Nucleic acid size detection method

• US 8,163,480 B2
• Filed: 10/05/2006
• Issued: 04/24/2012
• Est. Priority Date: 10/05/2006
• Status: Expired due to Fees

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated), U.S. Genomics, Inc (Toxic Reports LLC)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated), U.S. Genomics, Inc (Toxic Reports LLC)
• Inventors: Strom, Charles M., Huang, Donghui, Potts, Steven J., Rooke, Jenny Ellen
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US11/544,293
• Publication Number:
  US 20080124709A1
• Time in Patent Office: 2,028 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: Y10T 436/17 Nitrogen containing

Abstract

The present invention provides methods of determining the size of a particular nucleic acid segment of interest in a sample of nucleic acids through fragmentation of DNA, size fractionation, an optional second fragmentation, and identification using a marker sequence. In particular aspects, an expansion or reduction of tandem repeat sequences can be detected. In further aspects, carriers and individuals afflicted with fragile X syndrome or other diseases associated with tandem repeats can be distinguished from normal individuals.

Claims

1. A method of detecting a mutation in a tandem repeat segment of a gene in a human genomic nucleic acid sample comprising a nucleic acid segment and a marker sequence, wherein said mutation is characterized by a change in the number of repeats compared to the number of repeats in the wild type allele, said method comprising

a) fragmenting the nucleic acid sample into fragments, wherein said tandem repeat segment is associated with a marker sequence in at least one of said fragments;

b) separating said fragments from a) into liquid fractions according to size under conditions in which the fragment(s) containing the tandem repeat segment will be located in said liquid fractions according to the number of repeats in the tandem repeat segment;

c) subjecting said fragments from said one or more liquid fractions of b) to a second fragmentation such that all or a portion of the segment is cleaved from said marker sequence;

d) identifying the segment by amplifying and detecting the marker sequence within said one or more liquid fractions from c), wherein the number of repeats in the tandem repeat segment is determined by the liquid fraction in which it is identified in b), and

e) comparing the number of repeats in the tandem repeat segment determined in d) to the number in the corresponding wild type allele, wherein a number of repeats differing from the number in the wild type allele is indicative of a mutation.

2. A method according to claim 1, wherein said second fragmentation in c) is performed using a restriction endonuclease.

3. A method according to claim 2, wherein said restriction endonuclease used in c) is selected from the group consisting of BsaWI, HphI, BbvI, BstNI, Hpy1881, SmlI, and BmtI.

4. A method according to claim 1, wherein said amplification in d) is accomplished with polymerase chain reaction (PCR).

5. A method according to claim 4, wherein said PCR includes a labeled primer.

6. The method of claim 1, wherein said detecting in d) is accomplished using the Taqman PCR detection system.

7. A method according to claim 1, wherein said tandem repeats are trinucleotide tandem repeats.

8. A method of distinguishing between normal individuals, those that are carriers of fragile X syndrome, and those that are afflicted with fragile X syndrome, said method comprising,

a) fragmenting the nucleic acid in the sample from the individual into fragments, wherein said tandem repeat segment of the FMR1 gene is associated with a marker sequence in at least one of said fragments,

b) separating said fragments from a) into liquid fractions according to size under conditions in which the fragment(s) containing the tandem repeat segment having a normal number of repeats will be located in a first liquid fraction;

fragments containing a tandem repeat segment having a premutation will be located in a second liquid fraction; and

fragments having a tandem repeat region having a full mutation will be located in a third liquid fraction,

c) subjecting said fragments from each of said liquid fractions in b) to a second fragmentation such that all or a portion of the segment is cleaved from said marker sequence;

d) identifying the segment by amplifying and detecting the marker sequence within said liquid fractions from c), wherein the number of repeats in the tandem repeat segment is determined by the liquid fraction in which it is identified in b), and wherein

1) a positive result in only the first liquid fraction indicates the individual is normal;

2) a positive result in the second liquid fraction indicates the individual is a carrier; and

3) a positive result in the third liquid fraction indicates the individual is afflicted with fragile X syndrome.

9. A method according to claim 8, wherein said second fragmentation in c) is performed using a restriction endonuclease.

10. A method according to claim 9, wherein said restriction endonuclease used in c) is selected from the group consisting of BsaWI, HphI, BbvI, BstNI, Hpy1881, SmlI, and BmtI.

11. A method according to claim 8, wherein said amplification in d) is accomplished with polymerase chain reaction (PCR).

12. A method according to claim 11 wherein said PCR includes a labeled primer.

13. The method of claim 8, wherein said detecting in d) is accomplished using the Taqman PCR detection system.