Method and kits for detection of fragile X specific, GC-rich DNA sequences

• US 6,200,747 B1
• Filed: 01/28/1992
• Issued: 03/13/2001
• Est. Priority Date: 01/28/1992
• Status: Expired due to Term

• Current Assignee: North Shore University Hospital Research Corporation
• Original Assignee: North Shore University Hospital Research Corporation
• Inventors: Brown, W. Ted, Pergolizzi, Robert G., Erster, Susan H.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US07/827,691
• Time in Patent Office: 3,332 Days
• Field of Search: 435/6, 435/91.2, 536/27, 536/235, 536/24.31, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

A method is provided for amplifying and detecting specific GC-rich nucleic acid sequences contained in a nucleic acid or in a mixture of nucleic acids, which includes treating a separate nucleic acid containing the specific sequence with a molar excess of primers and a polymerase and extending the primers in the presence of dATP, dCTP, TTP, and an analogue of dGTP. In one application of the present invention, individuals who are carriers for, or afflicted by, the fragile X syndrome are detected.

Claims

1. A method for ascertaining whether an individual is a carrier for, or afflicted with Fragile X comprising:

a) obtaining a nucleic acid sample from said individual; and

b) amplifying a nucleic acid in said nucleic acid sample by performing a polymerase chain reaction on said sample in a reaction mixture that is substantially free of GTP and dGTP, said reaction mixture comprising;

(1) at least one primer selected from the group consisting of oligonucleotides that are capable of specifically hybridizing within the FMR-1 fragile site, and oligonucleotides capable of specifically hybridizing sufficiently near the FMR-1 GC-rich fragile site to yield a PCR product; and

(2) a nucleotide analogue selected from the group consisting of 7-deaza GTP, inosine, and 7-deaza inosine; and

c) determining the size of said amplified nucleic acid wherein the size indicates the number of CGG repeats in said amplified nucleic acid

d) correlating the size of the amplified nucleic acid and/or number of repeats to whether said individual is a carrier for or afflicted with Fragile X.

2. The method of claim 1 wherein said nucleic acid is selected from the group consisting of DNA and cDNA.

3. The method of claim 1 wherein said primer is present in a molar ratio of at least about 1000:

1 primer;

complementary strand.

4. The method of claim 1 wherein said analysis step further comprises hybridizing said amplified nucleic acid with a nucleic acid sequence comprising a plurality of CGG repeats.

5. The method of claim 1 which comprises at least five PCR cycles.

6. The method of claim 5 which comprises the use of 7-deaza-2′

-GTP.

7. The method of claim 5 which further comprises dispersing said triphosphates and primer in a buffer comprising 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 15 mM MgCl₂, 0.001% (w/v) gelatin, with the addition of 0.5-1 μ

g denatured genomic DNA, 50 pmoles of each oligonucleotide primer, 2.5 units of Taq polymerase, and 10% DMSO.

8. A method according to claim 1 which further comprises analyzing the products of said amplification to ascertain the number of base pairs therein.

9. A method according to claim 1 comprising a first and second primer, wherein the first primer is capable of binding to the first strand of a double stranded nucleic acid containing the FMR-1 gene and the second primer is capable of binding to the second nucleic acid strand of said double stranded nucleic acid, said primers binding to sites that straddle the GCC repeat region of said gene.

10. A kit for determining whether an individual is a carrier for or afflicted with Fragile X, comprising:

a) oligonucleotide primers capable of hybridizing to nucleic acid sequences selected from the group of sequences that consist of (I) sequences that are present within the FMR-1 fragile site, and (II) sequences that are sufficiently near the FMR-1 fragile site to provide an amplified product, wherein said oligonucleotide primers flank the FMR-1 fragile site;

b) a nucleotide analog selected from the group consisting of 7-deaza GTP, inosine, and 7-deaza-inosine; and

c) a PCR reaction mixture which is substantially free of added GTP and dGTP.

11. The kit according to claims 10 wherein said analogue is 7-deaza-2′

-GTP.

12. A kit according to claim 10 which further comprises a probe capable of hybridizing to FMR-1.

13. A kit according to claim 12 wherein said probe comprises a label.

14. A kit according to claim 11 which further comprises a probe consisting essentially of a plurality of 5′

3′

repeat units.

15. A kit according to claim 14 wherein said probe comprises a label.