A DNA sequencing strategy.
A modification of Lin's systematic DNA sequencing strategy is described. A method based on the religation of compatible cohesive ends generated by Sau3AI and BamHI was developed. The original procedure has been simplified and the yield of transfectant has been greatly improved. After complete digestion with BamHI and limited cleavage with Sau3AI, the single-cut linear DNA does not have to be separated from the supercoil or the open circular DNA on an agarose gel. After ligation, the DNA is digested with the restriction enzyme between the cloning site and BamHI site again. The original intact DNA is linearized, whereas the deleted subclone is not. Therefore the background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb cDNA fragment containing the haptoglobin-related sequences. It is not necessary to purify large amounts of RF DNA (500 ng is enough) to get enough subclones. A set of subclones was produced in 1 day and the yield of plaques was about sixfold higher than that published.
Lee
Y M
YM
Institute of Biological Chemistry, School of Medicine, National Taiwan University, Taiwan, Republic of China.
Lee
S C
SC
D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D010957:Plasmids; D015183:Restriction Mapping
D004247:DNA