Comparative of Forensic DNA Identification Using Cell Lysis Method and Magnetic Beads Method / 法医学杂志

OBJECTIVES@#To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification.@*METHODS@#The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios.@*RESULTS@#When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method.@*CONCLUSIONS@#The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.

Genetic Polymorphism of 16 X-STR Loci in Xinjiang Uygur Population / 法医学杂志

OBJECTIVES@#To study the genetic polymorphism and population genetic parameters of 16 X-STR loci in Xinjiang Uygur population.@*METHODS@#The Goldeneye® DNA identification system 17X was used to amplify 16 X-STR loci in 502 unrelated individuals (251 females and 251 males). The amplified products were detected by 3130xl genetic analyzer. Allele frequencies and population genetic parameters were analyzed statistically. The genetic distances between Uygur and other 8 populations were calculated. Multidimensional scaling and phylogenetic tree were constructed based on genetic distance.@*RESULTS@#In the 16 X-STR loci, a total of 67 alleles were detected in 502 Xinjiang Uygur unrelated individuals. The allele frequencies ranged from 0.001 3 to 0.572 4. PIC ranged from 0.568 8 to 0.855 3. The cumulative discrimination power in females and males were 0.999 999 999 999 999 and 0.999 999 999 743 071, respectively. The cumulative mean paternity exclusion chance in trios and in duos were 0.999 999 997 791 859 and 0.999 998 989 000 730, respectively. The genetic distance between Uygur population and Kazakh population was closer, and the genetic distance between Uygur and Han population was farther.@*CONCLUSIONS@#The 16 X-STR loci are highly polymorphic and suitable for identification in Uygur population, which can provide a powerful supplement for the study of individual identification, paternity identification and population genetics.

Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection / 法医学杂志

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

Application of ITO Method in Determination of Special Half-Sibling Relationship / 法医学杂志

OBJECTIVES@#To identify whether the relationship between Zhang A, Zhang B, Zhang C and Zhang X is the half-sibling relationship whose mother is sister (hereinafter referred to as the special half-sibling relationship) or the common first cousin relationship and discuss the application of ITO method in discriminating the special kinship.@*METHODS@#DNA was extracted from blood stain of four identified individuals, PowerPlex® 21 System and AGCU 21+1 STR kit were used to detect autosomal STR genetic markers. Investigator® Argus X-12 QS kit was used to detect the X chromosome STR genetic markers, the special half-sibling index (SHSI) and first cousin index (FCI) and their likelihood ratio (LR) were calculated by ITO method.@*RESULTS@#The LR results of SHSI to FCI, which were calculated based on autosomal STR genotyping and the analysis of X-STR genotyping results suggested that the relationship between Zhang A, Zhang B, Zhang C and Zhang X was inclined to be a special half-sibling relationship.@*CONCLUSIONS@#For the identification of special kinship, it is necessary to comprehensively apply various genetic markers according to the case. After the conclusion that shared alleles cannot be excluded from the analysis, ITO method can be further used to establish discriminant assumptions according to the specific case to obtain objective and reliable identification opinions.

Forensic Application of ForenSeqTM DNA Signature Prep Kit in Zhengjiang She Ethnic Group / 法医学杂志

OBJECTIVES@#To evaluate the ability of the ForenSeqTM DNA Signature Prep kit (ForenSeq kit) in analyzing the sequence information of STRs in Zhejiang She ethnic group and its forensic application efficacy.@*METHODS@#A total of 50 Zhejiang She ethnic group samples were sequenced with the ForenSeq kit on the MiSeq FGx platform. The data was analyzed using ForenSeqTM universal analysis software to obtain the motif structure and flank regions of the 58 STRs, then compared with PCR-CE typing results to test the consistency. At last, the allele frequency and population genetic parameters were calculated.@*RESULTS@#A total of 448 sequence polymorphic alleles were detected in 50 samples of Zhejiang She ethnic group. Compared with fragment length polymorphism detected by PCR-CE, 82 alleles were increased by MPS detection based on ForenSeq kit, and 7 SNPs variation were detected in the flanking regions of 6 loci. The 22 male individuals were genotyped, and total 19 haplotypes were detected in 24 Y chromosome STRs of these 22 males. The cumulative discrimination power of the 27 autosomal STRs was 1-8.87×10-30, the cumulative probability of exclusion of duo-testing was 0.999 999 962 640 657, the cumulative probability of exclusion of trios-testing was 0.999 999 999 999 633.@*CONCLUSIONS@#Based on MPS typing technology, using the ForenSeq kit greatly improves the detection efficiency. In addition, the 58 STRs have good genetic polymorphisms in Zhejiang She ethnic group, which are suitable for individual identification and paternity identification in forensic application.

