A method for isolation of sequences missing in one of two related genomes.
We describe a novel technique for isolation of sequences that are present in one genome (tracer), but absent in another (driver). Tracer DNA, cleaved with Sau 3A and capped with a single stranded PCR adapter, is allowed to hybridize with an excess of sheared biotinylated driver; biotinylated DNA and its hybrids with the tracer are removed by phenol/chloroform extraction after incubation with streptavidin. After several rounds of subtraction the ends of self-annealed tracer molecules from the nonextractable fraction are filled-in with Tag polymerase and amplified, using the single stranded PCR adapter as a primer. The method has been applied to purification of fragments from a 2.9 kb plasmid added to E. coli DNA at equimolar quantity. Plasmid derived fragments (250-1000 bp), initially comprising 1/1400th part of tracer DNA, were purified to homogeneity after two rounds of subtraction followed by PCR.
Lisitsyn
N A
NA
Institute of Molecular Genetics, Shemyakin Institute of Bioorganic Chemistry, Moscow.
Rosenberg
M V
MV
Launer
G A
GA
Wagner
L L
LL
Potapov
V K
VK
Kolesnik
T B
TB
Sverdlov
E D
ED
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial