METHODS AND COMPOSITIONS FOR NUCLEIC ACID ANALYSIS
This invention provides ultra-sensitive methods and compositions for detecting patient-specific mutations from cell free nucleic acids (cfDNA) without sequencing. Methods of the invention make use of fluidic partitions for multiplex amplification of cfDNA and thereby create a library of uniformly amplified amplicons. The uniformly amplified amplicons can be split into any number of different detection reactions (while maintaining detection sensitivity) for single-plex detection of mutations present in cfDNA. These methods provide substantially improved signal to noise ratio and easier discrimination of low-abundance mutations.
1 . A method for detecting a mutation, the method comprising:
preparing an aqueous solution comprising target nucleic acid and PCR primers; combining the aqueous solution with an oil to create a mixture;
shearing the mixture to form a plurality of partitions, wherein at least a portion of the partitions include a single target nucleic acid and PCR primers;
amplifying the target nucleic acid inside the partitions with the PCR primers to produce a library of amplicons;
splitting the library of amplicons into a plurality of different reaction vessels; and
detecting a mutation from an amplicon in one of the reaction vessels by PCR.
2 . The method of claim 1 , wherein detecting the mutation is performed with qPCR.
3 . The method of claim 1 , wherein, during amplification, a number of PCR cycles performed is greater than a number of pairs of PCR primers consumed inside a portion of the partitions.
4 . The method of claim 3 , wherein a number of unused PCR primers inside the portion of partitions reaches zero before a final PCR cycle is initiated.
5 . The method of claim 1 , wherein amplifying the target nucleic acid inside the portion of partitions involves at least one PCR cycle comprising zero amplification events.
6 . The method of claim 1 , wherein the target nucleic acid is uniformly amplified during amplification.
7 . The method of claim 1 , wherein the target nucleic acid is amplified by digital PCR.
8 . The method of claim 1 , wherein shearing the mixture comprises using template particles to template the formation of uniformly sized partitions comprising a substantially uniform number of PCR primers.
9 . The method of claim 1 , wherein the target nucleic acid is a cell free nucleic acid.
10 . The method of claim 9 , wherein the cell free nucleic acid is pre-identified as a recurrently protected genomic region.
11 . The method of claim 9 , wherein the cell free nucleic acid is isolated from a urine sample, a blood sample, or a sputum sample.
12 . The method of claim 1 , wherein the target nucleic acid is approximately 60-90 base pairs in length.
13 . The method of claim 1 , further comprising calculating a concentration of PCR primers to add to the aqueous solution such that a substantial number PCR primers are exhausted before amplification is complete.
14 . The method of claim 2 , wherein qPCR is performed with modified primers, the modified primers comprising one or more of a locked nucleic acid primer, a 2-tailed primer, or a light emitting primer.
15 . The method of claim 1 , wherein at least one of the PCR primers is fluorogenic.
16 . The method of claim 1 , wherein different ones of the PCR primers comprise sequences complementary to different molecules of target nucleic acid.
17 . The method of claim 1 , wherein the plurality of different reaction vessels contains reagents for detecting one or more cancer mutations.
18 . The method of claim 17 , wherein one of the plurality of different reaction vessels comprises a first reagent for detecting a first cancer mutation and a second one of the plurality of different reaction vessels comprises a second reaction for detecting a second cancer mutation.
19 . The method of claim 17 , wherein the reagents comprise primers for qPCR.
20 . The method of claim 19 , wherein, in the presence of a cancer mutation, the primers fail to hybridize with amplicons, thereby indicating the presence of mutation.