Simplified mutation detection

• US 6,451,526 B1
• Filed: 01/15/1999
• Issued: 09/17/2002
• Est. Priority Date: 01/20/1998
• Status: Expired due to Fees

• Current Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Original Assignee: Mayo Foundation For Medical Education And Research (Mayo Clinic)
• Inventors: Krajnik, Kelly L., OʼKane, Dennis J., Song, Lu, Heit, John A.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/232,273
• Time in Patent Office: 1,341 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/24.3, 536/24.33
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention involves methods and materials for detecting a mutation in the genome of an organism. Specifically, the invention provides compositions of oligonucleotides and diagnostic kits that can be used to amplify nucleic acid sequences such that if heterologous amplified fragments are present, then stable nucleic acid branching structures are formed and detected from a cell lysate mixture containing the amplified nucleic acid. The formation of stable nucleic acid branching structures is particularly useful for providing a mechanism to detect heterologous alleles verses homozygous alleles since homozygous amplified products do not form stable nucleic acid branching structures. In addition, the invention provides methods of detecting heterologous Factor V alleles as well as homozygous wild-type and homozygous mutant Factor V alleles.

Claims

1. A composition comprising four oligonucleotides, wherein said composition amplifies a region of Factor V genomic sequence during a nucleic acid amplification reaction such that amplified products that are heterologous form stable branching structures, wherein at least two of said four oligonucleotides hybridize to a Factor V genomic sequence between Factor V Leiden mutation site and the Factor V genomic sequence that hybridizes to Primer2, wherein the sequence of Primer2 is set forth in SEQ ID NO:

2, and wherein said region contains the Factor V Leiden mutation site.

2. The composition of claim 1, wherein two of said four oligonucleotides are identical in nucleic acid sequence, each of said two oligonucleotides having a different label attached at the 5′

end.

3. The composition of claim 2, wherein said label comprises a binding agent selected from the group consisting of biotin and digoxigenin.

4. The composition of claim 2, wherein each of said two oligonucleotides has a nucleic acid sequence as set forth in SEQ ID NO:

1.

5. The composition of claim 1, wherein two of said four oligonucleotides have a 5′

portion and a 3′

portion, wherein said 5′

portions of said two oligonucleotides are different in nucleic acid sequence, and said 3′

portions of said two oligonucleotides are identical in nucleic acid sequence, and wherein said 3′

portions hybridize to a sequence flanking said mutation site during said nucleic acid amplification reaction.

6. The composition of claim 5, wherein one of said two oligonucleotides has a nucleic acid sequence as set forth in SEQ ID NO:

4, 5, 6, or 7.

7. The composition of claim 1, wherein said amplified products are less than about 300 bases in length.

8. The composition of claim 1, wherein at least one of said at least two oligonucleotides has a nucleic acid sequence as set forth in SEQ ID NO:

4, 5, 6, or 7.

9. The composition of claim 1, wherein said four oligonucleotides are Dig-Primer1, Bt-Primer1, Primer4, and Primer5, wherein the sequence of Primer1 is set forth in SEQ ID NO:

1, the sequence of Primer4 is set forth in SEQ ID NO;

4, and the sequence of Primer5 is set forth in SEQ ID NO;

5.

10. An oligonucleotide, wherein said oligonucleotide consists of the sequence as set forth in SEQ ID NO:

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

11. A method of detecting heterologous Factor V alleles in an organism, said method comprising:

(a) amplifying a region of Factor V nucleic acid from said organism using oligonucleotides such that amplified products that are heterologous form stable branching structures, wherein said region contains the Factor V Leiden mutation site, and wherein at least two of said oligonucleotides hybridize to a Factor V genomic sequence between said Factor V Leiden mutation site and the Factor V genomic sequence that hybridizes to Primer2, wherein the sequence of Primer2 is set forth in SEQ ID NO;

2; and

(b) detecting the presence or absence of said stable branching structures, wherein the presence of said stable branching structures is indicative of the presence of said heterologous Factor V alleles in said organism.

12. The method of claim 11, wherein LOCI is used for said detecting step.

13. The method of claim 11, wherein a sandwich immunoassay is used for said detecting step.

14. A method of detecting homozygous mutant Factor V alleles in an organism, said method comprising:

(a) amplifying a region of Factor V nucleic acid from said organism using oligonucleotides to form amplification products, wherein said region contains the Factor V Leiden mutation site, and wherein at least two of said oligonucleotides hybridize to a Factor V genomic sequence between said Factor V Leiden mutation site and the Factor V genomic sequence that hybridizes to Primer2, wherein the sequence of Primer2 is set forth in SEQ ID NO;

2;

(b) contacting said amplification products with a solution to form a mixture, wherein said solution comprises homozygous wild-type Factor V products amplified using said oligonucleotides; and

(c) detecting the presence or absence of stable branching structures in said mixture, said stable branching structures being formed between said amplification products and said homozygous wild-type Factor V products, the presence of said stable branching structures being indicative of the presence of said homozygous mutant Factor V alleles in said organism.

15. The method of claim 14, wherein LOCI is used for said detecting step.

16. The method of claim 14, wherein a sandwich immunoassay is used for said detecting step.