Analysis for Nucleic Acids by Digital PCR

• US 20110183330A1
• Filed: 10/28/2010
• Published: 07/28/2011
• Est. Priority Date: 08/03/2007
• Status: Active Grant

• Current Assignee: Chinese University of Hong Kong
• Original Assignee: Chinese University of Hong Kong
• Inventors: LO, Yuk Ming Dennis, Chiu, Rossa Wai Kwun
• Granted Patent: US 8,722,334 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present invention provides a method for analyzing nucleic acids for their lengths and relative abundance in a sample, based on digital amplification of individual template molecules. This invention has many applications, including those in noninvasive prenatal diagnosis, transplantation monitoring, and the detection and monitoring of cancers and virus-associated diseases.

Claims

1. A method for analyzing nucleic acids in a sample, comprising the steps of:

(i) preparing multiple equal fractions from the sample, wherein more than 50% of the fractions contain no more than 1 target nucleic acid molecule per fraction;

(ii) performing amplification reactions in each fraction using at least one forward primer with at least two reverse primers, or using at least two forward primers with at least one reverse primer, wherein each of the forward or reverse primers has a distinct and definitive nucleotide sequence;

(iii) detecting in each fraction amplified nucleotide sequence from each pair of forward and reverse primers; and

(iv) counting the number of fractions in which different combinations of amplified nucleotide sequences from different pairs of forward and reverse primers are detected, thereby determining the relative amount of the target nucleic acids of different lengths in the sample.

2. The method of claim 1, wherein the multiple equal fractions are multiple equal dilutions from the sample.

3. The method of claim 1, wherein step (i) is performed by a microfluidics system.

4. The method of claim 1, wherein the amplification reactions are polymerase chain reactions (PCR).

5. The method of claim 4, wherein the PCR is real-time PCR.

6. The method of claim 4, wherein a fluorescent dye is present in the PCR.

7. The method of claim 6, wherein the fluorescent dye is SYBR Green or LC Green.

8. The method of claim 1, further comprising a step of reverse transcription prior to step (i) or step (ii).

9. The method of claim 1, wherein the amplified nucleotide sequences from different pairs of forward and reverse primers are of distinct lengths.

10. The method of claim 1, wherein step (ii) is performed by emulsion polymerase chain reaction.

11. The method of claim 1, wherein step (iii) is performed by melting curve analysis.

12. The method of claim 9, wherein step (iii) is performed by electrophoresis.

13. The method of claim 1, wherein step (iii) is performed by sequence-specific hybridization with probes with detectable labels, wherein each probe has a distinct detectable label and specifically hybridizes with an amplified nucleotide sequence from a pair of forward and reverse primers.

14. The method of claim 13, wherein the detectable labels are distinct fluorescent molecules.

15. The method of claim 1, wherein step (iii) is performed by primer extension reactions or by sequencing reactions.

16. The method of claim 15, wherein products of the primer extension reactions are detected by mass spectrometry.

17. The method of claim 1, wherein step (iii) is performed by flow cytometry.

18. The method of claim 1, wherein steps (ii) and (iii) are performed by BEAMing.

19. The method of claim 1, wherein the amplification reactions in step (ii) are performed consecutively using different pairs of forward and reverse primers.

20. The method of claim 1, wherein the amplification reactions in step (ii) are performed concurrently using different pairs of forward and reverse primers.

21. The method of claim 1, wherein the sample is from a pregnant woman.

22. The method of claim 21, wherein the sample is blood, plasma, serum, saliva, or a cervical lavage sample.

23. The method of claim 21, wherein each of the target nucleic acids comprises at least a portion of chromosome 13, 18, 21, X, or Y.

24. The method of claim 21, wherein each of the target nucleic acids comprises at least a portion of a gene related to a genetic disease or a genetic polymorphism.

25. The method of claim 24, wherein the gene is the β

-globin gene or the cystic fibrosis transmembrane conductance regulator gene.

26. The method of claim 24, wherein the genetic polymorphism is a single nucleotide polymorphism (SNP).

27. The method of claim 1, wherein the sample is from a cancer patient.

28. The method of claim 27, wherein the cancer is nasopharyngeal carcinoma, lymphoma, hepatocellular carcinoma, or cervical carcinoma.

29. The method of claim 27, wherein the sample is blood, plasma, serum, saliva, or tumor tissue.

30. The method of claim 27, wherein each of the target nucleic acids comprises at least a portion of an oncogene or a tumor suppressor gene.

31. The method of claim 30, wherein the oncogene or tumor suppressor gene is KRAS, erbB-2, p16, or RASSF1A.

32. The method of claim 1, wherein each of the target nucleic acids is from a virus genome.

33. The method of claim 32, wherein the virus is Epstein-Barr Virus, Human Papilloma Virus, or Hepatitis B Virus.