DNA sequencing method and DNA sample preparation method

摘要

The DNA sequencing of the present invention comprises:(1) a step of digesting a sample DNA with a restriction enzyme to obtain a DNA fragment;(2) a step of introducing an oligonucleotide having a definite base sequence into the DNA fragment at the 3' terminus thereof;(3) a step of performing a complementary strand extension reaction, using a labeled primer which comprises a first base sequence portion complementary to at least a part of the base sequence of the oligonucleotide, a second base sequence portion complementary to the recognition base sequence recognized by the restriction enzyme and a third base sequence of a possible combination of 1 to 4 bases, by using, as a template, a single strand of the DNA fragment having the oligonucleotide introduced thereinto to obtain a labeled extended primer having a base sequence complementary to the single strand of the DNA fragment;(4) a step of proceeding:(a) a step of performing a sequencing reaction using the labeled primer by using, as a template, the single strand of the DNA fragment having the oligonucleotide introduced thereinto, and,(b) a step of performing a sequencing reaction using the extended labeled primer by using, as a template, a part of the single strand of the sample DNA having the base sequence of the single strand of the DNA fragment and a contiguous sequence adjacent thereto, or the single strand of the sample DNA; and,(5) a step of subjecting products of the sequencing reaction to electrophoresis to determine the base sequence of the DNA fragment and at least a part of the base sequence of the sample DNA adjacent to the base sequence of the DNA fragment.