Bibliometric Analysis of Forensic Genetics Literatures in SCIE from 1989 to 2019 / 法医学杂志

Objective To conduct bibliometric analysis of forensic genetics literatures published by Chinese mainland scholars in SCIE journals from 1989 to 2019, to show the research achievements of the past three decades and predict future research fields and directions. Methods Microsoft Office Excel 2019 was utilized to analyze the general situation, research institutions, authors, funds, author keywords, etc. of the literatures. The status of research in forensic genetics in Chinese mainland was visualized by PlotDB, Gephi 0.9.2 software and literature interpretation. Results During the last three decades, 1 126 forensic genetics literatures were published by scholars from Chinese mainland on SCIE journals, mostly articles. The quantity and quality of the literatures were both on the increase. The number of literatures published in Forensic Science International-Genetics was the highest, and 60.83% of the literatures were funded, mainly by the National Natural Science Foundation of China （498 literatures）. The current research hotspots were STR, SNP, InDel polymorphisms, linkage genetic markers, mtDNA genetic markers, epigenetic markers, RNA genetic markers, chip technology and omics research method. Conclusion The forensic genetics in China has developed rapidly along with the promotion of forensic science in universities. The SCIE literatures on forensic genetics published by Chinese mainland scholars increased rapidly with the funding from the National Natural Science Foundation of China and Ministry of Science and Technology of the People's Republic of China, which positively contributes to the development of basic research and the improvement of overall level in forensic genetics in China.

An analysis of mutations at 41 short tandem repeat loci in Han Chinese population / 中国法医学杂志

Objective The aim of this study was to investigate mutations of 41 STR loci. Methods 4546 bloodstain samples were typed from 1932 father–mother–child trios by using AGCU_21+1, AGCU_EX22 and GlobalFiler_ExpressTM amplification Kit. Calculate the mutation rates of STR loci. Results 154 mutations were identified at 32 of the 41 loci. The average mutation rate was 1.0×10-3per locus(95%CI: 0.8~1.1×10-3), and the mutations of SE33 was highest. 152(98.7%) mutation events were one-step mutation, 2(1.3%) events were two-steps. The mutation events occurred in 150 father–mother–child triplets. The mutations in 146(97.3%) triplets occurred at single locus, 8 mutations were observed at two loci in 4(2.7%) triplets simultaneously. 104 paternal and 22 maternal mutations could be determined under 79212 paternal and maternal allelictransfers. The ratio of paternal versus maternal mutations was 4.7:1, and 28 unassigned mutations were observed. Conclusion STR mutation are common in paternity testing, and we should pay more attention to it.

Genetic polymorphisms of 19 STR loci in han popu-lation from Hainan and its application in paternity testing / 中国法医学杂志

Objective To construct a database for the genetic polymorphism of 19 STR loci in Han population from Hainan province. To investigate the application of 19 STR loci in the paternity testing. Methods The genotypes of 462 unrelated individuals in Hainan were detected with GoldeneyeTM 20A PCR Amplification Kit. 19-STR database was acquired, analyzed and evaluated in 283 paternity testing cases. Results No deviations of allele frequency from Hardy-Weinberg equilibrium expectations were found for Chi-square test (P>0.05). Observed heterozygosity (Hobs) varied between 0.603 and 0.914, total discrimination power (TDP) of 19 STR loci was more than 0.999999999999999, cumulative probability of exclusion (CPE) for triplet cases was 0.999999994. In all 283 paternity testing cases, triplets and duos were 170 and 113 respectively; there were 36 (12.7%) excluded cases comparing to 247 confirmed cases (87.3%). 14 mutation events were observed, and all were one-step mutation. Conclusion 14 out of 19 loci showed highly polymorphic in Han population from Hainan, and 19 STR system has high cumulative probability of exclusion and can meet the needs of paternity test of the local region. But mutation should be paid special attention to.

