Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis |
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| Authors: | Findlay, lan Ray, Pierre Quirke, Phil Rutherford, Anthony Lilford, Richard |
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| Affiliation: | 1Institute of Epidemiology and Health Services Research, University of Leeds 34 Hyde Terrace, Leeds LS2 9JT 2Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School Hammersmith Hospital, London 3Centre for Cancer Research and Molecular Pathology Algernon Firth Building, Leeds General Infirmary, Leeds LS1 3EX 4Assisted Conception Unit Leeds General Infirmary, Leeds LS1 3EX, UK |
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| Abstract: | Previously the diagnosis of sex and cystic fibrosis status hasbeen studied on single cells using the polymerase chain reaction(PCR). It has been suggested that allelic drop-out (PCR failureof one allele) and/or preferential amplification (hypo-amplificationof one allele) may contribute to poor reliability and misdiagnosis,although this remains controversial as some reports suggestthat allelic drop-out does not occur. We investigated an improvedmethod of diagnosing sex and cystic fibrosis in single cellsusing a new technology (fluorescent PCR) to determine the baselevel of PCR artefacts (allelic drop-out and preferential amplification)which, in combination with improved sensitivity, should improvePCR reliability and accuracy. Fluorescent PCR gives high reliability( 97%) and accuracy rates (97%) in somatic cells for both sexand cystic fibrosis diagnosis and its lower detection thresholdallows allelic drop-out and preferential amplification to beeasily distinguished. We also achieved high reliability andaccuracy in diagnosing cystic fibrosis in human blastomeres.This study confirms earlier reports of both allelic drop-outand preferential amplification in single cell analysis. We demonstratethat both allelic drop-out and preferential amplification occurin somatic cells and suggest these are separate phenomena. Preferentialamplification appeared common in single cell PCR while allelicdropout apparently occurred at random in each allele. Preferentialamplification was mainly amplification of the larger allele.We suggest that some inaccuracy/misdiagnosis may be due to bothpreferential amplification as well as allelic drop-out. Otherfindings were variability in drop-out between PCR and that amplificationof signals from human blastomeres may be linked to embryo quality.We suggest that allelic drop-out is dependent on the numberof cells within the sample. allelic drop-out/cystic fibrosis/preferential amplification/preimplantation diagnosis/sexing |
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