Typing of 24 mtDNA SNPs in a Chinese population using SNaPshot minisequencing.

Three SNaPshot multiplex assays were developed to test 23 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR)I and II, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both human population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 31 haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.

[Development of SNP multiplexes utilizing universal reporter primers for forensic purposes].

OBJECTIVE: To establish a simple, fast and economical technique for multiplex-typing SNPs and to explore its potential forensic application. METHODS: Five Y-SNP loci (IMS-JST164520, IMS-JST021354, IMS-JST003305, M119 and M134) were selected and the allele specific primers of each locus were designed with the universal reporter primers tailed at their 5' end. Alleles of these loci were amplified first by allele specific primers, then amplified by universal reporter primers tagged by fluorescent dye. RESULTS: A fluorescent-multiplex PCR system of the five Y-SNP loci was established. The typing results showed that two different colors of product peaks denoted two different alleles of a SNP locus, and the fragment sizes of alleles among different SNP loci were different. The haplotype diversity of these five loci was estimated to be 0.8655 in Wuhan Han population. CONCLUSION: The multiplex-typing SNPs based on the universal reporter primers is a simple, fast, and economical technique, and may have good application value in forensic medicine.

Typing of 24 mtDNA SNPs in a Chinese Population Using SNaPshot Minisequencing / 华中科技大学学报（医学）（英德文版）

Three SNaPshot multiplex assays were developed to test 23 coding region single nucleo-tide polymorphisms (SNPs) and one control region SNP outside hypervariable regions (HVR) Ⅰ and Ⅱ, which was aimed at increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and confirming haplogroup assignments of mtDNA profiles in both hu-man population studies and medical research. The selected SNPs targeted the East Asian phylogeny. These multiplex assays were validated by comparing with the sequencing analysis of samples chosen randomly. The mtDNA variations of 100 unrelated individuals from the Wuhan population in China were examined and classified into 3 i haplotypes, and the haplotype diversity was estimated to be 0.952. The multiplex SNaPshot method is rapid and robust, and suitable for large-scale screening studies of mtDNA variability.

Parentally imprinted allele (PIA) typing in the differentially methylated region upstream of the human H19 gene.

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study, the H19FR1 and H19FR2 haplotype polymorphisms including four and three SNPs, respectively, upstream of the H19 gene according to the GenBank sequence (accession no. AF125183) were investigated. Five haplotypes and nine genotypes were detected for H19FR1 in the Chinese Han population by means of PCR and subsequent denaturing gradient gel electrophoresis (DGGE). The power of discrimination (Dp), polymorphism information content (PIC) and probability of paternity exclusion (PE) were estimated to be 0.803, 0.58 and 0.322, respectively. For the H19FR2, two haplotypes and three genotyes were observed, and the Dp, PIC and PE were 0.626, 0.37 and 0.162, respectively. Sequencing results showed that only two of the four reported SNPs, a7342g and g7547a, were detected in H19FR1 in the Chinese Han population, and two new SNPs, g7351c and a7357g, were found. In the H19FR2 region, only one of the three reported SNPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FRs was examined by using two types of enzymes, the methylation-sensitive restriction enzyme (msRE) HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This parentally imprinted allele (PIA) typing method could be one of the useful techniques for discriminating the parental origin of alleles.

[The application of PCR-SSCP in forensic mtDNA typing].

OBJECTIVE: To study the application of PCR-SSCP in forensic mtDNA typing. METHODS: Primers flanking the mtDNA HV-I and HV-II regions were designed. By PCR-SSCP techniques, 70 family trios and 140 unrelated Wuhan Han individuals were investigated and analyzed. RESULTS: In 70 family trios, the SSCP profiles in region HV-I and HV-II of children were not same to that of their fathers in 98.57% and 97.13% respectively but were identical with their mothers. In 140 unrelated Wuhan Han individuals, 21 haplotypes were found in HVI, GD = 0.9556; 16 haplotypes were found in HVII, GD = 0.9356. CONCLUSION: PCR-SSCP technique may be useful in forensic mtDNA typing, especially for screening the suspects.

[Genetic polymorphism of 23 Y chromosome biallelic markers in Wuhan Han population].

To search polymorphic Y chromosome biallelic markers in Chinese Han population, and obtain their population genetic data. Genotyping of 23 biallelic markers on human Y chromosome (M7, M9, M50, M88, M89, M95, M111, M117, M119, M121, M122, M134, M159, M164, M175, M214, LINE1, MSY2, RPS4Y711, SRY465, IMS-JST164520, IMS-JST021354 and IMS-JST003305) were carried out in a sample of 160 unrelated Chinese male individuals living in Wuhan using fragment length discrepant allele specific PCR (FLDAS-PCR) and PAGE technique. In all 23 biallelic markers, genetic polymorphism were identified for 20 loci in Wuhan Han population except for M50, M159 and M164, and the ranges of gene diversity (GD) were 0.0126-0.4855. A total of 35 different haplogroups (Hg1-35) were observed and the haplogroup diversity (HD) was 0.9471. The haplogroups formed by 20 biallelic markers are highly polymorphic, and can be used in forensic science and population evolution studies.

Study on the application of parent-of-origin specific DNA methylation markers to forensic genetics.

In paternity test, especially in motherless cases, the allele inherited from father (obligatory gene, OG) often cannot be determined. The paternity exclusion probability (PE) of a genetic marker is reduced considerably. Therefore, it is necessary to develop a new technique, by which the parental origin of alleles can be determined without genealogical analysis. In this paper, we explored the possibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles, choosing the imprinted single nucleotide polymorphism (SNP) locus rs220028 (A/G) as a model system. We typed the SNP by mutagenically separated PCR (MS-PCR). The frequencies of alleles were A = 0.5085, G = 0.4915; the unbiased heterozygosity was 0.5020. In order to discriminate between the maternal allele and paternal allele, post-digestion MS-PCR, a novel PCR based methylation analysis and SNP typing technique was developed and performed on 18 heterozygous children, and the methylated maternal allele was detected specifically. As a pilot study on the use of epigenetic markers in forensic genetics, our results demonstrated the feasibility of using parent-of-origin specific DNA methylation markers to determine the parental origin of alleles.

[Observation and analysis on mutation of routine STR locus].

OBJECTIVE: To observe and analyze the characteristic of mutation at STR locus. METHODS: 27 mutant genes observed in 1211 paternity testing cases were checked by PAGE-silver stained and PowerPlex 16 System Kit and validated by sequencing. RESULTS: Mutant genes locate on 15 loci. The pattern of mutation was accord with stepwise mutation model. The mutation ratio of male-to-female was 8:1 and correlated to the age of father. CONCLUSION: Mutation rate is correlated to the geometric mean of the number of homogeneous repeats of locus. The higher the mean, the higher the mutation rate. These loci are not so appropriate for use in paternity testing.

[A simple and rapid modified--new method for SNP typing by fragment length discrepant allele specific PCR].

OBJECTIVE: To establish a new method for single nucleotide polymorphism (SNP) typing based on allele specific PCR: fragment length discrepant allele specific PCR (FLDAS-PCR), and study the influence on specific extension by introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers. METHODS: For SNP loci rs759117 and rs760887, two allele specific forward primers, with different length and a mismatch introduced at the third or fourth 3'-terminal base, and a public reverse primer were designed for SNP typing. The genotyping of SNP was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining. RESULTS: The different homozygote genotypes comprised a single band with different size respectively, and the heterozygote genotypes comprised two bands. Typing results were completely consistent with those by direct sequencing. Non-specific primer extension was decreased remarkably after introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers, and the stringency of PCR reaction was cut down. CONCLUSION: FLDAS-PCR is a simple, rapid and efficient new method for SNP typing. During FLDAS-PCR, specific primers with a mismatch at the third or fourth 3'-terminal base have more power to identify two alleles.

[Applications of DNA methylation markers in forensic medicine].

DNA methylation is a post-replication modification that is predominantly found in cytosines of the dinucleotide sequence CpG. Epigenetic information is stored in the distribution of the modified base 5-methylcytosine. DNA methylation profiles represent a more chemically and biologically stable source of molecular diagnostic information than RNA or most proteins. Recent advances attest to the great promise of DNA methylation markers as powerful future tools in the clinic. In the past decade, DNA methylation analysis has been revolutionized by two technological advances--bisulphite modification of DNA and methylation-specific polymerase chain reaction (MSP). The methylation pattern of human genome is space-time specific, sex-specific, parent-of-origin specific and disease specific, providing us an alternative way to solve forensic problems.

[The multiplex analysis of epigenetic markers and genetic markers by post-digestion mutagenically separated PCR].

OBJECTIVE: To establish a novel method for the multiplex analysis of the methylation and single nucleotide polymorphism (SNP). METHODS: The imprinted SNP rs220028 was chosen as a model. Genomic DNA, after being digested with methylation sensitive restriction enzyme, were typed by mutagenically separated PCR (MS-PCR). The polymorphism of restriction site was excluded by PCR-RFLP. RESULTS: By post-digestion MS-PCR, the methylated allele was detected selectively, the maternal origin of which was confirmed by pedigree analysis; A=0.5085, G=0.4915,PIC=0.3749. CONCLUSION: The multiplex analysis of methylation markers and SNP can be achieved by post-digestion MS-PCR. The imprinted SNP locus rs220028 is a potentially useful marker in screening Prader-Willi/Angelman syndrome.

[Application of chi-square test and exact test in Hardy-Weinberg equilibrium testing].

This article review the application of chi-square test of various data handling methods and exact test in Hardy-Weinberg equilibrium testing of human genetic marker in population genetics. The importance of HWE-exact test in multiallelic system was emphasized, especially in the study of forensic VNTR and STR typing.

Allele frequencies of six STR loci (D3S4536, D4S2633, D5S2500, D9S925, D9S1118 and D20S481) in Chinese Han population.

POPULATION: Over 265 unrelated individuals from Han population living in Wuhan, China.

[Application of the age-associated injure in mitochondrial DNA].

Nowadays, the injury in human mitochondrial DNA (mtDNA) is well known to accumulate in various tissues with age. It's significant to further investigate and then apply it to estimation of the age at parenchymas.

Development of the X-linked tetrameric microsatellite markers HumDXS6803 and HumDXS9895 for forensic purpose.

This paper presents sequence and population genetic data of the X-linked DXS6803 and DXS9895 short tandem repeat (STR). The tetranucleotide repeat polymorphism DXS6803 (also known as CHLC.GATA45H11) and DXS9895 (also known as CHLC.GATA124B04) are located at the Xq12-Xq21.33 and Xpter-Xp22.2 region, respectively. In kinship testing, DXS6803 and DXS9895 are suitable for concomitant use. Population genetic data were obtained by analyzing 182 unrelated females and 110 males from Chinese Han population. In this population, both DXS6803 and DXS9895 exhibited seven clearly distinguishable alleles ranging from 109bp to 128bp and 139bp to 163bp in length, respectively. Testing for Hardy-Weinberg equilibrium (HWE) showed no significant deviation for these two loci. The polymorphism information content (PIC), observed heterozygosity (H(obs)) and power of exclusion for parentage testing of a girl for trios (PE(trio)) and duos (PE(duo)) were 0.67, 0.687, 0.673 and 0.530 for DXS6803, and 0.69, 0.736, 0.688 and 0.547 for DXS9895, respectively. Seventy-eight families studies of these two loci confirmed X-linked codominant inheritance and mutations were not found.

[A study of genetic polymorphism of the STR loci D20S85 and D6S477 in Han population living in Wuhan].

OBJECTIVE: The genetic polymorphism of two STR loci, D20S85 and D6S477, were studied in 280 unrelated Chinese individuals in Wuhan. METHODS: The PCR amplified products were analyzed by PAGE and silver staining. RESULTS: 10 and 9 alleles were observed in these two STR loci, and the discriminating power (DP) were 0.9085 and 0.9127 respectively. No deviations from Hardy-Weinberg equilibrium were found. The two STR loci had been successfully applied to individual identification and paternity testing. CONCLUSION: The results demonstrated that the two loci were useful for forensic identification.

[PGM1 genotyping by PCR-SSCP].

OBJECTIVE: PGM1 genotyping by PCR-SSCP analysis. METHODS: Amplified genome DNA from 156 unrelated Han individuals living in Wuhan, PCR products for exon 4 and exon 8 of PGM1 were then analyzed by SSCP to detect the genotypes. RESULTS: 2 alleles and 3 genotypes were detected in exon 4 and 8 respectively. The discrimination power was 0.7318. PCR-SSCP analysis was suitable for determination of PGM1 genotypes from old blood and semen stains. CONCLUSION: PGM1 system typed by PCR-SSCP is useful for forensic identification.