Preimplantation Genetic Diagnosis of X-linked Severe Combined Immunodeficiency / 中山大学学报(医学科学版)

[Objective]To establish a reliable and accurate preimplantation genetic diagnosis (PGD)method using multiple dis?placement amplification (MDA), which can be applied to the diagnosis of X-linked severe combined immunodeficiency disease (X-SCID).[Methods]Haplotype analysis for the X-SCID family was performedusing five short tandem repeats (STR) markers flanking the both sides of the interleukin-2 (IL-2) receptor gamma chain (IL2RG) gene. MDA technique was used for single-cell whole genomic amplification. The products were used as template in polymerase chain reaction (PCR) of informative STR markers found by linkage analysis for haplotype analysis as well as sequencing of the IL2RG gene exon 5.The amelogenin (AMEL) locus was used to do sex diag?nosis.[Results]Linked analysis revealed 3 STR markers were informative. The method was evaluated with 10 single lymphocytes and 10 single blastomeres. MDA was successful in all single cell. The detection efficiency of gene sequencing of pathogenic IL 2RG exon5 was 100%. The PCR efficiency of 3 STR informative markers and AMEL was 96.3%(77/80)and the average allele drop-out (ADO) rate was 11.5%(7/61). A cycle of PGD was performed on the family, and seven embryos were diagnosed, two of which were normal embry?os. Twin pregnancy occurred after transplantation which were given a healthy baby boy and a healthy baby girl.[Conclusion]In this study, multiple displacement amplification combined with specific amplification/sequencing of pathogenic gene and haplotype analysis in the single cell level of X-linked severe combined immunodeficiency disease were performed. The protocol can avoid misdiagnosis caused by contamination and ADO, and improve the diagnostic efficiency of PGD.

Application of MiniSTR in the detection of fetal DNA in maternal plasma / 中国输血杂志

Objective To develop an efficient method for detecting the short tandem repeat(STR) of fetal DNA in maternal plasma by miniSTR technique.Methods A total of 9 blood samples form pregnant women from 11 to 27 weeks of gestation were collected.Each isolated total plasma DNA was amplified in single multiplex using the ABI MiniFilerTM kit,which could simultaneously genotype the 9 miniSTR loci,including D13S317,D7S820,D2S1338,D21S11,D16S539,D18S51,CSFIPO,FGA and Amelogenin,and the PCR products were detected by using ABI PrismTM 3100 DNA Sequencer.The allelic designation of each STR locus was accomplished using the GeneMapper ID 3.2 software.Results Father-origin fetal STR allele was detected in all the 9 plasma DNA samples.An average of 3.1 fetal STR alleles of the 8 autosomal STR loci was observed in each of the 9 plasma DNA samples.As for the Amelogenin locus,Amelogenin Y allele was detected in 5 plasma DNA samples from pregnancies with male fetus,and allelic peak height values were all over 50 RFU,according to ABI Mini FilerTM PCR conditions,and the ratio of Amelogenin Y allele peak height value to Amelogenin X allele peak height value was 8.45%.However,no Amelogenin Y allele was detected in other 4 plasma DNA samples from pregnant women with female fetus.Conclusion The miniSTR technique is suitable for STR genotyping using fetal DNA in maternal plasma,and it suggests a broad application in noninvasive molecular prenatal diagnosis.

A Case Related to Identifying the Missing Person / 대한법의학회지

The application of DNA typing method after amplification by the polymerase chain reaction (PCR) of DNA derived from bone from two missing persons. In this case, short tandem repeats (STR) are used. The AmpFlSTR Identifiler loci (amelogenin and 15 STRs) are analysed by multiplex-PCR and automated fluorescent detection using Perkin Elmer Prism 310 DNA sequencer.

