Supported PCR: an efficient procedure to amplify sequences flanking a known DNA segment.
We describe a novel modification of the polymerase chain reaction for efficient in vitro amplification of genomic DNA sequences flanking short stretches of known sequence. The technique utilizes a target enrichment step, based on the selective isolation of biotinylated fragments from the bulk of genomic DNA on streptavidin-containing support. Subsequently, following ligation with a second universal linker primer, the selected fragments can be amplified to amounts suitable for further molecular studies. The procedure has been applied to recover T-DNA flanking sequences in transgenic tomato plants which could subsequently be used to assign the positions of T-DNA to the molecular map of tomato. The method called supported PCR (sPCR) is a simple and efficient alternative to techniques used in the isolation of specific sequences flanking a known DNA segment.
Rudenko
G N
GN
Free University, Department of Genetics, Amsterdam, Netherlands.
Rommens
C M
CM
Nijkamp
H J
HJ
Hille
J
J
D001483:Base Sequence; D001710:Biotin; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012231:Rhizobium / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D001710:Biotin; D004247:DNA