virus-free, BKV-transformed hamster, rat, and mouse cells, which had been either maintained continuously in culture or reestablished in culture after one or more passage in the appropriate host, by Sendai virus-catalyzed fusion with permissive cells. Striking differences were observed among the 39 lines with respect to the efficiency of virus rescue. Fourteen of the lines were examined for the presence of nonintegrated viral DNA by dot-blot hybridization. The values obtained, which ranged from less than 1 to 2880 viral genome equivalents/cell, reveal a strong correlation between the efficiencies with which BKV can be rescued from these lines and the amounts of free viral DNA that they contain.

Guidelines and an illustration of the use of ecological data for seeking clues of excess risk.

Ecological studies have acquired a bad reputation as weak scientific studies suffering from the ecological fallacy and many intractable limitations of data and method. In order to reduce these problems, it is suggested that analyses be focused on specific populations-at-risk, diseases, risk factors and places. In addition, seven guidelines are suggested which will limit the possibility of false positive results and provide the best clues for expensive, follow-up research. These guidelines are testing to make sure that the results are consistent across different times, places and methods. The guidelines were applied to finding the best clues for occupational-related cancers among white males aged 35-64 in the State of Illinois during 1950-1975. The most interesting clue found was that most coal mining areas in Illinois during the late 1960s and 1970s manifested the highest cancer mortality rates of trachea, bronchus, and lung and among the highest rates of increase of this type of cancer. This finding was unexpected because many studies have shown low lung cancer rates among coal miners.

Analysis of DNA structure by hydroxyapatite columns and ethidium bromide fluorescence techniques. A comparative study and effect on DNA binding.

Seven duplex DNA preparations have been structurally analyzed by hydroxyapatite column chromatography and an ethidium bromide fluorescence technique. Significant contamination of one preparation with single-stranded DNA was detected by hydroxyapatite column chromatography. Five of the other six preparations were found to contain significant single-stranded regions by the ethidium bromide fluorescence technique. Synthetic poly dAT was found to be duplex in structure. The presence of single-stranded regions considerably influenced DNA binding results in a radioimmunoassay.

The purification and characterization of a DNA nicking-closing enzyme from Bacillus megaterium.

Although several eucaryote DNA nicking--closing (N--C) enzymes have been characterized, only the Escherichia coli enzyme has been extensively studied amongst procaryotes. The latter enzyme is distinctly different from the eucaryotic enzymes and we have therefore purified the N--C enzyme from Bacillus megaterium to determine if procaryotes form a distinctive class of N--C enzymes. The purified B. megaterium N--C enzyme has a molecular weight of 120,000, only partly relaxes negative supercoils, does not affect positive supercoils, requires Mg2+, and is inhibited by 0.2 M KCl. The enzyme is also inhibited by 1 mM nalidixic or oxolinic acids but unaffected by novobiocin. A crude N--C enzyme preparation from Micrococcus luteus shows very similar properties.

Evaluation of a standardised assay for the measurement of antibodies to double-stranded (native) DNA.

A standardised commercially available radioimmunoassay kit for the detection of antibodies to native DNA (N-DNA) has been evaluated in clinical practice. This test system is shown to be a reliably reproducible method of detecting these antibodies. In addition, evaluation of the purity of the radiolabelled test antigen in this assay has shown it to be almost entirely double stranded (native) DNA with virtually no contamination with single stranded (denatured) DNA, and with few areas of single stranded breaks or ends in the duplex molecule. The inclusion of known standards and precise characterisation of the DNA has partially overcome variability in results and provides for interlaboratory standardisation which is lacking in the techniques used at present.

The purification from Escherichia coli of a protein relaxing superhelical DNA.

The Escherichia coli omega protein was first described by Wang (Wang J.C.: J. Mol. Biol. 55, 523-533 (1971)) as having the ability to relax supercoiled covalently-closed circular DNA by changing the topological winding number, alpha. We have developed a rapid assay for omega activity which has allowed us to purify the protein to homogeneity. It appears to be an alphabeta-type subunit protein with a molecular weight of the intact protein of about 80,000 (determined by gel filtration) and of the individual subunits of 56000 and 31000 (sodium dodecyl sulfate polyacrylamide gels). We have confirmed Wang's observation that it only partly relaxes negative supercoils, and is not active on a positive supercoils. Its characteristics with respect to pH, salts, temperature and chromatography are described. A method for rapid screening of E. coli for omega mutants is described.