A Case Related to Identifying Missing Person by the floods in 2006 / 대한법의학회지

The fifteen persons from In-jae, Kang-won do, were missing by floods on July 2006. In relation to the this case, the two unidentified corpses were found nearby So-yang lake on June and September 2007, respectively. In these cases, autosome short tandem repeats (STR) and Y-STR were used for identification. The AmpFlSTR Identifiler kit (amelogenin and 15 STRs) and the AmpFlSTR Yfiler kit (16 Y-STRs) are analysed by multiplex-PCR and automated fluorescent detection using Perkin Elmer Prism 310 DNA sequencer. Finally we could found identify the two missing persons.

Frequency distributions of six short tandem repeat loci multiplexing in Jilin district,China / 临床检验杂志

Objective To analyze the genetic polymorphism of three short tandem repeat (STR) double loci,namely,six STR loci in the Chinese population of Han nationality in Jilin district and to obtain the corresponding genetic data.Methods DNA was extracted from the blood cells of one hundred and three unrelated individuals of Han nationality in Jilin district;Six loci were divided into three groups which were groupⅠ(TPOX+CSF1PO),groupⅡ(D3S1358+D13S317) and groupⅢ(D5S818+D19S400).The extracted DNA were amplified with PCR multiplexing method.The PCR products were analyzed by non-reduced PAGE,followed by silver staining.The allele frequency distributions of six STR loci were studied by statistical procedures.The heterozygosity (H),discrimination power (DP),probability of exclusion (PE) and polymorphic information content (PIC) were calculated.Results The allele frequencies of six loci were obtained.The genotypes distributions of the loci were consistent with Hardy-Weinberg equilibrium and had higher heterozygosities and PIC.The H,DP,PE and PIC were 0.7511-0.8376,0.8273-0.9247,0.5167-0.6718 and 0.7100-0.8195 respectively.Conclusions The three STR double loci exhibited higher polymorphism and were better genetic markers.The data on the allele frequencies of these six STR loci may be used in individual identification,paternity testing and in other population genetic researches.

Application of multiplex amplification of STRs for monitoring survival of engraftment after bone marrow transplantation / 临床检验杂志

Objective To evaluate the application of fluorescent multiplex amplification of short tandem repeat(STR)for monitoring survival of engraftment after bone marrow transplantation(BMT).Methods Three STR loci named D12S391,D18S865 and D20S161 in 56 cases were detected by fluorescent multiplex amplification.PCR products were separated and typed by DNA Sequencer.Results The genotypes of STR in 52 recipient after bone marrow transplantation were completely identical with those of the donors.In another 4 cases the evidences of mixed chimerism were observed.Conclusion The system of fluorescent multiplex amplification of STRs exhibited high capacity of discrimination and low cost.Its application in the detection of STR after BMT is reliable,sensitive and simple.Combined with the clinical manifestation it can be used to evaluate the effect of BMT.

A study of genetic polymorphisms of 11 Y chromosome specific STR loci / 中国法医学杂志

Objective To study the genetic polymorphisms of 11 Y chromosome specific STR loci in Guangzhou Han population. Method The DNA extracted from blood samples of unrelated individuals in Han population living in Guangzhou were amplified by PCR. The PCR products were analyzed by using PAGE. Results 3-5 alleles were detected in 11 Y - STR loci respectively in Han population in Guangzhou. The minimum GD value was 0.3037 (DYS434), while the maximum GD value was 0.8455 (DYS390) . Conclusion The 11 Y - specific STR loci are highly polymorphic and are suitable for personal identification and paternity testing.

