NUCLEIC ACID SIZE DETECTION METHOD

• US 20100167284A1
• Filed: 04/11/2007
• Published: 07/01/2010
• Est. Priority Date: 10/05/2006
• Status: Active Grant

• 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: Potts, Steven J., Rooke, Jenny Ellen, Huang, Donghui, Strom, Charles M.
• Granted Patent: US 8,697,399 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 for determining the size of a nucleic acid segment in a nucleic acid sample, comprising,

separating fragments of a nucleic acid, said fragments prepared from a nucleic acid-containing sample, wherein said fragments include some which contain said segment and a marker sequence, wherein the marker sequence is not contained within the segment, and said separating into fractions according to size under conditions in which a fragment containing said segment will be located in said fractions according to the size of the segment, and

identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the size of the segment is determined by the fraction in which it is identified.

2. The method of claim 1 wherein the nucleic acid segment is difficult to amplify by polymerase chain reaction.

3. The method of claim 2 wherein said nucleic acid segment is rich in guanine and cytosine bases.

4. A method according to claim 1, wherein said fragments are prepared using one or more restriction endonucleases.

5. A method according to claim 1, wherein said fractions are chosen to separate fragments containing segments having a normal size from fragments containing segments having an abnormal size.

6. A method according to claim 1, wherein said separating is into a number of fractions selected from the group consisting of 2-16.

7. method according to claim 1, wherein said separating is by electrophoresis.

8. A method according to claim 7, wherein said electrophoresis is capillary electrophoresis.

9. A method according to claim 1, wherein said separating is by chromatography.

10. A method according to claim 1, further comprising after said separating step, cleaving all or a portion of the segment from said marker sequence in fragments which contain said segment and said marker sequence.

11. A method according to claim 10, wherein said cleaving is accomplished using a restriction endonuclease.

12. A method according to claim 1, wherein said detecting further comprises amplifying said marker sequence.

13. A method according to claim 12, wherein said amplifying is accomplished with polymerase chain reaction (PCR).

14. A method according to claim 13, wherein said PCR includes a labeled primer.

15. The method of claim 1, wherein said detecting is accomplished using the Taqman PCR detection system.

16. A method according to claim 1, wherein said marker sequence is detected by hybridizing to two probes that hybridize simultaneously to the marker sequence.

17. A method according to claim 1, wherein said segment comprises a tandem repeat region.

18. A method according to claim 17, wherein the size determined is the number of repeats in said tandem repeat region.

20. A method according to claim 17, wherein said tandem repeats are trinucleotide tandem repeats.

21. A method according to claim 17, wherein said tandem repeat is associated with a gene selected from the group consisting of DRPLA, EPM1, FRAXA, FMR1, FRDA, huntingtin, JPH-3, AR, DM1, DM2, SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17, PABPN1 and HOXD13.

22. A method according to claim 21 wherein said tandem repeat is associated with a gene selected from the group consisting of FMR1, FRDA, and DM1.

23. method according to claim 22, wherein said gene is FMR1.

19. A method according to claim 19, wherein said fractions are chosen to separate fragments with a normal number of tandem repeats from fragments containing an abnormal number of tandem repeats.

24. A method of detecting a mutation in a tandem repeat segment of a gene in a nucleic acid sample, 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

separating fragments of a nucleic acid, said fragments prepared from a nucleic acid-containing sample, wherein said fragments include some which contain said tandem repeat segment and a marker sequence, wherein the marker sequence is not contained within the segment, and said separating into fractions according to size under conditions in which a fragment containing the tandem repeat segment will be located in said fractions according to the number of repeats in the tandem repeat segment;

identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, and

comparing the number of repeats in the tandem repeat segment from the nucleic acid sample 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.

25. A method according to claim 24, further comprising,

determining if a mutation is a premutation or a full mutation by comparing the number of repeats in the tandem repeat segment from the nucleic acid sample to the number in the corresponding full mutation allele, wherein a number of repeats greater than the wild type allele but less than the full mutation is indicative of a premutation allele, whereas a number of repeats greater than or equal to the full mutation is indicative of a full mutation allele.

26. A method according to claim 24, wherein said fragments are obtained using one or more restriction endonucleases.

27. A method according to claim 26, wherein said one or more restriction endonucleases are selected from the group consisting of AluI, SphI, BmtI, BlpI, MlyI, and BstNI.

28. A method according to claim 27, wherein said one or more restriction endonuclease is AluI.

29. A method according to claim 27, wherein said one or more restriction endonucleases are SphI and BmtI.

30. A method according to claim 27, wherein said one or more restriction endonucleases are BlpI and MylI.

31. A method according to claim 24, wherein said separating is by electrophoresis.

32. A method according to claim 33, wherein said electrophoresis is capillary electrophoresis.

33. A method according to claim 24, wherein said separating is by chromatography.

34. A method according to claim 24, wherein said fractions are chosen to separate fragments containing tandem repeat segments having a normal number of tandem repeats from fragments containing tandem repeat segments having an abnormal number of repeats.

