Methods and compositions for universal size-specific PCR

• US 9,404,150 B2
• Filed: 08/28/2008
• Issued: 08/02/2016
• Est. Priority Date: 08/29/2007
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Lee, Min Seob, Yang, Yanfeng
• Primary Examiner(s): Pande, Suchira
• Application Number:
  US12/674,403
• Publication Number:
  US 20110294699A1
• Time in Patent Office: 2,896 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/319 Exonuclease
• 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 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• 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 2549/113 using nested probes
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2561/101 Taqman

Abstract

Provided herein are products and processes for the amplification, detection and sequencing of short-stranded nucleic acid in the presence of a high background of long-stranded genomic material (e.g., host or maternal nucleic acids). The methods rely on the use of inside and outside primers introduced at varying concentrations, as well as universal amplification reactions that preferentially amplify short, low copy number nucleic acid.

Claims

1. A method of enriching for a target nucleic acid in a sample containing a mixture of target and non-target nucleic acid, comprising the steps of:

a) introducing to the sample a pair of forward and reverse inside primers that bind target and non-target nucleic acid, wherein;

(i) the target nucleic acid is a low copy number, relatively short nucleic acid of at least about 75 base pairs, but less than about 1200 base pairs and the non-target nucleic acid is an abundant copy number nucleic acid that is longer than the target nucleic acid, and

(ii) the inside primers comprise a common, universal domain and a sequence-specific domain complementary to both the target and non-target nucleic acids;

b) introducing to the sample an outside non-target binding primer at a concentration two times or more greater than the inside primer, wherein;

(i) the inside primers and outside primer are introduced to the sample at a concentration greater than the concentration of non-target nucleic acid, and

(ii) the outside non-target binding primer anneals upstream or downstream of the inside primer and is complementary to non-target nucleic acid, but not target nucleic acid;

c) introducing to the sample a universal primer capable of binding to the universal domain of the inside primers, wherein the universal primer is introduced at a concentration greater than the outside primer;

d) performing an amplification reaction using a polymerase having exonuclease activity, wherein;

(i) amplification of the non-target nucleic acid is initiated by the outside primer bound to one strand of the non-target nucleic acid, whereby the exonuclease activity digests the inside primer bound to the same strand of the non-target nucleic acid;

(ii) the outside primer does not bind to the target nucleic acid and amplification of the target nucleic acid is not initiated by the outside primer; and

(iii) amplification of the target nucleic acid is initiated by the inside primers, whereby the target nucleic acid is preferentially amplified relative to the non-target nucleic acid and whereby the target nucleic acid in the sample is enriched.

2. The method of claim 1, wherein a pair of forward and reverse outside primers are introduced, whereby both inside primers bound to the non-target nucleic acid are digested during amplification.

3. The method of claim 1, wherein the universal primer is introduced at a concentration about ten times greater than the inside primers, and the outside primer is introduced at a concentration about two times greater than the inside primer.

4. The method of claim 1, wherein multiple target nucleic acids are detected in a single, multiplexed reaction.

5. The method of claim 1, wherein the outside, non-target binding primers anneal to the non-target nucleic acid at least about 300 base pairs upstream of the inside primer.

6. The method of claim 1, wherein the inside primers contain a label.

7. The method of claim 1, wherein the inside primers are modified to facilitate their capture.

8. The method of claim 7, wherein the modifications are selected from the group consisting of capture mechanisms, compomers, tags, linkers and adapter molecules.

9. The method of claim 1, wherein the sample comprises cell-free nucleic acid.

10. The method of claim 1, wherein the target nucleic acid is an apoptotic product.

11. The method of claim 1, wherein the target nucleic acid is of fetal origin.

12. The method of claim 1, wherein the target nucleic acid comprises a locus of interest.

13. The method of claim 12, which further comprises determining the identity of at least one allele within the locus of interest.

14. The method of claim 12, wherein the inside primers are less than about 200 base pairs apart.

15. The method of claim 13, wherein the at least one allele falls between the inside primers.

16. The method of claim 13, wherein the outside primer is greater than about 250 base pairs 5′

upstream of the at least one allele.

17. The method of claim 1, wherein the non-target nucleic acid is of maternal origin.

18. The method of claim 1, wherein the sample is from a human.

19. The method of claim 1, wherein the sample is from a pregnant human.

20. The method of claim 19, wherein the sample is collected after the fifth week of gestation.

21. The method of claim 1, wherein the sample is selected from the group consisting of whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinal fluid, lavage fluid, biopsy sample, urine, feces, sputum, saliva, nasal mucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells.

22. The method of claim 1, wherein the sample is plasma.

23. The method of claim 1, wherein the sample is a previously isolated sample of nucleic acids.

24. The method of claim 1, wherein prior to (a), the sample is lysed in the presence of a lysis buffer, chaotropic substance and proteinase or protease, or any combination thereof.

25. A method for detecting a target nucleic acid, wherein the method of claim 1 is performed prior to, subsequent to, or simultaneously with another method for selectively detecting nucleic acid.

26. The method of claim 25, wherein the other method is selected from the group consisting of electrophoresis, liquid chromatography, size exclusion, microdialysis, electrodialysis, centrifugal membrane exclusion, organic or inorganic extraction, affinity chromatography, PCR, genome-wide PCR, sequence-specific PCR, methylation-specific PCR, restriction endonuclease enhanced polymorphic sequence detection, introducing a silica membrane or molecular sieve, and fragment selective amplification, or combinations thereof.

27. The method of claim 1, wherein the final relative percentage of target nucleic acid to non-target nucleic acid is at least about 25%.

28. The method of claim 1, further comprising quantifying the amplification products.

29. The method of claim 1, wherein the size of the target nucleic acid is about 500 base pairs or less and the size of the non-target nucleic acid is greater than about 500 base pairs.

30. The method of claim 29, wherein the target nucleic acid is of fetal origin.

31. The method of claim 29, wherein the non-target nucleic acid is of maternal origin.

32. The method of claim 30, wherein the non-target nucleic acid is of maternal origin.

33. The method of claim 30, wherein the sample is from a pregnant human.

34. The method of claim 1, further comprising detecting the preferentially amplified target nucleic acid.