Synthesis and enrichment of nucleic acid sequences

Inactive Publication Date: 2016-09-22
TROVAGENE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for quickly and accurately detecting small amounts of specific nucleic acid sequences in a biological sample. This method uses a relative increase in the amount of the target sequence by preferential amplification, resulting in an enrichment of about 700×-10000× fold and greater than other nucleic acids in the sample. This allows for the detection of as few as a single copy of the target sequence within a biological sample. This method has several technical advantages, including fewer steps, reduced reaction assay times, and increased sensitivity. It can be used with both short and long nucleic acid sequences, providing greater flexibility and accuracy in detecting mutations and other abnormalities in biological samples.

Problems solved by technology

Thus, such prior methods are restricted to conditions where the Tm of the double stranded target sequence is lower than the Tm for the double stranded reference sequence.

Method used

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  • Synthesis and enrichment of nucleic acid sequences
  • Synthesis and enrichment of nucleic acid sequences
  • Synthesis and enrichment of nucleic acid sequences

Examples

Experimental program
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Effect test

example 1

[0125]An example of a two-step PCR enrichment assay (EGFR Exon 19 deletions) is provided in FIG. 1. In this assay, a selective denaturation step precedes the annealing step. As the reaction ramps to the annealing temperature of 62.4° C., any complementary wildtype strands generated in the previous PCR cycle bind blocker before the primers anneal. The primers then anneal to the complementary mutant strand and bar the possibility of any blocker binding to that mutant strand due to sequence overlap. (For most deletions the likelihood of any blocker binding to the mutant is extremely low as there is actually very little common sequence). In this exemplary assay, at the preselected annealing step (62.4° C.), blocker:target and reference (wt):target heteroduplex formation is likely to be extremely low (below detectable or calculable levels) because only a minor percentage will be complementary.

TABLE 1Deletion-Specific Cycling Conditons (DSCC-3)StepTempTimeLid 98° C.  Singe 1 (32 cycles)85...

example 2

[0126]A schematic example of a four-step PCR enrichment assay (EGFR Exon 20 T790M) is provided in FIG. 2. In this assay, a 98° C. denaturation step ensures that all duplexes denature. During the second (optional) step (70° C.) blocker-wildtype duplexes form, but few blocker-mutant duplexes form (as 70° C. is above the blocker-mutant Tm). At step 3, selective denaturation, many of the blocker-wildtype will denature (along with any blocker-mutant duplexes that may exist). Just as in the two-step PCR (example 1), as the reaction ramps back to the annealing temperature of 64.0° C., complementary wildtype strands are bound by the excess blocker before the primers anneal. The primers then anneal to the complementary mutant strand and bar the possibility of blocker binding to that mutant strand. Short amplicon length allows extension without the need for an additional elongation step.

The level of enrichment for EGFR T790M_T is provided below in Table 6

TABLE 6FOLDENRICHMENTInputEGFR T790M_T...

example 3

Verification of a Single Copy Assay Sensitivity

[0137]FIG. 6 provides a Normal distribution histogram and Poisson probabilities table. The table on the right is a Poisson distribution table of probabilities. The columns of the table are the number of observed events in a discrete interval. The rows are the average number of events per interval (0.13 events per interval, 0.25 events per interval, etc.) Each cell in the table is the probability of observing a given number of events (columns) given an average expected number of events (rows) in a single interval. For example, with regards to a cancer mutation detection test, if we expect to detect 2 mutants DNA strands per milliliter (interval), a single milliliter would have a 14% chance of containing no mutant DNA strands at all.

[0138]FIG. 7 Curve fit and calculated input mutation level of a cancer patient was detected in a biological fluid sample from the patient containing the KRAS G12D mutation. The raw data plot of the enriched re...

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Abstract

The present disclosure relates to the enrichment of target nucleic acid sequences present in low-abundance relative to corresponding non-target or reference nucleic acid sequence in a sample. In particular, the methods allow for a substantially greater level of detection sensitivity of target sequence by orders of magnitude enrichment of a low-abundance sequence.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 893,283, filed Oct. 20, 2013, U.S. Provisional Application No. 61 / 904,141, filed Nov. 14, 2013, and U.S. Provisional Application No. 62 / 039,905, filed Aug. 20, 2014, all of which are incorporated by reference herein in their entirety.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. ______, 2014, is named 2-10230_SL.txt and is bytes in size.FIELD OF THE DISCLOSURE[0003]Mutations in BRAF and KRAS are examples of genetic alterations that confer a survival and growth advantage to cancer cells. Such genetic alterations can be used for selection of targeted therapies. But in a subject, the alterations are present with a large excess of non-altered, wild-type sequences.[0004]This disclosure relates to sy...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/6806C12Q1/686C12Q2527/107C12Q2537/163C12Q2563/159C12Q1/6886C12Q2600/156C12Q2600/16
Inventor POOLE, JASONHANDCOCK, SAEGEKOSCO, KARENAMELNIKOVA, VLADACROUCHER, PETERLU, TIMERLANDER, MARKSAMUELSZ, ERRIN
Owner TROVAGENE
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