Digital amplification

• US 7,915,015 B2
• Filed: 11/12/2009
• Issued: 03/29/2011
• Est. Priority Date: 08/02/1999
• Status: Expired due to Fees

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Kinzler, Kenneth W., Vogelstein, Bert
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US12/617,368
• Publication Number:
  US 20100209921A1
• Time in Patent Office: 502 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• 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 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The identification of pre-defined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. The exponential, analog nature of the polymerase chain reaction is transformed into a linear, digital signal suitable for this purpose. Single molecules can be isolated by dilution and individually amplified; each product is then separately analyzed for the presence of pre-defined mutations. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.

Claims

1. A method for determining an allelic imbalance in a biological sample, comprising the steps of:

amplifying template molecules within a set comprising a plurality of assay samples to form a population of amplified molecules in each of the assay samples of the set, wherein the template molecules are obtained from the biological sample;

analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain a first allelic form of a marker and a second number of assay samples which contain a second allelic form of the marker, wherein between 0.1 and 0.9 of the assay samples yield an amplification product;

comparing the first number to the second number to ascertain an allelic imbalance in the biological sample; and

identifying an allelic imbalance in the biological sample.

2. The method of claim 1 wherein the step of amplifying employs real-time polymerase chain reactions.

3. The method of claim 2 wherein the real-time polymerase chain reactions comprise a dual-labeled fluorogenic probe.

4. The method of claim 1 wherein between 0.1 and 0.9 of the assay samples yield an amplification product as determined by amplification of the first allelic form of the marker.

5. The method of claim 1 wherein between 0.1 and 0.9 of the assay samples yield an amplification product as determined by amplification of the second allelic form of the marker.

6. The method of claim 1 wherein the amplified molecules in each of the assay samples within the first and second numbers of assay samples are homogeneous such that the first number of assay samples do not contain the second allelic form of the marker and the second number of assay samples do not contain the first allelic form of the marker.

7. The method of claim 1 wherein the sample is from blood.

10. The method of claim 1 or 8 wherein between 0.1 and 0.6 of the assay samples yield an amplification product.

11. The method of claim 1 or 8 wherein between 0.3 and 0.5 of the assay samples yield an amplification product.

12. The method of claim 1 or 8 wherein the set comprises at least 500 assay samples.

13. The method of claim 1 or 8 wherein the set comprises at least 1000 assay samples.

8. A method for determining an allelic imbalance in a biological sample, comprising the steps of:

distributing nucleic acid template molecules from a biological sample to form a set comprising a plurality of assay samples;

amplifying the template molecules within the assay samples to form a population of amplified molecules in the assay samples of the set;

analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain a first allelic form of a marker and a second number of assay samples which contain a second allelic form of the marker;

comparing the first number of assay samples to the second number of assay samples to ascertain an allelic imbalance between the first allelic form and the second allelic form in the biological sample.

9. The method of claim 8 wherein the sample is from blood.

14. The method of claim 8 wherein the step of amplifying employs real-time polymerase chain reactions.

15. The method of claim 14 wherein the real-time polymerase chain reactions comprise a dual-labeled fluorogenic probe.

16. The method of claim 8 wherein between 0.1 and 0.9 of the assay samples yield an amplification product as determined by amplification of the first allelic form of the marker.

17. The method of claim 8 wherein between 0.1 and 0.9 of the assay samples yield an amplification product as determined by amplification of the second allelic form of the marker.

18. The method of claim 8 wherein the amplified molecules in each of the assay samples within the first and second numbers of assay samples are homogeneous such that the first number of assay samples do not contain the second allelic form of the marker and the second number of assay samples do not contain the first allelic form of the marker.