A partial-complementary adapter for an improved and simplified ligation-mediated suppression PCR technique.
Ligation-mediated suppression PCR (LMS-PCR) is a powerful tool for walking in unknown genomic DNA regions from known adjacent sequences. This approach has made it feasible to obtain promoter sequences and to enable researchers to identify full-length gene sequences or isoforms of multigene families. However, the advantages of LMS-PCR can be obviated by the presence of incomplete base modifications on the suppression adapters. We propose here that a 'partial-complementary adapter' is a more reliable suppression adapter, demanding only 5'-end phosphorylation. We also describe a simplified procedure for the easier preparation of PCR templates with very small quantities of DNA and a fast and direct characterization of the suppression-PCR products. A set of practical guidelines is proposed for pre-checking the efficiency of the adapter modification using two model systems: bacteriophage lambda (lambda) and Arabidopsis.
Jeung
Ji-Ung
JU
IRRI-Korea Office, NICS, RDA, Suwon 441-854, Korea.
Cho
Sung Ki
SK
Shin
Jeong Sheop
JS
D017360:Arabidopsis / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D005810:Multigene Family; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020033:Protein Isoforms
D017931:DNA Primers; D009841:Oligonucleotides; D020033:Protein Isoforms; D004247:DNA