Optimization and Development of an Efcient 13 X-STRs Multiplex PCR System for Paternity Testing

complex kinship cases. Objective: To develop a multiplex PCR system that consists of 13 X-chromosome STR markers, including GATA172D05, DXS8378, DXS6801, DXS6793, DXS6810, DXS7132, GATA31E08, DXS9902, HPRTB, DXS6789, DXS7423, DXS8377, DXS981 and sex-determining locus Amelogenin. Methods: Primer sequences of all X-STR markers were acquired from the Genome databases, and the original sequences for HPRTB, DXS6789, DXS7423, DXS8377 and DXS981 were modi�ed to eliminate primer-dimer formation and optimize melting temperatures to increase annealing e�ciency. All primer pairs were labelled with �uorescent dyes to support ampli�cation in a multiplex PCR, and the cycling conditions for multiplex PCR were optimized. Alleles for each locus were bi-directionally sequenced to determine the exact repeat size, and alleles generated in multiplex reactions were undistinguishable from alleles produced in a single marker PCR reaction. -8 Results: The combined power of discrimination of 13 X-STRs was 2.96 x 10-13 and 2.58 x10 in females and males, respectively. Conclusions: In conclusion, we have developed a 14-plex PCR system that can potentially be used for parentage testing and forensic casework studies.

[1]  P. Shrivastava,et al.  Direct PCR amplification from saliva sample using non-direct multiplex STR kits for forensic DNA typing , 2021, Scientific Reports.

[2]  A. A. Shahid,et al.  Development and characterization of a new 12-plex ChrX miniSTR system , 2014, International Journal of Legal Medicine.

[3]  Hu Zhao,et al.  Development and population study of the 12 X-STR loci multiplexes PCR systems , 2012, International Journal of Legal Medicine.

[4]  K. Minaguchi,et al.  Sixteen X-chromosomal STRs in two octaplex PCRs in Japanese population and development of 15-locus multiplex PCR system , 2010, International Journal of Legal Medicine.

[5]  P. Taberlet,et al.  DNA Barcoding for Honey Biodiversity , 2010 .

[6]  Erik T. Bieschke,et al.  X-chromosome STR sequence variation, repeat structure, and nomenclature in humans and chimpanzees , 2009, International Journal of Legal Medicine.

[7]  S. Riazuddin,et al.  Allele frequency distribution of 13 X-chromosomal STR loci in Pakistani population , 2008, International Journal of Legal Medicine.

[8]  D. De Leo,et al.  Development and forensic validation of a new multiplex PCR assay with 12 X-chromosomal short tandem repeats. , 2007, Forensic science international. Genetics.

[9]  Á. Carracedo,et al.  Genetic analysis of three US population groups using an X-chromosomal STR decaplex , 2007, International Journal of Legal Medicine.

[10]  C. Robino,et al.  Development of two multiplex PCR systems for the analysis of 12 X-chromosomal STR loci in a northwestern Italian population sample , 2006, International Journal of Legal Medicine.

[11]  K. Kobayashi,et al.  MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis , 2006, International Journal of Legal Medicine.

[12]  M. Poetsch,et al.  Development of two pentaplex systems with X-chromosomal STR loci and their allele frequencies in a northeast German population. , 2005, Forensic science international.

[13]  J. Edelmann,et al.  Validation of the X-linked STR DXS6801. , 2005, Forensic science international.

[14]  Sun Wha Park,et al.  Genetic analysis of 18 X-linked short tandem repeat markers in Korean population. , 2005, Forensic science international.

[15]  Myung Jin Park,et al.  Genetic characteristics and population study of 4 X-chromosomal STRs in Koreans: evidence for a null allele at DXS9898 , 2004, International Journal of Legal Medicine.

[16]  J. Ballantyne,et al.  SWGDAM developmental validation of a 19-locus Y-STR system for forensic casework. , 2004, Journal of forensic sciences.

[17]  A. Tagliabracci,et al.  Development of a heptaplex PCR system to analyse X-chromosome STR loci from five Italian population samples. A collaborative study. , 2004, Forensic science international.

