The use of unilateral PCR to identify prominent heteroduplexes formed during PCR of the mouse microsatellite locus D17Mit23

  • Mark A. Erhart
  • Taehoon Kim
  • Gladys M. Crews
  • Avani Pandya


Microsatellite markers are useful tools for understanding the evolutionary history of discrete segments of the mammalian genome. We used the microsatellite marker D17Mit23 to study the portion of the mouse genome known as the t complex, a naturally occurring variant of Chromosome 17. We identified an allelic variant of D17Mit23, which is shared by two forms of the t complex, the t haplotypes t w2 and t Lub2 . Polymerase chain reaction (PCR) analysis of DNA samples from mice that were heterozygous for either haplotype resulted in gel patterns with prominent bands of higher molecular weight in addition to the bona-fide D17Mit23 alleles. The appearance of these higher molecular weight bands, although consistent with heteroduplex formation, was not diminished through the use of reconditioning PCR. We used a modified form of asymmetric PCR, called “unilateral PCR”, to show that the higher molecular weight bands are heterodu-plexes and to identify their constituent strands. Certain microsatellite motifs may be especially prone to the production of prominent heteroduplex products, and this may lead to the erroneous genotyping of DNA samples.

Index Entries

Polymerase chain reaction (PCR) microsatellite heteroduplex unilateral PCR 


  1. 1.
    Edwards, A., Hammond, H. A., Jin, L., Caskey, C. T., and Chakraborty, R. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12, 241–253.PubMedCrossRefGoogle Scholar
  2. 2.
    Weber, J. L. and Wong, C. (1993) Mutation of human short tandem repeats. Hum. Mol. Genet. 2, 1123–1128.PubMedCrossRefGoogle Scholar
  3. 3.
    Brinkmann, B., Klintschar, M., Neuhuber, F., Huhne, J., and Rolf, B. (1998) Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat. Am. J. Hum. Genet. 62, 1408–1415.PubMedCrossRefGoogle Scholar
  4. 4.
    Dietrich, W. F., Miller, J., Steen, R., et al. (1996) A comprehensive genetic map of the mouse genome. Nature 380, 149–152.PubMedCrossRefGoogle Scholar
  5. 5.
    Ardlie, K. G. and Silver, L. M. (1998) Low frequency of t haplotypes in natural populations of house mice (Mus musculus domesticus). Evolution 52, 1185–1196.CrossRefGoogle Scholar
  6. 6.
    Huang, S.-W., Ardlie, K. G., and Yu. H.-T. (2001) Frequency and distribution of t-haplotypes in the southeast asian house mouse (Mus musculus castaneus) in Taiwan. Molecular Ecology 10, 2349–2354.PubMedCrossRefGoogle Scholar
  7. 7.
    Lai, F. and Artzt, K. (1992) Map positions of four dinucleotide repeats in the mouse t complex. Mamm. Genome 3, 476–477.PubMedCrossRefGoogle Scholar
  8. 8.
    Vernet, C. and Artzt, K. (1995) Mapping of 12 markers in the proximal region of mouse chromosome 17 using recombinant t haplotypes. Mamm. Genome 6, 219–221.PubMedCrossRefGoogle Scholar
  9. 9.
    Vernet, C., Abe, K., and Artzt, K. (1998) Genetic mapping of 10 microsatellites in thet complex region of mouse chromosome 17. Mamm. Genome 9, 472.PubMedCrossRefGoogle Scholar
  10. 10.
    Bennett, D., Dunn, L. C., and Rynerson, M. D. (1969) Genetical and embryological comparisons of semilethal t alleles from wild mouse populations. Genetics 61, 411–422.PubMedGoogle Scholar
  11. 11.
    Klein, J., Sipos, P., and Figueroa, F. (1984) Polymorphism of t-complex genes in European wild mice. Genet. Res. 44, 39–46.CrossRefGoogle Scholar
  12. 12.
    Selten, G., Cuypers, H. T., Boelens, W., et al. (1986) The primary structure of the putative oncogene pim-1 shows extensive homology with protein kinases. Cell 46, 603–611.PubMedCrossRefGoogle Scholar
  13. 13.
    Nadeau, J. H. and Phillips, S. J. (1987) The putative oncogene Pim-1 in the mouse: its linkage and variation among t haplotypes. Genetics 117, 533–541.PubMedGoogle Scholar
  14. 14.
    Hite, J. M., Eckert, K. A., and Cheng, K. C. (1996) Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n d(G-T)n microsatellite repeats. Nucl. Acids Res. 24, 2429–2434.PubMedCrossRefGoogle Scholar
  15. 15.
    Walsh, P. S., Fildes, N. J., and Reynolds, R. (1996) Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucl. Acids. Res. 24, 2807–2812.PubMedCrossRefGoogle Scholar