35. A method according to claim 24, wherein said separating is into a number of fractions selected from the group consisting of 2-16.

36. A method according to claim 24, further comprising after said separating step, cleaving all or a portion of the tandem repeat segment from said marker sequence in fragments which contain said tandem repeat segment and said marker sequence.

37. A method according to claim 36, wherein said cleaving is accomplished using a restriction endonuclease.

38. A method according to claim 37, wherein said restriction endonuclease is selected from the group consisting of BsaWI, HphI, BbvI, BstNI, Hpy1881, SmlI, and BmtI.

39. A method according to claim 24, wherein said detecting further comprises amplifying said marker.

40. A method according to claim 39, wherein said amplifying is accomplished with polymerase chain reaction (PCR).

41. A method according to claim 40 wherein said amplifying employs a detectably labeled primer.

42. A method according to claim 41 said detecting is accomplished with electrophoresis.

43. The method of claim 24, wherein said detecting is accomplished using the Taqman PCR detection system.

44. A method according to claim 24, wherein said marker sequence is detected by hybridizing to two probes that hybridize simultaneously to the marker sequence.

45. A method according to claim 24, wherein said tandem repeats are trinucleotide tandem repeats.

76. The method according to claim 24, wherein the gene is the FMR1 gene.

77. The method according to claim 76, wherein

the identification of a tandem repeat segment in the first fraction indicates the individual has an FMR1 allele with a normal number of tandem repeats;

the identification of a tandem repeat segment in the second fraction indicates the individual has a premutation FMR1 allele; and

the identification of a tandem repeat segment in the third fraction indicates the individual has a full mutation FMR1 allele.

46-64. -64. (canceled)

65. A method for screening male and female individuals for carrier status of mutations in the tandem repeat region of the FMR1 gene, said method comprising

assaying nucleic acids from an individual to determine gender;

and

assaying said nucleic acid to determine the length of the tandem repeat region of the FMR1 gene wherein said determining comprises

amplifying tandem repeat region,

detecting an amplification product, and

determining the number of tandem repeats in the amplification product, wherein

in male individuals;

the presence of an amplification product having less than 55 tandem repeats indicates the individual is not a carrier,

the presence of an amplification product having 55 or more tandem repeats indicates the individual is a carrier, or

in the absence of an amplification product, the carrier status is undetermined; and

in female individuals;

the presence of an amplification product having more than 55 tandem repeats indicates the individual is a carrier;

or

the presence of a single amplification product having less than 55 tandem repeats, the carrier status is undetermined.

66. A method according to claim 65, further comprising, analyzing undetermined individuals to determine carrier status, wherein said analyzing comprises,

separating fragments of a nucleic acid, said fragments prepared from a nucleic acid containing sample of the individual, wherein said fragments include some which contain a tandem repeat segment of the FMR1 gene and a marker sequence, wherein the marker sequence is not contained within the tandem repeat segment, said separating into fractions according to size under conditions in which a fragment containing the tandem repeat segment having a normal number of repeats will be located in a first fraction;

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

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

identifying those fraction(s) containing the segment by detecting the marker sequence, wherein the number of repeats in the tandem repeat segment is determined by the fraction in which it is identified, wherein

in male individuals;

a positive result in the first fraction indicates the individual is not a carrier,

a positive result in the second fraction indicates the individual is a premutation carrier,

a positive result in the third fraction indicates the individual is affected; and

in female individuals;

a positive result in only the first fraction indicates the individual is homozygous for the a normal allele;

a positive result in the second fraction indicates the individual is a premutation carrier; and

a positive result in the third fraction indicates the individual is a full mutation carrier.

67. A method according to claim 65, wherein said assaying nucleic acids to determine gender comprises nucleic acid amplification.

68. A method according to claim 67, wherein said nucleic acid amplification comprises

amplifying a region of the amelogenin gene which produces different sizes of amplification products from the amelogenin gene on the X chromosome and the amelogenin gene on the Y chromosome,

determining the size of the amplification product or products, wherein the presence of one product of a single size indicates the gender is female and the presence of two products of different sizes indicates the gender is male.

69. A method according to claim 67, wherein said amplifying of a region of the amelogenin gene is performed in multiplex with the amplifying of the tandem repeat region of the FMR1 gene.

70. A method according to claim 69, wherein said multiplex amplification further comprises amplifying one or more control sequences.

71. A method according to claim 66, further comprising after said separating step cleaving all or a portion tandem repeat segment from said marker sequence fragments in which contain said tandem repeat segment and said marker sequence.

72-75. -75. (canceled)