Molecular Human Reproduction Vol. 2, NUMBER 3 pp. 203-207, 1996
© European Society of Human Reproduction and Embryology 1996
research-article |
Identification of the five most common cystic fibrosis mutations in single cells using a rapid and specific differential amplification system
1Clinical Chemistry, Dept. of Clinical Laboratory Sciences, Queen's Medical Centre, University Hospital Nottingham NG7 2UH 2NURTURE, Nottingham University, Queen's Medical Centre Nottingham, UK
To whom correspondence should be addressed at: 3To whom correspondence should be addressed
We describe a rapid and specific differential amplification system which can detect five of the most common cystic fibrosis mutations from a single cell. In the first round of the polymerase chain reaction (PCR), regions of exons 4, 10 and 11 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing the mutations 
F508, G551D, R553X, G542X and 621÷1G>T were co-amplified in a single multiplex PCR. To identify potential contamination, we included external amplification primers for the polymorphic human tyrosine hydroxylase (HUMTH01) locus as a fingerprint for the sample. In the second round of PCR, detection of any of the five mutations was achieved using the amplification refractory mutation system (ARMS) in two separate reactions, each containing nested amplification primers for either wild type or mutant sequence. A separate second round PCR for the fingerprinting was performed with nested HUMTH01 PRIMERS. Using this procedure we have successfully and accurately detected five cystic fibrosis mutation in 30 single cells with a failed amplification rate of 7% and a contamination rate of 4.6% and that PCR failure or possible contamination will also be easily detected. This procedure allows detection of the five most common point mutations and small detetions responsible for cystic fibrosis from a single cell in <8 h which could be applicable to preimplantation diagnosis in human embryos.
cystic fibrosis/preimplantation diagnosis/lymphocytes
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