ABSTRACT
Massively parallel sequencing (MPS) has been used in forensic genetics in recent years owing to several advantages, e.g. MPS can provide precise descriptions of the repeat allele structure and variation in the repeat-flanking regions, increasing the discriminating power among loci and individuals. However, it cannot be fully utilized unless sufficient population data are available for all loci. Thus, there is a pressing need to perform population studies providing a basis for the introduction of MPS into forensic practice. Here, we constructed a multiplex PCR system with fusion primers for one-directional PCR for MPS of 15 commonly used forensic autosomal STRs and amelogenin. Samples from 554 unrelated Chinese Northern Han individuals were typed using this MPS assay. In total, 313 alleles obtained by MPS for all 15 STRs were observed, and the corresponding allele frequencies ranged between 0.0009 and 0.5162. Of all 15 loci, the number of alleles identified for 12 loci increased compared to capillary electrophoresis approaches, and for the following six loci more than double the number of alleles was found: D2S1338, D5S818, D21S11, D13S317, vWA, and D3S1358. Forensic parameters were calculated based on length and sequence-based alleles. D21S11 showed the highest heterozygosity (0.8791), discrimination power (0.9865), and paternity exclusion probability in trios (0.7529). The cumulative match probability for MPS was approximately 2.3157 × 10-20 .
Subject(s)
Asian People/genetics , Forensic Genetics/methods , High-Throughput Nucleotide Sequencing/methods , Microsatellite Repeats , Multiplex Polymerase Chain Reaction/methods , Polymorphism, Genetic , Amelogenin/genetics , China , DNA Fingerprinting/methods , Female , Gene Frequency , Genetics, Population , Genotyping Techniques/methods , Humans , MaleABSTRACT
UNLABELLED: To ensure the consistency of genotype results for PowerPlex 21 kit and Goldeneye 20A kit. METHODS: The STR loci were amplified in DNA samples from 205 unrelated individuals in Beijing Han population. And consistency of 19 overlap STR loci typing were observed. The genetic polymorphism of D1S1656 locus was obtained. RESULTS: All 19 overlap loci typing showed consistent. The proportion of peak height of heterozygous loci in two kits showed no statistical difference (P > 0.05). The observed heterozygosis of D1S1656 was 0.878. The discrimination power was 0.949. The excluding probability of paternity of triplet was 0.751. The excluding probability of paternity of diploid was 0.506. The polymorphism information content was 0.810. CONCLUSION: PowerPlex 21 kit and Goldeneye 20A kit present a good consistency. The primer design is reasonable. The polymorphism of D1S1656 is good. The two kits can be used for human genetic analysis, paternity test, and individual identification in forensic practice.
Subject(s)
DNA Fingerprinting , DNA Primers , DNA/analysis , Genotype , Heterozygote , Humans , Microsatellite Repeats , Paternity , Polymerase Chain Reaction , Polymorphism, GeneticABSTRACT
OBJECTIVE: To investigate the genetic polymorphisms of 16 non-CODIS loci (D6S477, D22-GATA198B05, D15S659, D8S1132, D3S3045, D17S1290, D14S608, D2S441, D18S535, D13S325, D10S1435, DlS2368, DIS1656, D7S3048, D10S1248 and D19S253) in Beijing Han population. METHODS: The DNA of 300 unrelated individuals in Beijing Han population were PCR amplified using GoldeneyeM DNA identification system 18NC kit, and the PCR products were analyzed by electrophoresis through 3130XL genetic analyzer. The fragment sizes of alleles were taken subsequently by GeneMapper v3.2. RESULTS: The distributions of genotype frequencies of 16 non-CODIS STR loci in Beijing Han population satisfied the Hardy-Weinberg equilibration. The population genetic parameters were obtained as followings: heterozygosity was 0.677-0.873; discrimination power, 0.890-0.967; probability of paternity exclusion, 0.393-0.741; and polymorphism information content, 0.706-0.853. CONCLUSION: These 16 non-CODIS STR loci show great genetic polymorphisms in Beijing Han population, and are useful for the research of population genetics and forensic application.
Subject(s)
Asian People/genetics , Genetics, Population , Microsatellite Repeats , Polymorphism, Genetic , Alleles , Asian People/ethnology , China , DNA Fingerprinting , Female , Forensic Genetics , Gene Frequency , Genetic Markers , Heterozygote , Humans , Male , Polymerase Chain ReactionABSTRACT
OBJECTIVE: To establish a new method for RNA and DNA co-extraction from the same sample by TRIzol reagent. METHODS: After the aqueous phase which contained total RNA was removed by traditional TRIzol method, the values of pH of the interphase phase and organic phase were adjusted. The DNA was precipitated with ethanol and purified with DNA IQ system. The purified DNA was measured in quality and quantity. As the template, it was amplified and typed by PCR-STR. The data was compared with that extracted by traditional TRIzol method. RESULTS: The DNA extracted by this modified method showed a better result of quality and quantity than that by traditional TRIzol method and a good STR typing. CONCLUSION: The modified TRIzol method is advisable and reliable to simultaneously extract both DNA and RNA from the same sample. It could be used for individual identification and paternity testing to satisfy the need of forensic science.
Subject(s)
Blood Chemical Analysis/methods , DNA/isolation & purification , Guanidines/chemistry , Phenols/chemistry , RNA/isolation & purification , DNA/blood , DNA Fingerprinting , Forensic Medicine , Humans , Hydrogen-Ion Concentration , Polymerase Chain Reaction , RNA/blood , Reagent Kits, DiagnosticABSTRACT
OBJECTIVE: To explore the tissue-specific gene expressions of the peripheral blood and the menstrual blood, and to search some specific factors to establish an effective method for identifying the peripheral blood and the menstrual blood. METHODS: The specific products of the peripheral blood and the menstrual blood were detected by RT-PCR and separated by electrophoretic technology. RESULTS: Beta-spectrin (SPTB) as one specific marker of peripheral blood and 18S rRNA as a kind of the housekeeping gene were expressed in both the peripheral blood and the menstrual blood. However, matrix metalloproteinase 7 (MMP7) as one specific marker of menstrual blood and human beta defensin 1 (HBD1) as one specific marker of vaginal discharge were only found in the menstrual blood. CONCLUSION: There are differences of specific gene expressions between the peripheral blood and the menstrual blood. They could be accurately distinguished from each other by using the combination of fluorescence technology and RT-PCR to detect the specific identification of mRNA.
Subject(s)
Blood/metabolism , Gene Expression Profiling , Menstruation/genetics , RNA, Messenger/genetics , Biomarkers , Female , Gene Expression , Humans , Matrix Metalloproteinase 7/genetics , Reverse Transcriptase Polymerase Chain Reaction , beta-DefensinsABSTRACT
OBJECTIVE: To assess the application value of laser capture microdissection (LCM) technique for isolating a small number of sperm cells from mixture sample. METHODS: Mixture samples were prepared with sperm cells and vaginal epithelia at different concentrations. Both LCM technique and the differential lysis method were employed to obtain sperm cells from the mixture samples, and DNA was extracted by magnetic beads method. STR genotyping was determined using Identifiler kit. RESULTS: The successful STR genotype rate of sperm cells isolated from mixture samples with LCM technique was 92.86% (13/14). The rate of differential lysis method was 7.14% (1/14). The successful rates between the two methods were statistically different (P < 0.05). CONCLUSION: LCM technique can effectively exclude the interference of female cell component and isolate a small number of sperm cells to obtain a single male STR genotyping. LCM technique is obviously better than the differential lysis method.
