Nucleic acid amplification with direct sequencing

A method of amplifying a sequence of interest present within a nucleic acid molecule which comprises:

• A) obtaining a sample of the nucleic acid molecule which contains the sequence of interest;
• B) if the nucleic acid molecule is a single-stranded RNA molecule, treating the sample from step (A) so as to prepare a sample containing a DNA molecule which contains a sequence complementary to the sequence of interest;
• C) treating the sample from step (A) if the nucleic acid molecule is a DNA molecule or the sample from step (B) if the nucleic acid molecule is a single-stranded RNA molecule so as to obtain a further sample containing a single-stranded DNA molecule which contains a sequence complementary to the sequence of interest;
• D) contacting the further sample from step (C) under hybridizing conditions with two or more oligonucleotide primers at least one of which includes at least (a) a promoter and (b) a nucleic acid sequence present within the nucleic acid molecule which contains the sequence of interest, which primer sequence is located adjacent to, and 5′ of, the sequence of interest, and at least one other of which includes a nucleic acid sequence complementary to a sequence present within the nucleic acid molecule which contains the sequence of interest, which primer, sequence is located adjacent to, and 5′ of, the nucleic acid sequence complementary to the sequence within the nucleic acid molecule which contains the sequence of interest, so that at least one of the oligonucleotide primers hybridizes with the single-stranded DNA molecule present in the sample which contains the sequence complementary to the sequence of interest, and at least one other of the oligonucleotide primers hybridizes with the single-stranded DNA molecule which contains the sequence of interest;
• E) treating the resulting sample containing the single-stranded DNA molecules to which the oligonucleotide primers are hybridized from step (D) with a polymerase under polymerizing conditions so that DNA extension products of the oligonucleotide primers are synthesized, some of which DNA extension products contain the sequence of interest and some of which DNA extension products contain the sequence complementary to the sequence of interest;
• F) treating the sample from step (E) so as to separate the DNA extension products from the single-stranded DNA molecules on which they were synthesized and thereby obtain single-stranded DNA molecules;
• G) contacting the resulting sample from step (F) containing the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest under hybridizing conditions, with two or more oligonucleotide primers at least one which includes at least (a) a promoter and (b) a nucleic acid sequence located adjacent to, and 5′ of, the sequence of interest, and at least one other of which includes a nucleic acid sequence complementary to a sequence present within the nucleic acid molecule which contains the sequence of interest, which primer sequence is located adjacent to, and 5′ of, the nucleic acid sequence complementary to the sequence within the nucleic acid molecule which contains the sequence of interest, so that at least one of the oligonucleotide primers DNA molecule present in the sample which contains the sequence complementary to the sequence of interest, and at least one other of the oligonucleotide primers hybridizes with the single-stranded DNA molecule which contains the sequence of interest;
• H) at least treating the sample containing the single-stranded DNA molecules to which the oligonucleotide primers are hybridized from step (G) with polymerase so as to synthesize further DNA extension products, some of which DNA extension products contain the sequence of interest and some of which DNA extension products contain the sequence complementary to the sequence of interest;
• I) repeating steps (F) through (H), as desired;
• J) contacting the sample from step (I) with an RNA polymerase which initiates polymerization from the promoter present, under polymerizing conditions, so as to obtain multiple RNA transcripts of each DNA extension product which contains the sequence complementary to the sequence of interest, thereby amplifying the sequence of interest.

A method of determining an internal nucleotide sequence present within a nucleic acid molecule which contains promoters at both ends of the nucleic acid molecule which comprises:

• a) cleaving the nucleic acid molecule under such conditions so as to generate fragments of the nucleic acid molecule;
• b) if the fragments of the nucleic acid. molecule do not have blunt ends, treating the fragments of the nucleic acid molecule so as to generate blunt ends;
• c) ligating a promoter to the blunt end of a fragment of the nucleic acid molecule obtained in step (a) or (b);
• d) amplifying a sequence of the fragment of the nucleic acid molecule containing the promoter obtained in step (c);
• e) transcribing the amplified fragment of the nucleic acid molecule obtained in step (d);
• and
• f) sequencing the transcript obtained in step (e) thereby determining an internal nucleotide sequence present within nucleic acid molecule.

A method of determining the nucleotide sequence of sequences present within a nucleic acid molecule which are adjacent to areas of known sequence which comprises:

• a) cleaving the nucleic acid molecule adjacent to the sequences of interest under conditions so as to generate fragments of the nucleic acid molecule which contain the sequences of interest;
• b) if the fragments of the nucleic acid molecule do not have blunt ends, treating the fragments of the nucleic acid molecule so as to generate blunt ends;
• c) contacting the fragments containing the sequences of interest obtained in step (a) or (b) with an oligonucleotide containing two different promoter sequences adjacent to each other by blunt end ligation under conditions such that the promoter sequence binds adjacent to the sequence of interest and it is unlikely that the fragment will bind a promoter at both ends;
• d) transcribing the fragments containing the sequences of interest and promoter sequence obtained in step (c) using a polymerase specific to the 5′ promoter sequence;
• e) degrading or removing the fragments which were generated in steps (a) and (b);
• f) synthesizing a nucleic acid sequence complementary to the first sequence to be determined using a downstream primer specific for the known sequence adjacent to the first sequence to be determined;
• g) amplifying the amount of fragments containing the sequence to be determined using a downstream primer specific for the known sequence adjacent to the second sequence to be determined and an upstream primer specific for the second promoter sequence;
• h) transcribing the fragments containing the sequence of interest using a polymerase specific to the second promoter sequence;
• i) sequencing using a downstream primer specific for the third known sequence.