Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.
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.
Pena
S D
SD
Núcleo de Genética Médica de Minas Gerais (GENE/MG), Belo Horizonte, Brazil.
Barreto
G
G
Vago
A R
AR
De Marco
L
L
Reinach
F C
FC
Dias Neto
E
E
Simpson
A J
AJ
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004252:DNA Mutational Analysis / Q000379:methods* / Q000706:statistics & numerical data; D017931:DNA Primers / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics; D003919:Diabetes Insipidus / Q000235:genetics; D005260:Female; D008040:Genetic Linkage; D014644:Genetic Variation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009218:Myosins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D017483:Receptors, Vasopressin / Q000235:genetics; D012680:Sensitivity and Specificity; D014960:X Chromosome
D017931:DNA Primers; D004272:DNA, Mitochondrial; D017483:Receptors, Vasopressin; D009218:Myosins