Detection of polymorphisms using thermal cycling with a single oligonucleotide on a DNA sequencing gel.
A method is described for the detection of restriction fragment length polymorphisms (RFLPs) in single copy genes in mammalian cells using one 5'-labelled oligonucleotide. This linear amplification (LA) method employs a single oligonucleotide as primer, which is extended by Taq DNA polymerase up to a restriction enzyme cleavage site. The products are arithmetically amplified by thermal cycling. The size of the products are determined by the sequence of the oligonucleotide and the position of the restriction enzyme cleavage site. Hence, an RFLP can be observed by measuring the size of the products. Polymorphisms which differ in size by a small number of base pairs, as are found in (CA)n repeats, are especially suitable for analysis by the LA procedure since the products are run on DNA sequencing gels. A number of genes were examined by the procedure and all produced a satisfactory signal including GC-rich template. It is proposed that the LA method would be suitable for large-scale genetic linkage analysis. The LA procedure has many advantages including the ability to multiplex signals under the same conditions, and lower cost since only one primer is needed.
Murray
V
V
School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, Australia.
Monchawin
C
C
Cairns
M J
MJ
England
P R
PR
Leigh
D
D
McDonald
B L
BL
D005069:Evaluation Studies as Topic; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
D009838:Oligodeoxyribonucleotides