Methods for single nucleotide polymorphism detection

• US 6,632,606 B1
• Filed: 06/12/2000
• Issued: 10/14/2003
• Est. Priority Date: 06/12/2000
• Status: Expired due to Fees

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Aclara Biosciences, Inc (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Ullman, Edwin F.
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/592,053
• Time in Patent Office: 1,219 Days
• Field of Search: 435/6, 435/91.2, 435/288.7, 536/25.32, 536/25.4, 422/61
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

Methods and compositions are provided for determining large numbers of single nucleotide polymorphisms in target DNA employing particles having (1) primers complementary to sequences in the target DNA where the next succeeding 3′-nucleotide is a potential single nucleotide polymorphism and coding composition members, where the members are unique for each primer, and (2) differentially labeled terminating nucleotides, where the label permits separation of the terminating nucleotides. Desirably the particles are separated into groups having a common prevalent next succeeding nucleotide. The particles and target DNA are combined under nucleotide extending conditions, the particles separated into groups in accordance with the terminating nucleotide and the coding members identified, so that one knows the sequence and the single nucleotide polymorphism. Various protocols are provided for the determination.

Claims

1. A method for determining a plurality of single nucleotide polymorphisms in a sample of target DNA, the method comprising the steps of:

providing a plurality of primers and coding members attached to particles such that each particle has a unique primer and a unique coding member indicative of the primer, the primer of each particle being complementary to a site on a target DNA in the sample, each site being adjacent to a nucleotide having a single nucleotide polymorphism, the coding member of each particle being attached by a releasable linkage to the particle and having a molecular weight of between 100 daltons and 20 kilodaltons, and the releasable linkage being thermally, photolytically, or chemically labile;

combining the particles with the sample such that each of the primers anneals to the target DNA and is enzymatically extended to form an extended primer by adding to such primer a label indicative of the nucleotide having a single nucleotide polymorphism;

separating particles having extended primers by the labels added to such primers;

releasing the coding members from the particles having extended primers after such separation by thermally, photolytically, or chemically cleaving the releasable linkages thereof; and

electrokinetically separating the coding members to form an electropherogram having a specific band for each coding member so that the plurality of single nucleotide polymorphisms is determined.

2. The method of claim 1 wherein said step of combining includes extending said primer by a polymerase to add a terminating nucleotide having said label.

3. The method of claim 2 wherein said label is a fluorophore.

4. The method of claim 3 wherein said step of separating said particles includes sorting said particles according to said fluorophore of said terminating nucleotide.

5. The method of claim 2 wherein said label is a binding member of a specific binding pair.

6. The method of claim 5 wherein said binding member is selected from the group consisting of biotin, digoxin, fluorescein, and 2,4-dinitrobenzene.

7. The method according to claim 1, 2, 3, 4, 5, or 6 wherein said plurality of said primers and said coding members is between 10 and 50.

8. The method of claim 7 wherein said step of electrokinetically separating said coding members includes separating said coding members by capillary electrophoresis.