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Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
Published on: April 2, 2016
Allele drop-out can occur in alleles differing by a single nucleotide and is not alleviated by preamplification or
S Hahn1, A M Garvin, E Di Naro
1Department of Obstetrics and Gynecology, University of Basel, Switzerland. shahn@uhbs.ch
Abstract:
The ability to analyze the genetic material of single cells by the PCR opens up new prospects for diagnostics. Because only two copies of the genetic template are available for amplification, a problem that frequently arises when examining heterozygous loci in single cells is allele drop-out (ADO). ADO results from the preferential amplification of one of a pair of heterozygous alleles, in which the other allele is totally under-represented. In examining single cells from carriers heterozygous for beta-thalassemia mutations, we have found ADO to occur in alleles differing by a single nucleotide, where either the normal or the mutant genotype was absent. We have found that ADO is not overcome by either increasing the amount of DNA template to 20 pg or by primer extension preamplification (PEP), but rather that the best diagnostic accuracy is obtained by examining multiple single cells and basing a diagnosis on the combined results of such an examination.
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