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

• US 5,658,764 A
• Filed: 06/07/1995
• Issued: 08/19/1997
• Est. Priority Date: 01/28/1992
• Status: Expired due to Term

• Current Assignee: North Shore-Long Island Jewish Research Institute
• 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:
  US08/488,235
• Time in Patent Office: 804 Days
• Field of Search: 435/6, 435/91.2, 536/24.33
• US Class Current: 435/91.2
• 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 a Fragile X mutation comprising:

a) obtaining a nucleic acid sample from the individual, said nucleic acid being DNA or RNA; and

b) amplifying a portion of said nucleic acid by performing a polymerase chain reaction using the nucleic acid as a template in a reaction mixture substantially free of GTP and dGTP and comprising;

(1) at least one oligonucleotide primer selected from the group consisting of primers that hybridize to sequence within the FMR-1 fragile site, primers that hybridize to sequence sufficiently near the FMR-1 GC-rich fragile site to yield a detectable PCR product, and primers that hybridize to both sequence within the FMR-1 fragile site and sequence that abuts the FMR-1 fragile site,

(2) at least one GTP or dGTP nucleotide analogue,

each nucleotide and nucleotide analogue used in the polymerase chain reaction is present at a final molar concentration in the range of from about 150 μ

M to about 320 μ

M;

c) analyzing the size of the PCR product; and

d) correlating the size of the PCR product with the absence of, carrier state of or presence of Fragile X in the individual.

2. The method of claim 1, wherein said reaction mixture further comprises 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1 μ

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

3. The method of claim 1 wherein said DNA is cDNA.

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

1 primer;

template.

5. The method of claim 1 wherein (c) further comprises hybridizing said amplified portion of said nucleic acid with a nucleic acid sequence comprising a plurality of 5'"'"'(CGG)3'"'"' repeat units.

6. A method according to claim 1 wherein said portion of said nucleic acid is amplified by performing at least five PCR cycles.

7. A method according to claim 1 comprising performing the polymerase chain reaction in the presence of a first and second primer, wherein the first primer binds to the first strand of a double stranded nucleic acid containing the FMR-1 gene and the second primer binds 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.

8. A method according to claim 1 wherein said at least one nucleotide primer comprises at least ten nucleotides.

9. A method according to claim 1 wherein said GTP or dGTP nucleotide analogue is selected from the group consisting of 7-deaza GTP, inosine and 7-deaza inosine.

10. A method according to claim 1 wherein said GTP or dGTP nucleotide analogue is 7-deaza-2'"'"' GTP.

11. A method according to claim 10 wherein the polymerase chain reaction is performed in a reaction mixture containing a final concentration of 320 uM of each of said nucleotides and nucleotide analogue.

12. The method according to claim 1 wherein the PCR product contains the entire GC-rich region present in the FMR-1 gene of the individual.

13. A kit for determining whether an individual carries a Fragile X mutation comprising:

a) at least one oligonucleotide primer selected from the group consisting of primers that hybridize to sequence within the FMR-1 fragile site, primers that hybridize to sequence sufficiently near the FMR-1 GC-rich fragile site to yield a detectable PCR product, and primers that hybridize to both sequence within the FMR-1 fragile site and sequence that abuts the FMR-1 fragile site,

b) at least one GTP or dGTP nucleotide analogue; and

c) a reaction mixture which is substantially free of GTP and dGTP, wherein the final molar concentration of each nucleotide and nucleotide analogue present in said reaction mixture is in the range of from about 150 μ

M to about 320 μ

M.

14. A kit according to claim 13 wherein said GTP or dGTP nucleotide analogue is selected from the group consisting of 7-deaza GTP, inosine and 7-deaza inosine.

15. A kit according to claim 14 wherein the final molar concentration of each of said nucleotide and nucleotide analogues is 320 uM.

21. The kit according to claim 13 wherein the PCR product contains the entire GC-rich region present in the FMR-1 gene of the individual.

16. A kit according to 13 wherein the GTP or dGTP nucleotide analogue is 7-deaza-2'"'"'-GTP.

17. A kit according to claim 16 which further comprises a probe capable of hybridizing to FMR-1 sequence.

18. A kit according to claim 17 wherein said probe comprises a label.

19. A kit according to claim 16 which further comprises a probe consisting essentially of a plurality of 5'"'"'(CGG)3'"'"' repeat units.

20. A kit according to claim 19 wherein said probe comprises a label.