Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.

AUTOR(ES)

Pena, S D

RESUMO

Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA.

Documentos Relacionados

• Nonenzymatic sequence-specific cleavage of single-stranded DNA.
• Nonenzymatic sequence-specific methyl transfer to single-stranded DNA.
• Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level
• Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars.
• Photoaffinity polyamines: sequence-specific interactions with DNA.