A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome

The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) can help resolve individuals with an identical HV1/HV2 mitotype. A set of 11 SNPs selected for distinguishing individuals of the most common Caucasian HV1/HV2 mitotype were incorporated in an allele specific primer extension assay. The assay was optimized for multiplex detection of SNPs at positions 3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477 and 14470 in the mtGenome. Primers were designed to allow for simultaneous PCR amplification of 11 unique regions in the mtGenome and subsequent primer extension. By enzymatically incorporating fluorescently labeled dideoxynucleotides (ddNTPs) onto the 3’ end of the extension primer, detection can be accomplished with a capillary-based electrophoresis (CE) platform common in most forensic laboratories. The electrophoretic mobility for the extension primers was compared in denaturing POP4 and POP6 CE running buffers. Empirical adjustment of extension primer concentrations resulted in even signal intensity for the 11 loci probed. We demonstrate that the assay performs well for heteroplasmy and mixture detection, and for typical mtDNA casework samples with highly degraded DNA.

[1]  C R Cantor,et al.  Oligonucleotide interactions. 3. Circular dichroism studies of the conformation of deoxyoligonucleotides. , 1970, Biopolymers.

[2]  Charles R. Cantor,et al.  Oligonucleotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleolides , 1970 .

[3]  T. Vulliamy,et al.  A new genetic polymorphism in the 16S ribosomal RNA gene of human motochondrial DNA , 1989, Annals of human genetics.

[4]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[5]  P. Rumsby PCR protocols—A guide to methods and applications: Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White. Academic Press Inc., CA, USA, 1990. pp. xviii + 482. $39.95. ISBN 0-12-372181-40 , 1991 .

[6]  S N Austad Forensic DNA typing. , 1992, Science.

[7]  G. Tully,et al.  Rapid detection of mitochondrial sequence polymorphisms using multiplex solid-phase fluorescent minisequencing. , 1996, Genomics.

[8]  D. Nickerson,et al.  AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns. , 1996, BioTechniques.

[9]  P. Vallone,et al.  Predicting sequence-dependent melting stability of short duplex DNA oligomers. , 1997, Biopolymers.

[10]  J. SantaLucia,et al.  A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Ross,et al.  High level multiplex genotyping by MALDI-TOF mass spectrometry , 1998, Nature Biotechnology.

[12]  M. Holland,et al.  Mitochondrial DNA Sequence Analysis - Validation and Use for Forensic Casework. , 1999, Forensic science review.

[13]  K. Livak,et al.  Allelic discrimination using fluorogenic probes and the 5' nuclease assay. , 1999, Genetic analysis : biomolecular engineering.

[14]  D. Turnbull,et al.  Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA , 1999, Nature Genetics.

[15]  T Foitzi,et al.  Allelic discrimination using fluorogenic probes and the 5' nuclease assay , 1999 .

[16]  A. Sajantila,et al.  Analysis of mtDNA HVRII in several human populations using an immobilised SSO probe hybridisation assay , 1999, European Journal of Human Genetics.

[17]  M. Uhlén,et al.  Single-nucleotide polymorphism analysis by pyrosequencing. , 2000, Analytical biochemistry.

[18]  M. Holland,et al.  A sensitive denaturing gradient-Gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region. , 2000, American journal of human genetics.

[19]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[20]  U Hammerling,et al.  Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. , 2000, Genome research.

[21]  M. Weiner,et al.  Flow cytometric platform for high-throughput single nucleotide polymorphism analysis. , 2001, BioTechniques.

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

[23]  N Risch,et al.  High-throughput genotyping with single nucleotide polymorphisms. , 2001, Genome research.

[24]  J. Butler,et al.  Quality control of PCR primers used in multiplex STR amplification reactions. , 2001, Forensic science international.

[25]  M. Holland,et al.  Improved MtDNA sequence analysis of forensic remains using a "mini-primer set" amplification strategy. , 2001, Journal of forensic sciences.

[26]  T. Parsons,et al.  Increasing the forensic discrimination of mitochondrial DNA testing through analysis of the entire mitochondrial DNA genome. , 2001, Croatian medical journal.

[27]  M. Weiner,et al.  Fluorescent microsphere‐based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification , 2001, Human mutation.

[28]  C. Tyler-Smith,et al.  Hierarchical high-throughput SNP genotyping of the human Y chromosome using MALDI-TOF mass spectrometry. , 2002, Nucleic acids research.

[29]  Ching Yu Austin Huang,et al.  SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomics and drug discovery. , 2002, BioTechniques.

[30]  I. Gut,et al.  Genotyping single-nucleotide polymorphisms by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[31]  Ivo G Gut,et al.  Genotyping single nucleotide polymorphisms by mass spectrometry. , 2002, Mass spectrometry reviews.

[32]  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.

[33]  Lars Kaderali,et al.  Primer-design for multiplexed genotyping. , 2003, Nucleic acids research.

[34]  T. Parsons,et al.  Rapid screening of mtDNA coding region SNPs for the identification of west European Caucasian haplogroups , 2003, International Journal of Legal Medicine.

[35]  R. Kapsa,et al.  Normal variants of human mitochondrial DNA and translation products: the building of a reference data base , 1991, Human Genetics.

[36]  T. Parsons,et al.  Single nucleotide polymorphisms over the entire mtDNA genome that increase the power of forensic testing in Caucasians , 2004, International Journal of Legal Medicine.

[37]  Mark R. Wilson,et al.  Validation of mitochondrial DNA sequencing for forensic casework analysis , 2005, International Journal of Legal Medicine.