DNA sequencing from single phage plaques using solid-phase magnetic capture.
Many operations encountered in molecular cloning are labor-intensive and time-consuming. One case that is often troublesome is the subcloning of cDNA clones from lambda gt11 phage into plasmid vectors. In situations where several clones have been isolated, time could be saved by a means of assessing insert size and sequence unambiguously without subcloning, particularly where degenerate PCR or low-stringency hybridization approaches are taken to identify multiple members of a gene family. We describe a simple and reliable strategy for efficient sequencing of small amounts of lambda phage DNA, lysates or individual phage plaques. The strategy combines the advantages of universal lambda phage primers, rapid air thermal cycling, streptavidin magnetic bead capture of highly purified single-stranded templates and the unparalleled clarity of T7 DNA polymerase sequence. We routinely obtain 350-500 bases of unambiguous sequence from each reaction. It takes only hours from lifting phage plaques to finishing the sequencing reactions. The method provides an alternative to thermal cycle sequencing that has comparable sensitivity and affords sequence data of much higher clarity.
Wang
S
S
Food and Drug Administration, Rockville, MD.
Krinks
M
M
Moos
M
M
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry*; D005784:Gene Amplification; D008280:Magnetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral