Methods and apparatus for determination of length polymorphisms in DNA

• US 6,395,493 B1
• Filed: 08/24/2000
• Issued: 05/28/2002
• Est. Priority Date: 02/25/1998
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Tu, Eugene, Sosnowski, Ronald G.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/645,757
• Time in Patent Office: 642 Days
• Field of Search: 435/6,7.1,91.1,91.2,283.1,285.2,287.2 536/22.1,23.1,24.3,24.31,24.32,24.33
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: H01L 29/6656 using multiple spacer layers, e.g. multiple sidewall spacers

Abstract

Methods and apparatus are provided for the analysis and determination of the nature of repeat units in a genetic target. In one method of this invention, the nature of the repeat units in the genetic target is determined by the steps of providing a plurality of hybridization complex assays arrayed on a plurality of test sites, where the hybridization complex assay includes at least a nucleic acid target containing a simple repetitive DNA sequence, a capture probe having a first unique flanking sequence and n repeat units, where n=0,1,2 . . . , or fractions thereof, being complementary to the target sequence, and a reporter probe having a selected sequence complementary to the same target sequence strand wherein the selected sequence of the reporter includes a second unique flanking sequence and m repeat units, where m=0,1,2 . . . , or fractions thereof, but where the sum of repeat units in the capture probe plus reporter probe is greater than 0 (n+m>0). Concordance and discordance among the hybridization complex assays at the test sites is determined at least in part by hybridization stability. Electronic stringency control may be utilized. Applications include paternity testing, forensic use, and disease diagnostics, such as for the identification of the existence of a clonal tumor.

Claims

1. A method for deterring the nature of repeat units in a genetic target, comprising the steps of:

providing in the presence of an applied electric field a plurality of hybridization complex assays arrayed on a plurality of test sites by;

providing a nucleic acid target containing repetitive DNA sequences,

providing a capture probe having a first unique flanking sequence and n repeat units, where n≧

0, complementary to the target sequence, and

providing a reporter probe having a selected sequence complementary to the same target sequence strand, the reporter including attributes selected from the group consisting of;

a) a second unique flanking sequence,

b) a sequence complementary to a variant region of the target sequence, and

c) a second unique flanking sequence and a sequence complementary to a variant region of the target sequence,

wherein the sequence complementary to a variant region may comprise one or more repeat units,

wherein the sum of the number of repeat units in the capture plus the reporter is greater than zero,

the sequence of the capture probe differing at at least two test sites of the array,

determining concordance and discordance among the hybridization complex assays at the test sites as determined at least in part by hybridization stability and

determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site.

2. The method of claim 1 wherein the location of the concordant test site indicates the nature of the target sequence repeat units by the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site.

3. The method of claim 2 wherein at the concordant test site, the number of repeat units in the target equals the sum of the number of repeat units in the capture probe and the number of repeat units in the reporter probe.

4. The method of claim 1 wherein the location of the concordant test site indicates the nature of the target sequence repeat units by the detection of microvariants in the target sequence.

5. The method of claim 1 wherein the location of the concordant test site indicates the nature of the target sequence repeat units by the number of repeat units and by the detection of microvariants in the target sequence.

6. The method of claim 1 wherein a test site is determined to be concordant when the hybridization complex present there is stable relative to other test sites for the same locus.

7. The method of claim 1 wherein the hybridization stability is determined, at least in part, by electronic stringency (ESC) control.

8. The method of claim 1 further including stringent conditions during the hybridizations of capture probe with the nucleic acid target.

9. The method of claim 1 further including electronic stringency conditions during the hybridization of the reporter probe with the capture probe nucleic acid target hybridization complex.

10. The method of claim 1 wherein the hybridization complex is labeled.

11. The method of claim 1 further including a statistical analysis step.

12. The method of claim 1 further including fluorescent perturbation analysis.

13. The method of claim 10 wherein the label is amplified.

14. The method of claim 1 wherein the hybridization complex is unlabeled.

15. The method of claim 1 further including a serial redundant assay which includes the following steps, after at least the step of determining concordance and discordance,

increasing denaturation stringency to remove said reporter probe at all sites, whether concordant or discordant,

hybridizing a second reporter probe where the number of repeat units in said second reporter probe differs from the number of repeat units in said reporter probe,

wherein the location of the concordant test site indicates the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site,

wherein at the concordant test site, the number of repeat units in the target equals the sum of the number of repeat number in the capture probe and the number of repeat units in the reporter probe,

determining concordant and discordant test sites among the hybridization complex assays at the test sites as determined at least in part by hybridization stability, and

comparing these results with the initial complex hybridization assay for confirmation of target repeat unit number.

16. The method of claim 1 wherein the plurality of hybridization complex assays arrayed at the test sites include at least one site for each allele of a locus.

17. The method of claim 1 wherein each concordant test site identifies the repeat unit number of the target.

18. The method of claim 1 wherein the group of discordant test sites indicates the repeat unit number of the target.

19. The method of claim 1 wherein the concordance and discordance is determined from gradient application of at least one stringency condition selected from the group consisting of:

electronic, chemical and thermal stringency conditions, and resulting change in the signal intensity of the hybridization complex assay.

