Keiji Tamaki

Human minisatellites, repeat DNA instability and meiotic recombination.

Minisatellites include some of the most variable loci in the human genome and are superb for dissecting processes of tandem repeat DNA instability. Single DNA molecule analysis has revealed different mutation processes operating in the soma and germline. Low‐level somatic instability results in simple intra‐allelic rearrangements. In contrast, high frequency germline instability involves complex gene conversions and is therefore recombinational in nature, almost certainly occurring at meiosis. To determine whether true meiotic crossovers occur at human minisatellites, we have used polymorphisms near the repeat array to recover recombinant DNA molecules directly from sperm DNA. Analysis of minisatellite MS32 has revealed an intense and highly localised meiotic crossover hotspot centred upstream of the array, the first example of a human hotspot defined at the molecular level. This hotspot extends into the beginning of the repeat array, resulting in unequal and equal crossovers. Array crossovers occur much less frequently than array conversions but appear to arise by a common process, most likely by alternative processing of a recombination initiation complex. The location of MS32 at the boundary of a recombination hotspot suggests that this locus has evolved as a by‐product of localised meiotic recombination activity, and that minisatellites might in general mark recombinationally proficient hotspots or hot domains in the genome. Finally, sperm crossover analysis makes it possible to explore the molecular rules that govern human meiotic recombination, and to detect phenomena such as meiotic drive that could provide a possible connection between recombination and DNA sequence diversity itself.

Minisatellite mutation frequency in human sperm following radiotherapy

Screening pedigrees for inherited minisatellite length changes provides an efficient means of monitoring repeat DNA instability but has given rise to apparently contradictory results regarding the effects of radiation on the human germline. To explore this further in individuals with known radiation doses and to potentially gain information on the timing of mutation induction, we have used an extremely sensitive single molecule approach to quantify the frequencies of mutation at the hypervariable minisatellites B6.7 and CEB1 in the sperm of three seminoma patients following hemipelvic radiotherapy. Scattered radiation doses to the testicles were monitored and pre-treatment sperm DNA was compared with sperm derived from irradiated pre-meiotic, meiotic and post-meiotic cells. We show no evidence for mutation induction in any of the patients and discuss this finding in the context of previous population studies using minisatellites as reporter systems, one of which provided evidence for radiation-induced germline mutation.

Deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese

The deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs is described. HLA-DQA1 genotypes could be determined from single plucked hair roots. However, it was not easy to type HLA-DQA1 with hair shaft portions. Increase in the specimens of hair shaft portions (over 10 cm in length) to get sufficient DNA caused inhibition of polymerase chain reaction (PCR). Synthetic melanin as well as the one extracted from hairs inhibited the PCR of the genomic DNA template when added to the PCR reaction at the concentrations over than 15 ng/100 microL. Therefore, typability of hair shaft portions seems to depend on the delicate balance of the concentrations of DNA and the contaminated melanin in the final DNA extracts.

Frequency of HLA-DQA1 alleles in the Japanese population

One of the HLA class II genes, HLA-DQA1, was typed from 290 unrelated healthy Japanese using the oligonucleotide typing method. The HLA-DQA1 gene was enzymatically amplified and typed by dot-blot hybridizations with 10 sequence-specific oligonucleotide probes labeled nonradioactively. Using this method, the HLA-DQA1 genotype was theoretically classified into 36 genotypes: 8 homozygous and 28 heterozygous ones. Actually, 26 genotypes were observed in the present study, and the gene frequency of each allele was calculated. The observed numbers were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. The HLA-DQA1 genotype was also determined in aged bloodstains. Since the genotype is polymorphic in the Japanese population and a very small amount of blood is required for determination, this typing is particularly useful for forensic analysis.

