Universal primer sequence for multiplex DNA amplification

• US 6,207,372 B1
• Filed: 02/10/1998
• Issued: 03/27/2001
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Esoterix Genetic Laboratories LLC (Laboratory Corporation Of America Holdings)
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Shuber, Anthony P.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/983,466
• Time in Patent Office: 1,141 Days
• Field of Search: 536/24.3, 435/91.2, 435/91.1, 435/6
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention provides primer that allow simultaneous amplification of multiple DNA target sequences present in a DNA sample. Further provided are methods for detecting multiple defined target DNA sequences in a DNA sample. Methods for high-throughput genetic screening are also provided. In yet another aspect, the present invention provides single-stranded oligonucleotide DNA primers for amplification of a target DNA sequence in a multiplex polymerase chain reaction.

Claims

1. A multiplicity of single-stranded oligonucleotide DNA primers for simultaneous amplification of multiple target DNA sequences under a single set of reaction conditions in a multiplex polymerase chain reaction (PCR), said primers having a 5′

X domain and a 3′

Y domain, wherein

a) each said 5′

X domain comprises a sequence that does not hybridize to said multiple target sequences;

b) the melting temperature of a hybrid between X and its complement in the absence of other sequences is greater than about 60°

C.; and

c) Y comprises a sequence contained within or flanking said target sequence or its complement

d) each of said primers being capable of annealing specifically with it cognate target sequence under uniform high stringency annealing conditions during said amplification.

2. The primer of claim 1, wherein X comprises the sequence 5′

-GCGGTCCCAAAAGGGTCAGT-3′

(SEQ ID NO;

1).

3. The primer of claim 1, wherein X comprises the sequence 5′

-CGCCAGGGTTTTCCCAGTCA-3′

(SEQ ID NO;

2).

4. The primer of claim 1, wherein X comprises the sequence 5′

-CGCCAGGGGGGGCCCAGTCA-3′

(SEQ ID NO;

3).

5. The primer of claim 1, wherein X comprises the sequence 5′

-CGGCAGCGGGGCCCAGTCCA-3′

(SEQ ID NO;

4).

6. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGGCCGGGGCCCATCCCA-3′

(SEQ ID NO;

5).

7. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGGCCGGGGCCATCTCAA-3′

(SEQ ID NO;

6).

8. The primer of claim 1, wherein X comprises the sequence 5′

-GAGGCCGGTGGCCATGTCAA-3′

(SEQ ID NO;

7).

9. The primer of claim 1, wherein X comprises the sequence 5′

-TAGGCGCGTGGCCATGTCAA-3′

(SEQ ID NO;

8).

10. The primer of claim 1, wherein X comprises the sequence 5′

-TAGGCCCGTGGCCATGTTAA-3′

(SEQ ID NO;

9).

11. The primer of claim 1, wherein X comprises the sequence 5′

-TAGGCCCGTGGCAATATTAA-3′

(SEQ ID NO;

10).

12. The primer of claim 1, wherein X comprises the sequence 5′

-CCGGTCCGTGGCAATATTAA-3′

(SEQ ID NO;

11).

13. The primer of claim 1, wherein X comprises the sequence 5′

-CCGGCTCGTGGCGATGTTAA-3′

(SEQ ID NO;

12).

14. The primer of claim 1, wherein X comprises the sequence 5′

-CCGGCGTGTGCCGATATTAA-3′

(SEQ ID NO;

13).

15. The primer of claim 1, wherein X comprises the sequence 5′

-CCATGCGTGTGCCGATATTA-3′

(SEQ ID NO;

14).

16. The primer of claim 1, wherein X comprises the sequence 5′

-CAATGCGGGCGCCGATATTA-3′

(SEQ ID NO;

15).

17. The primer of claim 1, wherein X comprises the sequence 5′

-CGATGCGGGAGCCAATATAA-3′

(SEQ ID NO;

16).

18. The primer of claim 1, wherein X comprises the sequence 5′

-AGATGCGGTAGCCAATATAA-3′

(SEQ ID NO;

17).

19. The primer of claim 1, wherein X comprises the sequence 5′

-GGCGTGCTGAGCCAATATGG-3′

(SEQ ID NO;

18).

20. The primer of claim 1, wherein X comprises the sequence 5′

-GGCGCGCTGAGCCAATATGG-3′

(SEQ ID NO;

19).

21. The primer of claim 1, wherein X comprises the sequence:

5′

-GGCGCGCCGAGCCAATATGG-3′

(SEQ ID NO;

20).

22. The primer of claim 1, wherein X comprises the sequence 5′

-GGCGCGCCGAGCTAATATAT-3′

(SEQ ID NO;

21).

23. The primer of claim 1, wherein X comprises the sequence 5′

-AGCTCGGCGAGCTAATATAT-3′

(SEQ ID NO;

22).

24. The primer of claim 1, wherein X comprises the sequence 5′

-AGCGCGGCCAGCTAAGAGAT-3′

(SEQ ID NO;

23).

25. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGCGGCCGGCTGGAGAGA-3′

(SEQ ID NO;

24).

26. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGAGGCCGGCTGTAGAGG-3′

(SEQ ID NO;

25).

27. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGAGGCCAGCGGCCGAGG-3′

(SEQ ID NO;

26).

28. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGAGGCCAGCGGTCGAGG-3′

(SEQ ID NO;

27).

29. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGAGGCCAGCGGTCGAGG-3′

(SEQ ID NO;

28).

30. The primer of claim 1, wherein X comprises the sequence 5′

-CGCGGGGCCCGCGGCCGCGG-3′

(SEQ ID NO;

29).

31. The primer of claim 1, wherein X comprises the sequence 5′

-CGCCCGCCGCGCCCCGCGCC-3′

(SEQ ID NO;

30).

32. The primer of claim 1, wherein X comprises the sequence 5′

-GGCGCTCCATTAGCGTGAGT-3′

(SEQ ID NO;

31).

33. The primer of claim 1, wherein X and Y each comprise from 17 to 20 bases.

34. The primer of claim 1, wherein the melting temperature of a hybrid formed between said primer and its complement in a solution of 0.5 M NaCl is at least 72°

C.