20. The method of claim 1 wherein the number of test sites is fewer than required to provide a test site for each allele.

21. The method of claim 1 wherein the target material constitutes more than one allele per locus for a mixed sample.

22. The method of claim 1 further including a mixed sample which includes tumor tissue mixed with normal tissue.

23. The method of claim 1 wherein the method is used for identification.

24. The method of claim 1 wherein the method is used for disease diagnostics.

25. The method of claim 1 wherein the method is used for breeding.

26. The method of claim 1 further including a mixed sample having monoclonal and polyclonal cell sources.

27. The method of claim 13 wherein the label is amplified by enzymatic label amplification.

28. A method for determining the nature of repeat units in a genetic target, comprising the steps of:

providing a plurality of hybridization complex assays arrayed on a plurality of test sites by;

providing a nucleic acid target containing repetitive DNA sequences,

providing a capture probe having a first unique flanking sequence and n repeat units, where n≧

0, complementary to the target sequence, and

providing a reporter probe having a selected sequence complementary to the same target sequence strand, the reporter including attributes selected from the group consisting of;

a) a second unique flanking sequence,

b) a sequence complementary to a variant region of the target sequence, and

c) a second unique flanking sequence and a sequence complementary to a variant region of the target sequence,

wherein the sequence complementary to a variant region may comprise one or more repeat units,

wherein the sum of the number of repeat units in the capture plus the reporter is greater than zero,

the sequence of the capture probe differing at at least two test sites of the array,

determining concordance and discordance among the hybridization complex assays at the test sites as determined at least in part by hybridization stability as determined at least in part by applied electronic stringency, and

determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site.

29. The method of claim 28 wherein the location of the concordant test site indicates the nature of the target sequence repeat units by the number of repeat units present in the target, based upon the knowledge of the predetermined sequences of the probes located at that test site.

30. The method of claim 29 wherein at the concordant test site, the number of repeat units in the target equals the sum of the number of repeat units in the capture probe and the number of repeat units in the reporter probe.

31. The method of claim 28 wherein the location of the concordant test site indicates the nature of the target sequence repeat units by the detection of microvariants in the target sequence.

32. The method of claim 28 wherein the location of the concordant test site indicates the nature of the target sequence repeat units by the number of repeat units and by the detection of microvariants in the target sequence.

33. The method of claim 28 wherein a test site is determined to be concordant when the hybridization complex present there is stable relative to other test sites for the same locus.

34. The method of claim 28 further including stringent conditions during the hybridizations of capture probe with the nucleic acid target.

35. The method of claim 28 further including electronic stringency conditions during the hybridization of the reporter probe with the capture probe nucleic acid target hybridization complex.

36. The method of claim 28 wherein the hybridization complex is labeled.

37. The method of claim 28 further including a statistical analysis step.

38. The method of claim 28 further including fluorescent perturbation analysis.

39. The method of claim 36 wherein the label is amplified.

40. The method of claim 28 wherein the hybridization complex is unlabeled.

41. The method of claim 28 further including a serial redundant assay which includes the following steps, after at least the step of determining concordance and discordance,

increasing denaturation stringency to remove said reporter probe at all sites, whether concordant or discordant,

hybridizing a second reporter probe where the number of repeat units in said second reporter probe differs from the number of repeat units in said reporter probe,

wherein the location of the concordant test site indicates the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site,

wherein at the concordant test site, the number of repeat units in the target equals the sum of the number of repeat number in the capture probe and the number of repeat units in the reporter probe,

determining concordant and discordant test sites among the hybridization complex assays at the test sites as determined at least in part by hybridization stability, and

comparing these results with the initial complex hybridization assay for confirmation of target repeat unit number.

42. The method of claim 28 wherein the plurality of hybridization complex assays arrayed at the test sites include at least one site for each allele of a locus.

43. The method of claim 28 wherein each concordant test site identifies the repeat unit number of the target.

44. The method of claim 28 wherein the group of discordant test sites indicates the repeat unit number of the target.

45. The method of claim 28 wherein the concordance and discordance is determined from gradient application of at least one stringency condition selected from the group consisting of:

electronic, chemical and thermal stringency conditions, and resulting change in the signal intensity of the hybridization complex assay.

46. The method of claim 28 wherein the number of test sites is fewer than required to provide a test site for each allele.

47. The method of claim 28 wherein the target material constitutes more than one allele per locus for a mixed sample.

48. The method of claim 28 further including a mixed sample which includes tumor tissue mixed with normal tissue.

49. The method of claim 28 wherein the method is used for identification.

50. The method of claim 28 wherein the method is used for disease diagnostics.

51. The method of claim 28 wherein the method is used for breeding.

52. The method of claim 28 further including a mixed sample having monoclonal and polyclonal cell sources.

53. The method of claim 36 wherein the label is amplified by enzymatic label amplification.