[Colony and phage-plaque direct sequencings by dye-terminator methods].
Direct sequencing using lambda phage DNA and E. coli colonies with plasmid DNA is a very powerful technique. Almost all of the reported direct sequencing methods involve either radioactive sequencing or fluorescent dye-primer sequencing. We present a direct colony sequencing strategy that uses a dye terminator (BigDye terminator kit) together with dye primer sequencing. We found that single-colony sequencing with the terminator yielded about 500 base pairs of sequence information. Signal strength was not improved when the number of cycles increased to 40. The colony used for the sequencing was estimated to contain about 5.6 x 10(7) cells. In addition, although a single plaque consisted of 2 x 10(6) cells, the pfu was not high enough to read with single-cycle sequencing, and only about 300 base pairs of sequence information were obtained from a single plaque using two cycle-sequencing reactions (re-cycle sequencing). The optimal amounts of the template were 500 ng of purified lambda DNA and 1 x 10(7) pfu of the lambda phage suspension, but with BigDye terminator it was possible to detect as little as 50 ng of purified lambda DNA and 2 x 10(6) pfu for lambda phage suspensions. Thus, colony direct sequencing and plaque direct sequencing are estimated to be very useful for rapid and high-throughout screening of genomic and cDNA libraries.
Sasho
C
C
Center for Research and Education, Room C10, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Obata
H
H
Tanaka
T
T
Okuto
K
K
Yamaguchi
Y
Y
Suzuki
K
K
D004396:Coloring Agents; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007202:Indicators and Reagents; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D017906:Siphoviridae / Q000235:genetics
D004396:Coloring Agents; D004269:DNA, Bacterial; D007202:Indicators and Reagents