Objective To evaluate the efficiency of REPLI-g® Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g® Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. Goldeneye® DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g® Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony® DNA Investigator® Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g® Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g® Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.
DNA ; DNA Fingerprinting ; Humans ; Microsatellite Repeats ; Nucleic Acid Amplification Techniques/standards* ; Sequence Analysis, DNA/methods*

OBJECTIVETo analyze the possible reason for HLA-C allele dropout in routine sequence-based typing (SBT) and improve the accuracy of HLA-C SBT test.

METHODSA total of 620 randomly selected samples from healthy voluntary blood donors in Shenzhen were typed at HLA-C locus by sequence-based typing using the AlleleSEQR HLA-C plus sequence-based typing kit. Samples with no full match result were subjected to cloning and haplotype sequencing of the full-length HLA-C gene. If no novel mutations were found, samples were then retyped, using our self-designed PCR primer pair and PCR conditions replacing the AlleleSEQR HLA-C PCR reagents in the PCR set-up procedure so as to analyze the potential reasons for causing abnormal SBT result.

RESULTSIn the 620 samples typed at HLA-C locus using the AlleleSEQR HLA-C SBT commercial kit, 5 samples with no full match result were identified. The closest genotype showed one nucleotide mismatch with many different allele groups at different nucleotide position. Based on the PCR-SBT nucleotide sequence, heterozygous nucleotides were determined only in exon 4, whereas the nucleotides in exon 2 and 3 were all homozygotes. The results showed that HLA-Cw*0706 allele dropout existed in all the 5 samples with abnormal SBT results initially identified by AlleleSEQR HLA-C SBT kit, no novel mutation was found.

CONCLUSIONThe results indicate that the PCR primer pair incompatible with DNA template may result in allele dropout in HLA-C SBT test. Based on the characterization of HLA-C full-length, it is essential to develop HLA-C SBT kit suitable for Chinese population in the future.

Alleles ; Amino Acid Sequence ; Base Sequence ; DNA Fingerprinting ; methods ; standards ; HLA-C Antigens ; genetics ; Humans ; Molecular Sequence Data ; Mutation ; Sequence Analysis, DNA ; methods ; standards

OBJECTIVE: To establish miniSTR fluorescent detection system with all detected fragments below 150 bp and to enhance the efficiency of detecting the degraded DNA samples. METHODS: All candidate primers were designed by Primer Premier 5 and screened by FastPCR 6.0. The miniSTR multiplex system was established by these selected loci labeling by four fluorescent dye. The parameters of PCR and primer concentrations were subsequently optimized. The electrophoresis was fulfilled under POP4 on 3100-Avant and the typing data was validated by standard DNA 9947A and 007. Fresh blood samples and difficult degraded DNA samples were tested to evaluate the usefulness of the system. RESULTS: All amplicons in the established miniSTR fluorescent detection system (D12ATA63, D2S1776, D1GATA113, D4S2408, D17S974, D20S482, D3S3053, Amelogenin, D6S474, D9S1122) were less than 150bp. The profile showed a balanced peak height without extra stutter by optimal protocol. Allele frequencies showed no deviations from Hardy-Weinberg equilibrium. The system showed accumulated probability of discrimination 0.999 999 983 and accumulated triplet excluding probability of paternity 0.996 8. It could detect corrupt muscle tissue, low copy number DNA samples and human tissues fixed by 40% formaldehyde solution for 12 days. CONCLUSION The miniSTR fluorescent detection system could be solely used for personal identification of degraded DNA samples or complementally used for paternity tests. And the system could enhance the ability of detecting the trace and degraded DNA.
DNA/chemistry* ; DNA Fingerprinting ; DNA Primers/genetics* ; Electrophoresis, Agar Gel ; Forensic Genetics ; Gene Frequency/genetics* ; Genetic Markers/genetics* ; Genetics, Population ; Humans ; Polymerase Chain Reaction/methods* ; Reference Standards ; Sequence Analysis, DNA/methods*

OBJECTIVE: To perform the validation and analysis of forensic parameters of Goldeneye DNA ID 26Y system. METHODS: Based on the validation rules of Scientific Working Group on DNA Analysis Methods (SWGDAM), the kit was assessed from several parts, as test of PCR system, reproducibility, accuracy, and sensitivity, etc. And Y-STR loci of 517 unrelated healthy individuals from Eastern China were genotypes by this kit. The distribution and frequency of haplotype were calculated and forensic parameters of the kit were assessed. RESULTS: The complete profiles can be obtained even when the PCR reaction volume with 6.25 microL. And correct profile was obtained with DNA down to 125 pg. No reproducible peaks were detected with the DNA of common animals and microorganism with the kit. For the male-male mixture testing, average 70% of the minor alleles were obtained when the ratios of 1:19 and 19:1. For the male-female mixture testing, results showed that the sensitivity of the kit was no compromised with the addition of female samples. CONCLUSION The validation studies demonstrated that Goldeneye DNA ID 26Y system has good sensitivity and specificity, and suitable for mixture testing. The polymorphism of 26 Y-STR loci included in this kit are good for forensic application.
Alleles ; Animals ; Asian People/genetics* ; China ; Chromosomes, Human, Y ; DNA ; DNA Fingerprinting/standards* ; Female ; Forensic Genetics/methods* ; Genotype ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVE: To test and estimate the forensic application of Goldeneye™ DNA ID 25A Kit. METHODS: The kit was validated by a series of tests for accuracy, sensitivity, consistency, peak height balance, stability, and mixed samples through measured blood samples and other samples in routine casework. RESULTS: The peak height balance of the different loci was ≥ 42%. The genotyping results of the positive control DNA was accurate. The complete STR genotyping result could be obtained from 0.125 ng positive control DNA. CONCLUSION Goldeneye™ DNA ID 25A Kit is suitable for criminal cases and DNA database in forensic practice.
Asian People/genetics* ; China ; Chromosomes, Human, Y ; DNA/genetics* ; DNA Fingerprinting/standards* ; Databases, Nucleic Acid ; Female ; Forensic Genetics/methods* ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Reproducibility of Results ; Sensitivity and Specificity