Microsatellite genotyping by primer extension and MALDI-ToF mass spectrometry |
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Authors: | Maxime Paris Michael G K Jones |
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Institution: | (1) Western Australian State Agricultural Biotechnology Centre, Murdoch University, 6150 Perth, WA, Australia |
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Abstract: | A novel method for genotyping microsatellite alleles using primer extensions and mass spectrometry analysis has been developed.
Following PCR amplification of the target region, a genotyping primer, with its 3′ end directly flanking the microsatellite
repeats, was extended by a mixture of dNTPs complementary to the nucleotides composing the microsatellite. The length and
molecular weight of extended primers vary with the number of repeats present in the allele(s) under examination. The weights
of extension products were determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF
MS) and used to identify genotypes on the basis of differential primer extension. This is a platform that is not gel based
and is amenable to multiplexing and automation. The technique enables identification of heterozygous progeny in which alleles
differ by a single trinucleotide repeat. The method is illustrated by genotyping a polymorphic microsatellite identified in
an intron of the barleyMlo gene. |
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Keywords: | barley MALDI-ToF microsatellite primer extension simple sequence repeats |
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