Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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.
Weber, J. L. and Wong, C. (1993) Mutation of human short tandem repeats. Hum. Mol. Genet. 2, 1123–1128.
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.
Dietrich, W. F., Miller, J., Steen, R., et al. (1996) A comprehensive genetic map of the mouse genome. Nature 380, 149–152.
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.
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.
Lai, F. and Artzt, K. (1992) Map positions of four dinucleotide repeats in the mouse t complex. Mamm. Genome 3, 476–477.
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.
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.
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.
Klein, J., Sipos, P., and Figueroa, F. (1984) Polymorphism of t-complex genes in European wild mice. Genet. Res. 44, 39–46.
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.
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.
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.
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.
Miller, M. J. and Yuan, B.-Z. (1997) Semiautomated resolution of overlapping stutter patterns in genomic microsatellite analysis. Anal. Biochem. 251, 50–56.
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.
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.
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.
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.
Phillips, S. J. and Nadeau, J. H. (1984) Personal communication. Mouse Newslet 70, 83.
Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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.
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.
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.
Schimenti, J. and Hammer, M. (1990) Rapid identification of mouse t haplotypes by PCR polymorphism (PCRP). Mouse Genome 87, 108.
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.
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.
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.
Levinson, G. and Gutman, G. A. (1987) Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203–221.
Luty, J. A., Guo, Z., Willard, H. F., Ledbetter, D. H., Ledbetter, S., and Litt, M. (1990) Am. J. Hum. Genet. 46, 776–783.
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.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Erhart, M.A., Kim, T., Crews, G.M. et al. The use of unilateral PCR to identify prominent heteroduplexes formed during PCR of the mouse microsatellite locus D17Mit23 . Mol Biotechnol 33, 37–48 (2006). https://doi.org/10.1385/MB:33:1:37
Issue Date:
DOI: https://doi.org/10.1385/MB:33:1:37