ALLELE DISTRIBUTION AT NINE STR LOCI-D3S1358, VWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 AND D7S820-IN THE JAPANESE POPULATION BY MULTIPLEX PCR AND CAPILLARY ELECTROPHORESIS

Nine tetranucleotide short tandem repeat (STR) loci, D3S1358, vWA, FGA TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820, were analyzed in the Japanese population with a newly released kit for personal identification using multiplex PCR with fluorescent-labeled primers following capillary electrophoresis. The observed heterozygosities were 0.67, 0.77, 0.82, 0.61, 0.62, 0.73, 0.78, 0.81 and 0.74, respectively, and the combined discrimination power of the nineplex was 0.9999999991. None of the nine loci deviated from Hardy-Weinberg equilibrium expectations using the chi-square test, homozygosity test, likelihood ratio test and exact test after the grouping of the alleles. The nine STR loci allele frequencies were significantly different from those of other ethnic populations.

Identification of saliva stains by determination of the specific activity of amylase

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.

Purification of nuclear DNA from single hair shafts for DNA analysis in forensic sciences

The typing of nuclear DNA from hair shafts has often been unsuccessful to date. We tried to type one of the nuclear DNA loci, HLA-DQA1, from hair shafts, using an efficient cetyl-trimethyl ammonium bromide (CTAB) precipitation for DNA purification and a sensitive semi-nested PCR. After thorough washing with ethanol and water, hair shafts were digested by proteinase K in the presence of dithiothreitol, followed by a purification step including CTAB-DNA precipitation. The specific region of HLA-DQA1 gene was amplified by the semi-nested PCR, and the amplified products were cloned and sequenced. The HLA-DQA1 genotype was determined by comparing the sequence to the known sequence of each allele. All genotypes of HLA-DQA1 were successfully typed with hair shafts from six known heterozygotes, although one of them showed the predominant appearance of one allele. For correct typing, a template DNA equivalent to a hair shaft of 5 or 10 cm in length was necessary. Without the CTAB-DNA precipitation step, DNA extract from such hair shafts inevitably contains enough melanin to inhibit PCR. The present results suggest that hair shafts can be used for the typing of nuclear DNA loci.

Maternal Identification from Skeletal Remains of an Infant Kept by the Alleged Mother for 16 Years with DNA Typing

This is a case study concerning maternal identification by DNA typing at various loci. An infant skeleton was found in the alleged mothers apartment after it was kept for 16 years. We obtained the skeletal remains as well as saliva stains from the alleged mother. DNA typing was conducted for three loci in the HLA class II region (HLA-DQA1, -DPB1, and DRB1), five loci with the AmpliType PM kit (LDLR, GYPA, HBGG, D7S8, and GC), five STR loci (LPL, vWA, F13B, TH01, and TPOX) and D-loop region in mtDNA for maternal identification. Sex determination was accomplished using fluorescent DNA capillary electrophoresis typing. Approximately 5 ng of human DNA was recovered from 1 g of femur bone retrieved from the infant skeletal remains. The probability of two unrelated Japanese sharing the same genotypes was estimated as 7.2 x 10(-11). The combined probability of exclusion that an individual is not the mother was also calculated at 0.998. We therefore conclude that the skeleton is from a female infant, and that there is no inconsistency in the claim that the infant was a daughter of the alleged mother.

Minisatellite variant repeat mapping: Application to DNA typing and mutation analysis

Most DNA typing systems assay allele length variation at tandemly repeated loci such as minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at minisatellite loci and of the mutational mechanisms that generate ultravariability.

Usefulness of a toothbrush as a source of evidential DNA for typing.

We investigated the usefulness of a toothbrush as a source of DNA for an unidentified cadaver. Ten toothbrushes were obtained from ten individuals along with their peripheral blood. We recovered from 10 to 430 ng of DNA from all but one of the toothbrushes. All ten toothbrushes, including the one containing no detectable DNA by fluorometry, were typed correctly at all of the loci tested, including nine STRs. Three toothbrushes obtained in two actual deaths also identified two victims and one suspect. Therefore, toothbrushes seem to be useful as a source of evidential DNA for personal identification.