Method for amplifying nucleotide sequence and sequence determination

A method for amplifying a sequence adjacent to a specific sequence in a nucleotide strand, comprising the following steps (1) to (6):

• (1) a step of annealing a first forward primer to the specific sequence, performing an extension reaction with the first forward primer as a starting point, and synthesizing a complementary strand containing a sequence complementary to an adjacent sequence at a 3-end;
• (2) a step of polymerically adding a first deoxynucleotide to the 3-end of the complementary strand obtained in step (1);
• (3) a step of further polymerically adding a second deoxynucleotide to a 3′-end of a polydeoxynucleotide strand comprising the first deoxynucleotide added in step (2);
• (4) a step of annealing a first reverse primer to a site spanning a junction of the 3′-end of the complementary strand and the polydeoxynucleotide strand in a single-stranded DNA formed in step (3), performing an extension reaction with the first reverse primer as a starting point, and synthesizing a double-stranded DNA;
• (5) a step of performing a polymerase chain reaction with the double-stranded DNA synthesized in step (4) as a template by using a second forward primer complementary to the specific sequence and the first reverse primer;
• and
• (6) a step of further performing a polymerase chain reaction with an amplification product obtained in step (5) as a template by using a third forward primer complementary to the specific sequence and a second reverse primer, wherein
• the second forward primer is located closer to the adjacent sequence than the first forward primer in the specific sequence,
• the third forward primer is located closer to the adjacent sequence than the second forward primer in the specific sequence,
• the first reverse primer is a primer containing an adapter primer sequence and an oligonucleotide comprising a third deoxynucleotide in order from a 5′-end,
• the second reverse primer is a primer containing an adapter primer sequence at a 3′-end,
• the first to third deoxynucleotides are each one type of deoxynucleotide selected from four types consisting of deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine,
• the second deoxynucleotide is a deoxynucleotide different from the first deoxynucleotide, and
• the third deoxynucleotide is a deoxynucleotide complementary to the first deoxynucleotide.