Methods and compositions for nucleotide analysis

• US 20030082572A1
• Filed: 04/16/2002
• Published: 05/01/2003
• Est. Priority Date: 04/16/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Spier, Eugene, Boyd, Victoria L.
• Granted Patent: US 6,825,010 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2535/138 Amplified fragment length polymorphism [AFLP]
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The invention relates generally to the field of nucleic acid sequence analysis. In certain embodiments, the analysis is genotyping. In certain embodiments, the analysis involves detecting single nucleotide polymorphisms (SNPs). The invention also relates to methods, kits, and computer software for nucleic acid analysis.

Claims

1. A method for identifying at least one target nucleotide within a target sequence comprising:

generating multiple fragments of the target sequence by digesting the target sequence with at least one restriction enzyme;

amplifying the multiple fragments using a first set of primers to generate multiple amplified fragments, wherein the first set of primers comprises a first primer and a second primer;

amplifying at least one subset of the multiple amplified fragments to generate at least one amplified subset of the multiple amplified fragments using a first set of extended primers, wherein the first set of extended primers comprises a first extended primer which comprises the sequence of the first primer and one additional nucleotide on the 3′

end and a second extended primer which comprises the sequence of the second primer and one additional nucleotide on the 3′

end; and

identifying the at least one target nucleotide within a target sequence by identifying the at least one target nucleotide in at least one of the amplified subsets of the multiple amplified fragments.

2. The method according to claim 1, further comprising amplifying the at least one subset of the multiple amplified fragments to generate at least one second subset of amplified fragments using a second set of extended primers, wherein the second set of extended primers comprises a third extended primer which comprises the sequence of the first extended primer and one additional nucleotide on the 3′

end and a fourth extended primer which comprises the sequence of the second extended primer and one additional nucleotide on the 3′

end.

3. The method according to claim 2, further comprising amplifying the at least one second subset of the multiple amplified fragments to generate at least one third subset of amplified fragments using a third set of extended primers, wherein the third set of extended primers comprises a fifth extended primer which comprises the sequence of the third extended primer and one additional nucleotide on the 3′

end and a sixth extended primer which comprises the sequence of the fourth extended primer and one additional nucleotide on the 3′

end.

4. The method of claim 1, further comprising ligating an adaptor to each end of the multiple fragments of the target sequence after the generating the multiple fragments of the target sequence and before the amplifying the multiple fragments, and wherein the first primer comprises a sequence complementary to the adaptor or one end of the multiple fragments of the target sequence and the second primer comprises a sequence complementary to the adaptor on the other end of the multiple fragments of the target sequence.

5. The method according to claim 1, further comprising asymmetrically amplifying the at least one amplified subset of the multiple amplified fragments to generate single-stranded template prior to the identifying the at least one target nucleotide within a target sequence.

6. The method according to claim 5, wherein the asymmetrically amplifying comprises amplifying with a primer comprising the sequence of one of the primers of the first set of primers, and at least one primer which comprises a sequence corresponding to an area immediately adjacent to a nucleotide to be identified;

wherein there is an excess of the primer comprising the sequence of one of the primers of the first set of primers compared to the at least one primer which comprises a sequence corresponding to an area immediately adjacent to a nucleotide to be identified;

to generate a long oligonucleotide primer of a known length, wherein the 3′

end of the long primer is immediately adjacent to the nucleotide to be identified.

7. The method of claim 6, further comprising performing a single base extension reaction using the long oligonucleotide primer and sequence terminators having an indicator specific for the specific nucleotide of the sequence terminator, to generate a single base extension product;

and

identifying nucleotide to be identified.

8. The method according to claim 7, further comprising resolving the single base extension product according to the length of the long oligonucleotide primer.

9. The method according to claim 7, further comprising attaching a mobility modifier to the single base extension product and resolving the single base extension product by its mobility.

10. The method according to claim 7, wherein the single base extension reaction is performed isothermally.

11. A kit for the amplification of amplified fragment length polymorphisms comprising:

a first set of primers comprising a first primer and a second primer used to generate amplified fragment length polymorphisms; and

a first set of extended primers comprising a third extended primer which comprises the sequence of the first extended primer and one additional nucleotide on the 3′

end and a fourth extended primer which comprises the sequence of the second extended primer and one additional nucleotide on the 3′

end.

12. A method for identifying a nucleotide at a SNP site comprising:

digesting genomic DNA with two restriction enzymes to generate multiple fragments;

ligating an adaptor to each end of the multiple fragments of the target sequence;

amplifying the multiple fragments to generate multiple amplified fragments using a first set of primers, wherein the first set of primers comprises a first primer and a second primer, wherein the first primer comprises a sequence complementary to the adaptor on one end of the multiple fragments of the target sequence and the second primer comprises a sequence complementary to the adaptor on the other end of the multiple fragments of the target sequence.

adding a first set of extended primers, wherein the first set of extended primers comprises a first extended primer which comprises the sequence of the first primer and one additional nucleotide on the 3′

end and a second extended primer which comprises the sequence of the second primer and one additional nucleotide on the 3′

end;

amplifying a subset of the multiple amplified fragments using the first set of extended primers to generate a first amplified subset of the multiple amplified fragments;

adding a second set of extended primers, wherein the second set of extended primers comprises a third extended primer which comprises the sequence of the first extended primer and one additional nucleotide on the 3′

end and a fourth extended primer which comprises the sequence of the second extended primer and one additional nucleotide on the 3′

end;

amplifying a second subset of the multiple amplified fragments employing the second set of extended primers to generate a second amplified subset of the multiple amplified fragments;

performing an asymmetric amplification of the second amplified subset of the multiple amplified fragments to generate multiple single stranded fragments;

performing a single base extension reaction using at least one of the multiple single stranded fragments, a single base extension primer, and sequence terminators having an indicator specific for the specific nucleotide of the sequence terminator, wherein the single base extension primer comprises (i) at its 3′

end a sequence complementary to nucleotides immediately adjacent to the SNP site and (ii) a unique sequence identifier, to obtain a single base extension product; and

identifying the nucleotide at the SNP site from the specific indicator.