United States Patent 6,475,736
Stanton, Jr. November 5, 2002
--------------------------------------------------------------------------------
Methods for genetic analysis of DNA using biased amplification of polymorphic sites
Abstract
Methods for determining genotypes and haplotypes of genes are described. Also described are single nucleotide polymorphisms and haplotypes in the ApoE gene and methods of using that information.
--------------------------------------------------------------------------------
Inventors: Stanton, Jr.; Vincent P. (Belmont, MA)
Assignee: Variagenics, Inc. (Cambridge, MA)
Appl. No.: 696998
Filed: October 25, 2000
Current U.S. Class: 435/6; 435/91.2; 536/24.33; 536/22.1
Intern'l Class: C12Q 001/68; C12P 019/34; C07H 021/04; C07H 019/00
Field of Search: 435/6,91.2 536/24.33,22.1
--------------------------------------------------------------------------------
References Cited [Referenced By]
--------------------------------------------------------------------------------
U.S. Patent Documents
5573905 Nov., 1996 Lerner et al. 435/6.
5695937 Dec., 1997 Kinzler et al. 435/6.
5876933 Mar., 1999 Perin 435/6.
Foreign Patent Documents
WO 00/31300 Jun., 2000 WO.
Other References
Laken SJ et al. Genotyping by mass spectrometric analysis of short DNA fragments. Nature Biotechnology, vol. 16 : 1352-1356, 1998.
Primary Examiner: Benzion; Gary
Assistant Examiner: Chunduru; Suryaprabha
Attorney, Agent or Firm: Fish & Richardson, P.C.
--------------------------------------------------------------------------------
Parent Case Text
--------------------------------------------------------------------------------
RELATED APPLICATION
This application claims the benefit of Stanton et al., U.S. Provisional Application No. 60/206,613, filed May 23, 2000, entitled METHODS FOR GENETIC ANALYSIS OF DNA, which is hereby incorporated by reference in its entirety, including drawings.
--------------------------------------------------------------------------------
Claims
--------------------------------------------------------------------------------
What is claimed is:
1. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, the method comprising:
a) amplifying a portion of the target nucleic acid molecule comprising the selected polymorphic site using a first primer and a second primer, the second primer containing a recognition site for a first restriction enzyme and a recognition site for a second restriction enzyme, to generate amplification product containing a recognition site for the first restriction enzyme and a recognition site for the second restriction enzyme such that digestion of the amplification product with the first restriction enzyme and the second restriction enzyme generates a nucleic acid fragment containing the selected polymorphic site;
b) digesting the amplification product with the first restriction enzyme and the second restriction enzyme to generate a nucleic acid fragment containing the selected polymorphic site; and
c) analyzing the nucleic acid fragment to identify the nucleotide present at the selected polymorphic site.
2. The method of claim 1 wherein the second primer comprises at least one nucleotide sequence that is not present in the target nucleic acid molecule.
3. The method of claim 2 wherein the second primer comprises 5' nucleotide sequence that is complementary to a first portion of the target nucleic acid molecule, a 3' nucleotide sequence that is complementary to a second portion of the target nucleic acid molecule, and a nucleotide sequence that is not complementary to the target nucleic acid molecule.
4. The method of claim 1 wherein the first restriction enzyme is a type IIS restriction enzyme.
5. The method of claim 1 wherein the step of analyzing the nucleic acid fragment to identify the nucleotide present at the selected polymorphic site comprises subjecting the nucleic acid fragment to mass spectrometry. |