[18]  P. Schneider,et al.  Preparation of degraded human DNA under controlled conditions. , 2004, Forensic science international.

[19]  P. Gill,et al.  STR analysis of artificially degraded DNA-results of a collaborative European exercise. , 2004, Forensic science international.

[20]  P. Betz,et al.  Development of a 13-locus PCR multiplex system for paternity testing , 2004, International Journal of Legal Medicine.

[21]  L. Roewer,et al.  Cell line DNA typing in forensic genetics--the necessity of reliable standards. , 2003, Forensic science international.

[22]  Weibo Liang,et al.  Two X-chromosome STR loci DXS6803 and XS6793 frequency data in Chinese population. , 2003, Journal of forensic sciences.

[23]  M. Krawczak,et al.  Use of X-linked markers for forensic purposes , 2003, International Journal of Legal Medicine.

[24]  W. Parson,et al.  New sensitive amplification primers for the STR locus D2S1338 for degraded casework DNA , 2003, International Journal of Legal Medicine.

[25]  Richard Schoske,et al.  Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci , 2003, Analytical and bioanalytical chemistry.

[26]  J. Riancho,et al.  Sequence structure and population data of two X-linked markers: DXS7423 and DXS8377 , 2002, International Journal of Legal Medicine.

[27]  J. Edelmann,et al.  Sequence variation and allele nomenclature for the X-linked STRs DXS9895, DXS8378, DXS7132, DXS6800, DXS7133, GATA172D05, DXS7423 and DXS8377. , 2002, Forensic science international.

[28]  J. Riancho,et al.  A new pentaplex system to study short tandem repeat markers of forensic interest on X chromosome. , 2002, Forensic science international.

[29]  L. Roewer,et al.  16 X-chromosome STR loci frequency data from a German population. , 2001, Forensic science international.

[30]  R. Szibor,et al.  Development of the X-linked tetrameric microsatellite marker HumDXS6789 for forensic purposes. , 2001, Forensic science international.

[31]  Christian M. Ruitberg,et al.  Capillary electrophoresis as a tool for optimization of multiplex PCR reactions , 2001, Fresenius' journal of analytical chemistry.

[32]  Joseph B. Rayman,et al.  Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. , 1996, BioTechniques.

[33]  P. Walsh,et al.  Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. , 1996, Nucleic acids research.

[34]  N. Morling,et al.  Analysis of the locus D1S80 by amplified fragment length polymorphism technique (AMP-FLP). Frequency distribution in Danes. Intra and inter laboratory reproducibility of the technique. , 1993, Forensic science international.

[35]  R Higuchi,et al.  Preferential PCR amplification of alleles: mechanisms and solutions. , 1992, PCR methods and applications.

[36]  C. M. Hearne,et al.  Tetranucleotide repeat polymorphism at the HPRT locus. , 1991, Nucleic acids research.

[37]  Á. Carracedo,et al.  A method for the analysis of 32 X chromosome insertion deletion polymorphisms in a single PCR , 2011, International Journal of Legal Medicine.

[38]  Yih-Yuan Chang,et al.  Genetic analysis of eight population groups living in Taiwan using a 13 X-chromosomal STR loci multiplex system , 2009, International Journal of Legal Medicine.

[39]  Ha Eun-Ho,et al.  State of Individual Identification Using Genetic Polymorphism in Korea: Step to Criminal DNA Bank. , 2006 .

[40]  J. Ballantyne,et al.  A highly discriminating 21 locus Y-STR "megaplex" system designed to augment the minimal haplotype loci for forensic casework. , 2004, Journal of forensic sciences.

[41]  K. Alt,et al.  Development of a quadruplex PCR system for the genetic analysis of X-chromosomal STR loci , 2003 .

[42]  P. Lincoln.  DNA recommendations--further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. , 1997, Forensic science international.

[43]  C. Kimpton,et al.  Highly discriminating heptaplex short tandem repeat PCR system for forensic identification. , 1995, BioTechniques.

[44]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.