Multiplex amplification and separation of nucleic acid sequences using ligation-dependant strand displacement amplification and bioelectronic chip technology

• US 6,238,868 B1
• Filed: 04/12/1999
• Issued: 05/29/2001
• Est. Priority Date: 04/12/1999
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen/Becton Dickinson Partnership
• Inventors: Diver, Jonathan M., Gerrue, Louis O., Carrino, John J.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TAYLOR, JANELL E
• Application Number:
  US09/290,577
• Time in Patent Office: 778 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/25.32, 536/25.4, 536/27, 436/501, 436/94
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00614 Delimitation of the attachment areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• 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: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to devices, methods, and compositions of matter for the multiplex amplification and analysis of nucleic acid sequences in a sample using ligation-dependent strand displacement amplification technologies in combination with bioelectronic microchip technology.

Claims

1. A method for the amplification of one or more target nucleic acid sequences of interest comprising:

a. contacting first and second oligonucleotide ligation probes to adjacent nucleic acid sequences of any one of said target sequences such that a 3′

terminus of one of said probes is juxtaposed to a 5′

terminus of the other of said probes while both the first and second probes are in contact with said target sequence,

b. ligating together said oligonucleotide probes at their respective juxtaposed termini to form a ligated target probe template;

c. using said ligated target probe template in a strand displacement amplification reaction to form amplicons of said ligated target probe template wherein no bumper primers are used in said strand displacement amplification.

2. A method of claim 1 wherein said ligation probe that is not the probe having a juxtaposed 3′

terminus further having a 3′

terminus that is modified such that it cannot have added thereto additional nucleotide triphosphates.

3. A method of claim 1 wherein said strand displacement amplification reaction uses a first amplification primer capable of hybridizing to all ligated target probe templates and a second amplification primer having nucleotide sequences identical to a portion of nucleotide sequences contained on said ligated target probe templates.

4. A method of claim 1 wherein no bumper primers are used in said strand displacement amplification.

5. A method of claim 1 wherein amplicons of said ligated target probe template are detected.

6. A method of claim 5 wherein detection of amplicons is by at least one of fluorescence, chemiluminescence, and electrochemiluminescence.

23. A method of claim 1 wherein the first and second ligation probes are incapable of being ligated together to form the ligated probe template due to a mutation in said target sequence.

7. A method for the multiplex amplification of one or more target nucleic acid sequences of interest comprising:

a. contacting first and second oligonucleotide ligation probes to adjacent nucleic acid sequences of any one of said target sequences such that a 3′

terminus of one of said probes is juxtaposed to a 5′

terminus of the other of said probes while both the first and second probes are in contact with said target sequence,

b. ligating together said oligonucleotide probes at their respective juxtaposed termini to form a ligated target probe template;

c. using said ligated target probe template in a strand displacement amplification reaction to form amplicons of said ligated target probe template;

d. wherein said ligation probe that is not the probe having a juxtaposed 3′

terminus further having a 3′

terminus that is modified such that it cannot have added thereto additional nucleotide triphosphates;

e. wherein no bumper primers are used in said strand displacement amplification reaction;

f. wherein said strand displacement amplification reaction uses a first amplification primer capable of hybridizing to said ligated target probe templates and a second amplification primer having nucleotide sequences identical to a portion of nucleotide sequences of said ligated target probe templates.

8. A method of claim 7 wherein amplicons of said ligated target probe template are detected.

9. A method of claim 8 wherein detection of amplicons is by at least one of fluorescence, chemiluminescence, and electrochemiluminescence.

10. A method for the multiplex amplification of a multiplicity of target nucleic acid sequences of interest using an electronically addressable microchip comprising:

a. for each target nucleic acid sequences of interest,

b. contacting first and second oligonucleotide ligation probes to adjacent nucleic acid sequences of any one of said target sequences such that a 3′

terminus of one of said probes is juxtaposed to a 5′

terminus of the other of said probes while both the first and second probes are in contact with said target sequence,

c. ligating together said oligonucleotide probes at their respective juxtaposed terminal to form a ligated target probe template;

d. using said ligated target probe template in a strand displacement amplification reaction to form amplicons of said ligated target probe template.

11. A method of claim 10 wherein any of said target sequence, ligated target probe template, and amplicons are electronically addressed to any of a plurality of capture sites on said microchip.

12. A method of claim 10 wherein said ligation probe that is not the probe having a juxtaposed 3′

terminus further having a 3′

terminus that is modified such that it cannot have added thereto additional nucleotide triphosphates.

13. A method of claim 10 wherein said strand displacement amplification reaction uses a first amplification primer capable of hybridizing to all ligated target probe templates and a second amplification primer having nucleotide sequences identical to a portion of nucleotide sequences of said ligated target probe templates.

14. A method of claim 10 wherein no bumper primers are used in said strand displacement amplification.

15. A method of claim 10 wherein amplicons of said ligated target probe template are detected.

16. A method of claim 15 wherein detection of amplicons is by at least one of fluorescence, chemiluminescence, and electrochemiluminescence.

17. A method of claim 16 wherein said amplification and detection are carried out either consecutively or simultaneously in relation to one another.

