Automated sanger DNA sequencing with one label in less than four lanes on gel

Abstract

Novel Sanger dideoxy sequencing with only one fluorescent dye label for the four bases of one clone and sequence determination in two lanes on polyacrylamide gel is presented, loading A > G in one lane and T > C in the other. Sequencing reactions for the two bases in each lane carried out in one tube. At present the ratio of ddATP:ddGTP and ddTTP:ddCPT is set to 5 : 1 in the two tubes. Distinction between the two bases in one lane is done by comparing the different magnitudes of the perks. This method increases the capacity since more clones may be run simultaneously on one gel, while keeping the reliability and simplicity that comes with the use of only one fluorescent dye for the four bases of one clone. At present about 200 bases are determined with the one-dye two-lane method on the EMBL's automated fluorescent DNA sequencer, using T7 DNA polymerase. The error rate in the deduced sequence is about 1%. The technique is used for the determination of overlaps in mapping projects. In principle, it is possible to determine the sequence with one dye in only one lane on the gel by choosing the proper ddNTP ratios for all four bases, carrying out reactions in one tube and applying the product in one lane, but the error rate for this one-lane method seems too high at present and further improvements in the uniformity of peaks obtainable with the T7 DNA polymerase or other enzymes are required.