  16. 16.
    Miller, M. J. and Yuan, B.-Z. (1997) Semiautomated resolution of overlapping stutter patterns in genomic microsatellite analysis. Anal. Biochem. 251, 50–56.PubMedCrossRefGoogle Scholar
  17. 17.
    Cohen, B. B., Anderson, V. A., and Gillespie, K. (1998) Artefactual allotyping related to DNA source, concentration, and the number of PCR cycles. Dis. Markers 14, 165–167.PubMedGoogle Scholar
  18. 18.
    Bovo, D., Rugge, M., and Shiao, Y.-H. (1999) Origin of spurious multiple bands in the amplification of microsatellite sequences. J. Clin. Pathol. 52, 50–51.CrossRefGoogle Scholar
  19. 19.
    Thompson, J. R., Marcelino, L. A., and Polz, M. F. (2002) Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucl. Acids Res. 30, 2083–2088.PubMedCrossRefGoogle Scholar
  20. 20.
    McCabe, P. C. (1990) Production of single-stranded DNA by asymmetric PCR, in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Srinsky, J. J., and White, T. T., eds.), Academic Press, San Diego, CA, pp. 76–83.Google Scholar
  21. 21.
    Phillips, S. J. and Nadeau, J. H. (1984) Personal communication. Mouse Newslet 70, 83.Google Scholar
  22. 22.
    Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  23. 23.
    You, Y., Bergstram, R., Klemm, M., Nelson, H., Jaenisch, R., and Schimenti, J. (1998) Utility of C57BL/6J ×129/SvJae embryonic stem cells for generating chromosomal deletions: tolerance to gamma radiation and microsatellite polymorphism. Mamm. Genome 9, 232–234.PubMedCrossRefGoogle Scholar
  24. 24.
    Winking, H. and Silver, L. M. (1984) Characterization of a recombinant mouse t haplotype that expresses a dominant lethal maternal effect. Genetics 108, 1013–1020.PubMedGoogle Scholar
  25. 25.
    Dunn, L. C. and Bennett, D. (1967) Maintenance of gene frequency of a male sterile, semi-lethal T-aliele in a confined population of wild mice. Am. Nat. 101, 535–538.CrossRefGoogle Scholar
  26. 26.
    Schimenti, J. and Hammer, M. (1990) Rapid identification of mouse t haplotypes by PCR polymorphism (PCRP). Mouse Genome 87, 108.Google Scholar
  27. 27.
    Hsieh, C.-H. and Griffith, J. D. (1989) Deletions of bases in one strand of duplex DNA, in contrast to single-base mismatches, produce highly kinked molecules: Possible relevance to the folding of single-stranded nucleic acids. Proc. Natl. Acad. Sci. USA 86, 4833–4837.PubMedCrossRefGoogle Scholar
  28. 28.
    Hammer, M. F. and Silver, L. M. (1993) Phylogenetic analysis of the alpha-globin pseudogene-4 (Hba-ps4) locus in the house mouse species complex reveals a stepwise evolution of t haplotypes. Mol. Biol. Evol. 10, 971–1001.PubMedGoogle Scholar
  29. 29.
    Dod, B., Litel, C., Makoundou, P., Orth, A., and Boursot, P. (2003) Identification and characterization of t haplotypes in wild mice populations using molecular markers. Genet. Res. Camb. 81, 103–114.Google Scholar
  30. 30.
    Levinson, G. and Gutman, G. A. (1987) Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203–221.PubMedGoogle Scholar
  31. 31.
    Luty, J. A., Guo, Z., Willard, H. F., Ledbetter, D. H., Ledbetter, S., and Litt, M. (1990) Am. J. Hum. Genet. 46, 776–783.PubMedGoogle Scholar
  32. 32.
    Qiu, X. Y., Wu, L. Y., Huang, H. S., et al. (2001) Evaluation of PCR-generated chimeras: mutations and heteroduplexes with 16S rRNA gene-based cloning. Appl. Environ. Microbiol. 67, 880–887.PubMedCrossRefGoogle Scholar
  33. 33.
    Speksnijder, A., Kowalchuk, G. A., De Jong, S., Kline, E., Stephen J. R., and Laanbroek, H. J. (2001) Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences. Appl. Environ. Microbiol. 67, 469–472.PubMedCrossRefGoogle Scholar
  34. 34.
    von Wintzingerode, F., Gobel, U. B., and Stackebrandt, E. (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21, 213–229.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • Mark A. Erhart
    • 1
  • Taehoon Kim
    • 2
  • Gladys M. Crews
    • 3
  • Avani Pandya
    • 1
  1. 1.Department of Biological SciencesChicago State UniversityChicago
  2. 2.Department of Molecular Microbiology and ImmunologySaint Louis UniversityLouis
  3. 3.Evanston Research ParkAstellas CorporationEvanston

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