18. A method for the multiplex amplification and detection of a multiplicity of target nucleic acid sequences of interest using an electronically addressable microchip comprising:

a. contacting first and second oligonucleotide ligation probes to adjacent nucleic acid sequences of any one of said target sequences such that a 3′

terminus of one of said probes is juxtaposed to a 5′

terminus of the other of said probes while both the first and second probes are in contact with said target sequence, wherein said ligation probe that is not the probe having a juxtaposed 3′

terminus further having a 3′

terminus that is modified such that it cannot have added thereto additional nucleotide triphosphates;

b. ligating together said oligonucleotide probes at their respective juxtaposed termini to form a ligated target probe template;

c. using said ligated target probe template in a strand displacement amplification reaction to form amplicons of said ligated target probe template, wherein no bumper primers are used in said strand displacement amplification reaction and said strand displacement amplification reaction uses a first amplification primer capable of hybridizing to all ligated target probe templates and a second amplification primer having nucleic acid sequences identical to a portion of nucleic acid sequences of said ligated target probe templates; and

d. detecting said amplicons.

19. A method of claim 18 wherein any of said target sequence, ligated target probe template, and amplicons are electronically addressed to any of a plurality of capture sites on said microchip.

20. A method of claim 18 wherein said amplification and detection are carried out either consecutively or simultaneously in relation to one another.

21. A method of claim 20 wherein detection of amplicons is by at least one of fluorescence, chemiluminescence, and electrochemiluminescence.

22. A method of claim 19 wherein said capture sites have capture probes that comprise any of:

a ligation probe;

a ligation probe having a 3′

terminus that is modified such that it cannot have added thereto additional nucleotide triphosphates;

an amplification primer;

a branched oligonucleotide comprised of a first and second amplification primer where said first amplification primer is capable of hybridizing to all ligated target probe templates and said second amplification primer has a nucleotide sequence identical to a portion of nucleotide sequences of said ligated target probe templates.

24. A method for the amplification of one or more target nucleic acid sequences of interest comprising:

a. contacting first and second oligonucleotide ligation probes to adjacent nucleic acid sequences of any one of said target sequences such that a 3′

terminus of one of said probes is juxtaposed to a 5′

terminus of the other of said probes while both the first and second probes are in contact with said target sequence, wherein said first and second oligonucleotide ligation probes are initially incapable of being ligated together;

b. rendering said first and second oligonucleotide ligation probes which are initially incapable of being ligated together, capable of being ligated together;

c. ligating together said oligonucleotide probes at their respective juxtaposed termini to form a ligated target probe template;

d. using said ligated target probe template in a strand displacement amplification reaction to form amplicons of said ligated target probe template.

25. A method of claim 24 wherein said rendering comprises removing one or more terminal nucleotides from said oligonucleotide probes.

26. A method of claim 25 wherein one or more terminal nucleotides are removed from the 3′

terminus of said second oligonucleotide ligation probe.

27. A method of claim 26 wherein one or more terminal nucleotides are removed by an endonuclease.

28. A method of claim 27 wherein said endonuclease is Endonuclease IV.

29. A method of claim 25 wherein one or more terminal nucleotides are removed from the 5′

terminus of said first oligonucleotide ligation probe.

30. A method of claim 29 wherein one or more terminal nucleotides are removed by an exonuclease.

31. A method of claim 30 wherein said exonuclease is a DNA polymerase.

32. A method of claim 30 wherein one or more terminal nucleotides that are removed are non-complementary to said target sequences.

33. A method for the multiplex amplification of a multiplicity of target nucleic acid sequences of interest using an electronically addressable microchip comprising:

a. for each target nucleic acid sequences of interest;

b. contacting first and second oligonucleotide ligation probes to adjacent nucleic acid sequences of any one of said target sequences such that a 3′

terminus of one of said probes is juxtaposed to a 5′

terminus of the other of said probes while both the first and second probes are in contact with said target sequence, wherein said first and second oligonucleotide ligation probes are initially incapable of being ligated together;

c. rendering said first and second oligonucleotide ligation probes which are initially incapable of being ligated together, capable of being ligated together;

d. ligating together said oligonucleotide probes at their respective juxtaposed termini to form a ligated target probe template;

e. using said ligated target probe template in a strand displacement amplification reaction to form amplicons of said ligated target probe template.

34. A method of claim 33 wherein said rendering comprises removing one or more terminal nucleotides from said oligonucleotide probes.

35. A method of claim 34 wherein one or more terminal nucleotides are removed from the 3′

terminus of said second oligonucleotide ligation probe.

36. A method of claim 35 wherein one or more terminal nucleotides are removed by an endonuclease.

37. A method of claim 36 wherein said endonuclease is Endonuclease IV.

38. A method of claim 34 wherein one or more terminal nucleotides are removed from the 5′

terminus of said first oligonucleotide ligation probe.

39. A method of claim 38 wherein one or more terminal nucleotides are removed by an exonuclease.

40. A method of claim 39 wherein said exonuclease is a DNA polymerase.

41. A method of claim 39 wherein one or more terminal nucleotides that are removed are non-complementary to said target sequences.

42. A kit for carrying out ligation-based SDA reactions for use on a bioelectronic microchip comprising:

a. one or more oligonucleotides for use on a bioelectronic microchip specific for Factor V, Hemochromotosis, or a bacterium, which oligonucleotides comprise amplification primers, bumper primers, capture probes, and/or signal probes selected from the group consisting of Seq. Id. Nos. 20, 21, 31, 32, 45, 46, 51, 52, 57, and 58.

43. A kit according to claim 42 wherein said signal probes are labeled with a detectable label.