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Haplotyping pp 3–22Cite as

Haplotyping of Heterozygous SNPs in Genomic DNA Using Long-Range PCR

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1551))

Abstract

To study meiotic recombination products, cis- or trans-association of disease polymorphisms, or allele-specific expression patterns, it is necessary to phase heterozygous polymorphisms separated by several kilobases. Haplotyping using long-range polymerase chain reaction (PCR) is a powerful, cost-effective method to directly obtain the phase of multiple heterozygous sites with standard laboratory equipment in a handful of loci for many samples. The method is based on the amplification of large genomic DNA regions (up to ~40 kb) with a reaction mixture that combines a proofreading polymerase with allele-specific primer pairs that preferentially amplify matched templates. The analysis of two heterozygous SNPs requires four reactions, each containing one of the four possible allele-specific primer combinations (two forward and two reverse primers), with the mismatches occurring at the 3′ ends of the primers. The two correct primer combinations will more efficiently elongate the matching alleles than the alternative alleles, and the difference in amplification efficiency can be monitored with real-time PCR.

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Acknowledgements

This work was supported by the “Austrian Science Fund” (FWF) P25525-B13 and P23811-B12 to I.T-B., and DOC Fellowships of the Austrian Academy of Sciences to B.A. and A.H.

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Authors and Affiliations

  1. Institute of Biophysics, Johannes Kepler University, Gruberstraße 40, Linz, 4020, Austria

    Barbara Arbeithuber, Angelika Heissl & Irene Tiemann-Boege

Authors
  1. Barbara Arbeithuber
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  2. Angelika Heissl
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  3. Irene Tiemann-Boege
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Corresponding author

Correspondence to Irene Tiemann-Boege .

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Editors and Affiliations

  1. Johannes Kepler University Institute of Biophysics, Linz, Austria

    Irene Tiemann-Boege

  2. Vetmeduni Vienna InstitutPopulationsgenetik, Wien, Austria

    Andrea Betancourt

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Arbeithuber, B., Heissl, A., Tiemann-Boege, I. (2017). Haplotyping of Heterozygous SNPs in Genomic DNA Using Long-Range PCR. In: Tiemann-Boege, I., Betancourt, A. (eds) Haplotyping. Methods in Molecular Biology, vol 1551. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6750-6_1

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  • DOI: https://doi.org/10.1007/978-1-4939-6750-6_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6748-3

  • Online ISBN: 978-1-4939-6750-6

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