Compositions and methods for repetitive use of genomic DNA

• US 6,913,884 B2
• Filed: 08/16/2001
• Issued: 07/05/2005
• Est. Priority Date: 08/16/2001
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Chee, Mark S., Stuelpnagel, John R.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/931,285
• Publication Number:
  US 20030036064A1
• Time in Patent Office: 1,419 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.1, 435/287.2, 435/91.51, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention relates to detection or genotyping (or other sample analysis) of target nucleic acids following immobilization of the target nucleic acids onto a surface. The target nucleic acids can be re-used multiple times, thus conserving sample materials and simplifying sample preparation.

Claims

1. A method comprising:

a) providing a composition camprising first primers and at least 10 different target nucleic acids, wherein either said first primers or said at least 10 different target nucleic acids are immobilized to at least one solid support;

b) performing a first analysis of said at least 10 different target nucleic acids, said first analysis comprising;

i) contacting said first primers with said at least 10 different target nucleic acids whereby at least one of said first primers hybridizes with said at least 10 different target nucleic acids;

ii) removing unhybridized first primers; and

iii) contacting said hybridized first primers with an enzyme such that said hybridized first primers are modified forming first modified primers, whereby a sinnal is obtained from said first analysis and whereby said at least 10 different target nucleic acids is not consumed, and

c) performing a second analysis of said at least 10 different target nucleic acids, comprising;

i) contacting second primers with said at least 10 different target nucleic acids whereby at least one of said second primers hybridizes with said at least 10 different target nucleic acids;

ii) removing unhybridized second primers; and

iii) contacting said hybridized second primers with an enzyme such that said hybridized second primers are modified forming second modified primers, whereby a signal is obtained from said second analysis and wherein a signal obtained from said second analysis is not diminished more than 40% compared to a signal obtained from said first analysis.

2. The method according to claim 1, further comprising detecting said first and second modified primers.

3. The method according to claim 1, further comprising amplifying said first and second modified primers to form first and second amplicons.

4. The method according to claim 3, further comprising detecting said first and second amplicons.

5. The method according to claim 4, wherein said first and second amplicons comprise labels.

6. The method according to claim 5, wherein said first and second amplicons are labeled during said amplification.

7. The method according to claim 1 , wherein said target nucleic acid comprises genomic DNA.

8. The method according to claim 7, wherein said genomic DNA comprises at least one copy of the genomic DNA from an organism.

9. The method according to claim 8, wherein said organism is selected from humans, mice, pigs, cows, bacteria, viruses or plants.

10. The method according to claim 1, wherein at least one of said first and second primers comprises an adapter sequence.

25. The method according to claim 1, 11 or 22, wherein said target nucleic acid is immobilized on at least one solid support.

26. The method according to claim 1, 11 or 22, wherein said first primers are immobilized on at least one solid support.

27. The method according to claim 1, 11 or 22, wherein at least 10 different target nucleic acids are analyzed in a single reaction.

29. The method according to claim 1, 11 or 22, wherein at least 100 different target nucleic acids are analyzed in a single reaction.

11. A method comprising:

a) providing a composition comprising first primers and a target nucleic acid wherein said first primers are ligation primers;

b) hybridizing said first ligation primers with said target nucleic acid to form first ligation complexes, whereby said first ligation primers hybridize to said target nucleic acid comprising a first target sequence;

c) removing unhybridized ligation primers;

d) contacting said first ligation complexes with a ligation enzyme, whereby when said first ligation primers are complementary to said first target sequence, said ligation enzyme ligates said first ligation primers generating first ligation products, whereby a signal is obtained from said first ligation products;

e) removing said first ligation products from said target nucleic acid;

f) hybridizing said target nucleic acid with second ligation primers to form second ligation complexes, whereby said second ligation primers hybridize to said target nucleic acid comprising a second target sequence, and

g) contacting said second ligation complex with a ligation enzyme, whereby when said second ligation primers are complementary to said second target sequence, said ligation enzyme ligates said second ligation primers generating second ligation products, whereby a signal is obtained from said second ligation products and wherein a signal obtained from said second ligation products is not diminished more than 40% compared to a signal obtained from said first ligation products.

12. The method according to claim 11, further comprising:

h) contacting said first and second ligation products with amplification primers, nucleotides and an amplification enzyme to form first and second amplicons; and

i) detecting said first and second amplicons.

13. The method according to claim 12, wherein said amplification enzyme is a DNA polymerase and said nucleotides are dNTPs.

14. The method according to claim 12, wherein said amplification enzyme is an RNA polymerase and said nucleotides are NTPs.

15. The method according to claim 11 further comprising amplifying said first and second ligation products to form first and second amplicons.

16. The method according to claim 15, further comprising detecting said first and second amplicons.

17. The method according to claim 16, wherein said first and second amplicons comprise labels.

18. The method according to claim 17, wherein said first and second amplicons are labeled during said amplification.

19. The method according to claim 11, wherein said target nucleic acid comprises genomic DNA.

20. The method according to claim 19, wherein said genomic DNA comprises at least one copy of the genomic DNA from an organism.

21. The method according to claim 20, wherein said organism is selected from humans, mice, pigs, cows, bacteria, viruses or plants.

24. The method according to claim 11 or 22, wherein said target nucleic acid is genomic DNA.

28. The method according to claim 11 or 22, wherein at least 50 different target nucleic acids are analyzed in a single reaction.

22. A method of reusing a target nucleic acid comprising:

a) providing a composition comprising first primers and a target nucleic acid, wherein either said first primers or said target nucleic acid are immobilized on at least one solid support;

b) performing a first analysis of said target nucleic acid, said first analysis comprising;

i) contacting said first primers with said target nucleic acid whereby at least one of said first primers hybridizes with said target nucleic acid;

ii) removing unhybridized first primers; and

iii) contacting said hybridized first primers with an enzyme such that said hybridized first primers are modified forming first modified primers, whereby a signal is obtained from said first analysis and whereby said target nucleic acid is not consumed; and

c) reusing said target nucleic acid in a second analysis, whereby a signal is obtained from said second analysis and wherein a signal obtained from said second analysis is not diminished more than 40% compared to a signal obtained from said first analysis.

23. The method according to claim 22, wherein said target nucleic acid is reused at least five times.

30. The method according to claim 22, further comprising amplifying said first modified primers to form first amplicons.

31. The method according to claim 30, further comprising detecting said first amplicons.

32. The method according to claim 31, wherein said first amplicons comprise labels.

33. The method according to claim 32, wherein said first amplicons are labeled during said amplification.

34. The method according to claim 32, wherein said target nucleic acid comprises genomic DNA.

35. The method according to claim 34, wherein said genomic DNA comprises at least one copy of the genomic DNA from an organism.

36. The method according to claim 35, wherein said organism is selected from humans, mice, pigs, cows, bacteria, viruses or plants.