A robust method for detecting single-nucleotide changes as polymorphic markers by PCR.
Numerous techniques in plant molecular genetic analysis, such as mapping and positional cloning techniques, rely on the availability of molecular markers that can differentiate between alleles at a particular locus. PCR-based cleaved amplified polymorphic sequences (CAPS) markers have been widely used as a means of rapidly and reliably detecting a single-base change that creates a unique restriction site in one of a pair of alleles. However, the majority of single-nucleotide changes do not create such sites and thus cannot be used to create CAPS markers. In this paper, a modification of the CAPS technique that allows detection of most single-nucleotide changes by utilizing mismatched PCR primers is described. The mismatches in the PCR primers, in combination with the single-nucleotide change, create a unique restriction site in one of the alleles.
Michaels
S D
SD
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Amasino
R M
RM
D001483:Base Sequence; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific; D005819:Genetic Markers; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015183:Restriction Mapping
D017931:DNA Primers; D005819:Genetic Markers; C060413:endodeoxyribonuclease MwoI; D015252:Deoxyribonucleases, Type II Site-Specific