Polymorphism and haplotype analysis of three novel short tandem repeat loci in the p11.4 region of human X chromosome.

X-chromosomal short tandem repeats (X-STRs) are useful for the identification of absent single parents and complex blood relations. In the present study, we aimed to identify novel STR loci for use as DNA markers by conducting polymorphism and haplotype analyses. We detected three novel STR loci (LC552061, LC552062, and LC552063, with repetitive structures of (GGAA)n(GGGA)m, (CCTT)n(CCCT)m, and (ATTT)n, respectively) in the p11.4 region of the X chromosome. For these X-STRs, the polymorphism information content values ranged from 0.5766 to 0.6377 and the power of discrimination in males and females ranged from 0.6269 to 0.6844 and from 0.8105 to 0.8537, respectively. The linkage disequilibrium analysis revealed p values of < 0.0001, < 0.0001, and 0.00909 between LC552061 and LC552062, LC552061 and LC552063, and LC552062 and LC552063, respectively. Additional linkage disequilibrium analysis including seven previously analyzed loci (LC149476, LC149479, LC149480, LC149484, LC317283, LC317284, and LC317285) revealed a p value of < 0.001 among each of the five loci (LC149476, LC149479, LC149480, LC149484, and LC317283) and between LC317284 and LC317285, indicating that they were a linked group. These results indicate that, in addition to the seven previously detected loci, the three novel X-STR loci identified in the present study might be useful DNA markers for complex kinship analysis and might support the Investigator® Argus X-12 kit.

Genetic polymorphism analyses of three novel X chromosomal short tandem repeat loci in the Xp22.3 region.

X chromosomal short tandem repeats (X-STRs) can be useful for haplotype analysis in DNA testing, particularly for complex kinship testing or when one parent is absent. We searched downstream of four previously detected loci in the Xp22.3 region (LC149476, LC149479, LC149480, and LC149484) and detected and analyzed three novel short tandem repeats (STRs), LC317283, LC317284, and LC317285, with the repeat sequences TATAA, TTTA, and TATC, respectively. The forensic statistical values in Japanese subjects were confirmed to be noninferior to existing loci, with values for polymorphism information content, the power of discrimination in males (PDm), and the power of discrimination in females (PDf) of 0.5606-0.7448, 0.6078-0.7774, and 0.7990-0.9178, respectively. Haplotype analysis also revealed linkage disequilibrium between LC317283 and the four known loci (LC149476, LC149479, LC149480, and LC149484) and between two other novel loci (LC317284 and LC317285). Analysis of three family samples suggested that these STRs could be useful in complex kinship testing, so we developed an X-STR multiplex polymerase chain reaction (PCR) system for the seven loci and confirmed its ability to provide favorable amplification. We anticipate that the identified loci and developed multiplex PCR system will be beneficial to the field of forensic medicine.

Analysis of four novel X-chromosomal short tandem repeats within 71 kb of the Xp22.3 region.

DNA testing using X-chromosomal short tandem repeat (X-STR) polymorphisms has been used in maternity/paternity and complex kinship cases. Analyses of repeat sequences, surveys on racial statistics, and development of practical applications for DNA testing continue to be reported. In this study, we identified four novel tetranucleotide STR loci located in the X chromosome, which is the basis of X-STR research. These four tetranucleotide STRs were located within 71 kb of the chromosome Xp22.3 region. Using sequence analysis of the structure of repeat sequences, we identified simple repeat sequences of TAAA, CTTT, TATC, and GATA with rare insertions. We then calculated forensic statistical parameters using base length analysis. In the Japanese population, the polymorphism information content was 0.597-0.687, power of discrimination in females was 0.829-0.884, and power of discrimination in males was 0.635-0.729. As these tetranucleotide STRs are closely linked, we conducted haplotype analysis and detected that three loci (LC149476, LC149479, and LC149480) were in linkage disequilibrium. We demonstrated that the simultaneous analysis of these loci may be useful in complex kinship cases. Because these four loci can be detected by multiplex PCR, the detection of alleles at these loci can be rapidly and easily achieved. We conclude that the X-STR loci detected in this study may be useful tools in complex kinship cases and may increase the reliability of genetic testing.

Detection of a novel X-chromosomal short tandem repeat marker in Xq28 in four ethnic groups.

DNA testing of X-chromosomal short tandem repeat (X-STR) polymorphisms has been the focus of attention in several studies, mainly due to its applicability in the investigation of complex kinship cases. Studies of X-STR in analyses of DNA sequences, population studies and DNA testing applications have been reported. We performed detection and population genetic study of a novel tetranucleotide X-STR locus in the present study. We identified a unique X-STR locus consisting of two tetranucleotides in Xq28. Although the STR is a simple tetranucleotide, its polymorphism was comparatively high [polymorphism information content (PIC)=0.7140] in Japanese subjects. In addition, the STR varied in structure among ethnic groups. We conclude that this locus will be useful for forensic DNA testing and anthropological studies.

DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies.

DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples.

Vanadium accelerates polymerase chain reaction and expands the applicability of forensic DNA testing.

Polymerase chain reaction (PCR) has been rapidly established as one of the most widely used techniques in molecular biology. Because most DNA analysis is PCR-based, the analysis of unamplifiable DNA of poor quality or low quantity is nearly impossible. However, we observed that if an appropriate concentration of vanadium chloride is added to the standard reaction mixture, the enzymatic amplification of DNA could be enhanced. Using multiplex PCR with the addition of vanadium, DNA typing was possible from even trace amounts of DNA that we were unable to amplify using normal reaction conditions. This method might be an effective tool for not only criminal investigations and ancient DNA analysis, but also for nearly all fields using DNA technology.

Application of a novel multiplex polymerase chain reaction system for 12 X-chromosomal short tandem repeats to a Japanese population study.

The analysis of X-chromosomal short tandem repeat (X-STR) polymorphisms has been the focus of attention in several researches, mainly due to its applicability in the investigation of complex kinship cases. A new 12 X-STR multiplex system (GATA172D05, DXS7423, DSX6809, DXS10134, DXS7132, DXS9902, DXS6789, DXS10074, DXS8378, DXS9898, DXS10147, and GATA31E08) was developed and applied to a Japanese population study. DNA samples from 290 males and 160 females were successfully analyzed using the 12 X-STR multiplex system. No mutation was detected in the kinship cases involving 34 family trios. The combined powers of discrimination of the 12 X-STR loci in males and females were 0.999997 and 0.9999999996, respectively. We conclude that the combined analysis of 12 X-STR loci using this single multiplex polymerase chain reaction system is a powerful tool in forensic DNA testing.