A study on genetic polymorphism and rare alleles of 15 STR loci in 10071 individuals in Guangdong Han population / 中国法医学杂志

Objective To study the genetic polymorphism and frequencies of 15 STR loci in 10071 unrelated individuals of Han nationality, which are compared with the data reported previously. Methods 15 STR loci were amplified in DNA samples from 10071 unrelated individuals in Guangdong Han population using PowerPlex~(TM) 16 system, which were genotyped with ABI 377 or 3100 Genetic Analyzer. Frequencies for 15 STR loci were obtained. Results Except D8S1179 locus, rare alleles were found in the other 14 STR loci. The number of rare alleles ranged froml to 7 in the 14 STR loci ,which increased up to 34 rare alleles, including D21 S11 (32.1 and 36.2), D18S51 (15.2 and 17.2), Penta E (15.2, 17.4, 18.4, 19.4, 26 and 27), D7S820 (9.2, 10.1, 11.1 and 15), Penta D (18, 19 and 20), TPOX (14) and FGA (13) which were detected the first time and D21S11 (30.3), D7S820(9.1 and 9.2) which were frequently seen in the population although infrequent in European population. Conclusion More rare alleles can be detected with genetic polymorphism data from a lager number of individuals and more credible frequencies of alleles be obtained.

Application of Y-chromosome specific short tandem repeat loci in noninvasive prenatal diagnosis / 中国输血杂志

Objective To establish a noninvasive method for prenatal genetic analysis by using maternal serum and apply the method in fetal sex determination,paternity testing. Methods Samples of maternal serum from 53 pregnant women (11 to 36 weeks of gestation) were collected. The DNA extracted from each sample was amplified by using"Y-PLEX 6" amplification kit .which enabled the simultaneous analysis of six Y-STR loci including DYS393.DYS19.DYS389 II, DYS390, DYS391 and DYS385. The PCR products were detected by using ABI PrismTM 377 Sequencer and genotyped by related analysis software. Results (1) Y-STR specific alleles were detected in the maternal sera of all 29 mothers bearing male babies. Among the six Y-STR loci,specific alleles were detected in 29/29 at DYS393 locus,in 18/29 at DYS19 locus and in 10/29 at DYS390 locus. (2) Y-STR specific alleles were not detected in maternal sera of 24 pregnant women bearing female babies. (3) According to the presence of specific alleles at DYS393 locus and the value of allelic peak height and peak area, the accuracy of fetal sex determination was 100% . (4)The observed Y-STR alleles of each prenatal specimen from pregnant women with male fetuses were the same as the results of their husbands. Conclusion The assay of highly polymorphic Y-STR genotyping system developed by the authors provided a sensitive, accurate and non-invasive method to prenatal diagnosis. Our results demonstrate that fetal sex can be accurately determined and imply that paternity testing could be performed for pregnant women carrying male fetuses.

Application of autosomal STR genetic markers in siblings identification / 中国法医学杂志

Objective To discuss the probability of siblings identification by autosomal short tandem repeat (STR). Methods 150 pairs of full siblings and 150 pairs of unrelated individuals were genotyped by the 15 STR loci of Power Plex?6 system. Paternity index of full siblings (Pips) and paternity probability of full siblings (WFS) were calculated with the method of ITO. WFS and allelic matching of the two groups were compared, and three sorts of allelic matching in each pair were tested by chi-square test. Results Wps of 100 pairs of siblings (66.67%) were more than 0.9995; WFS of unrelated individuals were less than 0.8, and that of 100 pairs (66.67%) were less than 0.27. The locus number of the entire-same ranged from 1 to 10, averaged 5.49 in siblings, while in unrelated individuals ranged from 0 to 5 and averaged 1.33; that of the entire-different ranged from 0 to 6, averaged 1.66 in siblings but ranged from 2 to 11 and averaged 6.57 in unrelated individuals; that of the half-same ranged from 3 to 13, averaged 7.85 in siblings, and ranged from 1 to 13, averaged 7.11 in unrelated individuals. By chi-square test, there was significant difference (P0.05) in the half-same between the two groups. Conclusion It is effective to identify siblings by Power-Plex?6 system. Testing of 15 STR loci of PowerPlex?6 system, it suggested that the pair were unrelated individuals when the locus number of the entirely-different is not less than 6 or that of the entirely-same is zero, and that the pair were siblings when the locus number of entirely-different is not more than 1 or that of the entirely-same is not less than 6.