Methods for genetic analysis of DNA to detect sequence variances

• US 7,659,054 B1
• Filed: 10/25/2000
• Issued: 02/09/2010
• Est. Priority Date: 05/23/2000
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Nuvelo, Inc. (Arca Biopharma, Inc.)
• Inventors: Olson, Jeffrey, Stanton, Vincent P. Jr.
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US09/697,028
• Time in Patent Office: 3,394 Days
• Field of Search: 435/6, 435/91.2, 536/24.33
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides

Abstract

The presently claimed invention concerns a method for biasing the amplification of the DNA molecules in a sample such that a nucleic acid molecule having a specific nucleotide at a selected position (e.g., a polymorphic site) is preferentially amplified relative to an otherwise identical nucleic acid molecule not having the specific nucleotide at the selected position. The method entails amplification of target nucleic acid molecules using a pair of primers one of which causes the incorporation into the amplification product of a sequence that interferes with amplification of nucleic acid molecules not having the specific nucleotide at the selected position. The method is useful for preferentially amplifying at least a portion of one allele of a gene relative to another, different allele of the gene in a sample containing both alleles of the gene.

Claims

1. A method for biasing a DNA amplification reaction such that a first nucleic acid molecule having a first nucleotide present at a polymorphic site is amplified to a greater extent than a second nucleic acid molecule having a second, different nucleotide present at the polymorphic site, comprising

(a) contacting a sample of DNA comprising at least the first nucleic acid molecule with two amplification primers that hybridize to both the first nucleic acid molecule and the second nucleic acid molecule at locations which flank the polymorphic site such that neither the first primer nor the second primer hybridizes to the polymorphic site, one of the two primers including a 5′

portion which, when incorporated into an amplification product, will upon further amplification yield products that form a stable stem-loop structure, the stem of which is perfectly matched and includes the polymorphic site only when the second nucleotide is present at the polymorphic site, but not when the first nucleotide is present at the polymorphic site; and

(b) carrying out an amplification reaction, whereby the first nucleic acid molecule is amplified to a greater extent than the second nucleic acid molecule.

2. The method of claim 1, wherein the DNA is single-stranded DNA.

3. The method of claim 1, wherein the DNA is double-stranded DNA.

4. The method of claim 1 further comprising separately carrying out steps (a) and (b) for each of a plurality of polymorphic sites.

5. The method of claim 1 wherein the sample of DNA comprises mammalian DNA.

6. The method of claim 5 wherein the sample of DNA comprises human DNA.

7. A method for haplotyping a first nucleic acid molecule having a first nucleotide present at a polymorphic site present in a sample of DNA comprising at least the first nucleic acid molecule and a second nucleic acid molecule having a second, different nucleotide present at the polymorphic site, comprising:

(a) contacting a sample of DNA comprising at least the first nucleic acid molecule with two amplification primers that hybridize to both the first nucleic acid molecule and the second nucleic acid molecule at locations which flank the polymorphic site such that neither the first primer nor the second primer hybridizes to the polymorphic site, one of the two primers including a 5′

portion which, when incorporated into an amplification product, will upon further amplification yield products that form a stable stem-loop structure, the stem of which is perfectly matched and includes the polymorphic site only when the second nucleotide is present at the polymorphic site, but not when the first nucleotide is present at the polymorphic site;

(b) carrying out an amplification reaction, whereby the first nucleic acid molecule is amplified to a greater extent than the second nucleic acid molecule to create an amplified DNA sample; and

(c) determining the nucleotide sequence of at least a portion of the DNA present in the amplified DNA sample.