The reverse DNA sequencing using Bst DNA polymerase.

The reverse DNA sequencing (RDS) [1] is a rapid method used to check the DNA sequences by sequencing them from the opposite orientation. Because the RDS is basically a double stranded sequencing, the quality of the sequence patterns so obtained generally is not as good as those obtained by the single stranded sequencing, and extra bands and higher background are produced more frequently. This paper shows that the RDS could now generate as good sequence patterns as those obtained by sequencing on the single stranded DNA template if Bst DNA polymerase instead of the conventional enzymes, such as the Klenow enzyme, was used in the RDS. Bst DNA polymerase is heat stable (optimum reaction temperature 65 degrees C) and has recently been successfully used in the conventional DNA sequencing. The RDS has recently been further simplified to meet the need of large DNA sequencing projects such as the human genome project. The combination of the simplified RDS and the use of Bst polymerase should be expected to facilitate greatly the work on sequence confirmation and correction.