A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis |
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Authors: | El-Hashemite N; Delhanty JD |
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Institution: | Human Genetics Group, The Galton Laboratory, University College London, UK. |
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Abstract: | Advances in techniques of molecular biology have made possible the
amplification of specific genes from single cells. This has a major
clinical application in preimplantation diagnosis of monogenic disorders.
However, the incidence of allele specific amplification failure (allele
drop out) in heterozygous single cells can lead to misdiagnosis and the
transfer of affected embryos. Few studies have been done to investigate the
actual cause of allele drop out, although some investigators have succeeded
in reducing but not eliminating it. Here we report the efficiency of
amplifying both alleles in heterozygous cells lysed according to two
different protocols. A total of 177 heterozygous cells from carriers of
cystic fibrosis (CF) and haemoglobin C (HbC) were lysed using two different
lysis buffers. Interestingly none of the cells that were lysed with sodium
dodecyl sulphate/proteinase K showed any example of allele specific
amplification failure whereas in those lysed by KOH/dithiothreitol it was
present in 17.6 and 4.7% of the CF and HbC cells respectively. Our results
suggest that the phenomenon of allele specific amplification failure is at
least in part dependent on the lysis buffer used.
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