Identification of the five most common cystic fibrosis mutations in single cells using a rapid and specific differential amplification system

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 regulato...

Full description

Saved in:
Bibliographic Details
Published in:Molecular human reproduction 1996-03, Vol.2 (3), p.203-207
Main Authors: Scobie, Graeme, Woodroffe, Bridget, Fishel, Simon, Kalsheker, Noor
Format: Article
Language:English
Subjects:
cystic fibrosis
Cystic Fibrosis - diagnosis
Cystic Fibrosis - genetics
Cystic Fibrosis Transmembrane Conductance Regulator - genetics
DNA Fingerprinting
DNA Primers
Heterozygote
Homozygote
Humans
lymphocytes
Mutation
Polymerase Chain Reaction - methods
preimplantation diagnosis
Sensitivity and Specificity
Tyrosine 3-Monooxygenase - genetics
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 δF508, 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
ISSN:1360-9947
1460-2407
DOI:10.1093/molehr/2.3.203