Determining microsatellite genotyping reliability and mutation detection ability: an approach using small-pool PCR from sperm DNA

Anna MacDonald, Stephen Sarre, Nancy FitzSimmons, Nicola Aitken

    Research output: Contribution to journalArticle

    10 Citations (Scopus)

    Abstract

    Microsatellite genotyping from trace DNA is now common in fields as diverse as medicine, forensics and wildlife genetics. Conversely, small-pool PCR (SP-PCR) has been used to investigate microsatellite mutation mechanisms in human DNA, but has had only limited application to non-human species. Trace DNA and SP-PCR studies share many challenges, including problems associated with allelic drop-out, false alleles and other PCR artefacts, and the need to reliably identify genuine alleles and/or mutations. We provide a framework for the validation of such studies without a multiple tube approach and demonstrate the utility of that approach with an analysis of microsatellite mutations in the tammar wallaby (Macropus eugenii). Specifically, we amplified three autosomal microsatellites from somatic DNA to characterise efficiency and reliability of PCR from low-template DNA. Reconstruction experiments determined our ability to discriminate mutations from parental alleles. We then developed rules to guide data interpretation. We estimated mutation rates in sperm DNA to range from 1.5 ÿ 10⿿2 to 2.2 ÿ 10⿿3 mutations per locus per generation. Large multi-step mutations were observed, providing evidence for complex mutation processes at microsatellites and potentially violating key assumptions in the stepwise mutation model. Our data demonstrate the necessity of actively searching for large mutation events when investigating microsatellite evolution and highlight the need for a thorough understanding of microsatellite amplification characteristics before embarking on SP-PCR or trace DNA studies
    Original languageEnglish
    Pages (from-to)1-18
    Number of pages18
    JournalMolecular Genetics and Genomics
    Volume285
    Issue number1
    DOIs
    Publication statusPublished - 2011

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    Microsatellite Repeats
    Spermatozoa
    Polymerase Chain Reaction
    Mutation
    DNA
    Macropodidae
    Alleles
    Forensic Genetics
    Validation Studies
    Mutation Rate
    Artifacts
    Medicine

    Cite this

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    abstract = "Microsatellite genotyping from trace DNA is now common in fields as diverse as medicine, forensics and wildlife genetics. Conversely, small-pool PCR (SP-PCR) has been used to investigate microsatellite mutation mechanisms in human DNA, but has had only limited application to non-human species. Trace DNA and SP-PCR studies share many challenges, including problems associated with allelic drop-out, false alleles and other PCR artefacts, and the need to reliably identify genuine alleles and/or mutations. We provide a framework for the validation of such studies without a multiple tube approach and demonstrate the utility of that approach with an analysis of microsatellite mutations in the tammar wallaby (Macropus eugenii). Specifically, we amplified three autosomal microsatellites from somatic DNA to characterise efficiency and reliability of PCR from low-template DNA. Reconstruction experiments determined our ability to discriminate mutations from parental alleles. We then developed rules to guide data interpretation. We estimated mutation rates in sperm DNA to range from 1.5 {\~A}¿ 10{\^a}¿¿2 to 2.2 {\~A}¿ 10{\^a}¿¿3 mutations per locus per generation. Large multi-step mutations were observed, providing evidence for complex mutation processes at microsatellites and potentially violating key assumptions in the stepwise mutation model. Our data demonstrate the necessity of actively searching for large mutation events when investigating microsatellite evolution and highlight the need for a thorough understanding of microsatellite amplification characteristics before embarking on SP-PCR or trace DNA studies",
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    author = "Anna MacDonald and Stephen Sarre and Nancy FitzSimmons and Nicola Aitken",
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    Determining microsatellite genotyping reliability and mutation detection ability: an approach using small-pool PCR from sperm DNA. / MacDonald, Anna; Sarre, Stephen; FitzSimmons, Nancy; Aitken, Nicola.

    In: Molecular Genetics and Genomics, Vol. 285, No. 1, 2011, p. 1-18.

    Research output: Contribution to journalArticle

    TY - JOUR

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    AU - MacDonald, Anna

    AU - Sarre, Stephen

    AU - FitzSimmons, Nancy

    AU - Aitken, Nicola

    PY - 2011

    Y1 - 2011

    N2 - Microsatellite genotyping from trace DNA is now common in fields as diverse as medicine, forensics and wildlife genetics. Conversely, small-pool PCR (SP-PCR) has been used to investigate microsatellite mutation mechanisms in human DNA, but has had only limited application to non-human species. Trace DNA and SP-PCR studies share many challenges, including problems associated with allelic drop-out, false alleles and other PCR artefacts, and the need to reliably identify genuine alleles and/or mutations. We provide a framework for the validation of such studies without a multiple tube approach and demonstrate the utility of that approach with an analysis of microsatellite mutations in the tammar wallaby (Macropus eugenii). Specifically, we amplified three autosomal microsatellites from somatic DNA to characterise efficiency and reliability of PCR from low-template DNA. Reconstruction experiments determined our ability to discriminate mutations from parental alleles. We then developed rules to guide data interpretation. We estimated mutation rates in sperm DNA to range from 1.5 ÿ 10⿿2 to 2.2 ÿ 10⿿3 mutations per locus per generation. Large multi-step mutations were observed, providing evidence for complex mutation processes at microsatellites and potentially violating key assumptions in the stepwise mutation model. Our data demonstrate the necessity of actively searching for large mutation events when investigating microsatellite evolution and highlight the need for a thorough understanding of microsatellite amplification characteristics before embarking on SP-PCR or trace DNA studies

    AB - Microsatellite genotyping from trace DNA is now common in fields as diverse as medicine, forensics and wildlife genetics. Conversely, small-pool PCR (SP-PCR) has been used to investigate microsatellite mutation mechanisms in human DNA, but has had only limited application to non-human species. Trace DNA and SP-PCR studies share many challenges, including problems associated with allelic drop-out, false alleles and other PCR artefacts, and the need to reliably identify genuine alleles and/or mutations. We provide a framework for the validation of such studies without a multiple tube approach and demonstrate the utility of that approach with an analysis of microsatellite mutations in the tammar wallaby (Macropus eugenii). Specifically, we amplified three autosomal microsatellites from somatic DNA to characterise efficiency and reliability of PCR from low-template DNA. Reconstruction experiments determined our ability to discriminate mutations from parental alleles. We then developed rules to guide data interpretation. We estimated mutation rates in sperm DNA to range from 1.5 ÿ 10⿿2 to 2.2 ÿ 10⿿3 mutations per locus per generation. Large multi-step mutations were observed, providing evidence for complex mutation processes at microsatellites and potentially violating key assumptions in the stepwise mutation model. Our data demonstrate the necessity of actively searching for large mutation events when investigating microsatellite evolution and highlight the need for a thorough understanding of microsatellite amplification characteristics before embarking on SP-PCR or trace DNA studies

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    KW - STR

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