Methods for detecting a target polynucleotide in a sample
AssigneeNexgen Cancer Detection LLC
InventorMatthew NELSON
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Abstract
Disclosed are methods for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide. The methods may be utilized for detecting with high selectivity a target polynucleotide in a mixture of the reference polynucleotide and the target polynucleotide in which the target polynucleotide represents a low percentage of the mixture. The methods may be adapted for diagnosing, prognosing, and treating subjects having a disease or disorder associated with the genetic alteration.
Claims32
A method for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide, the method comprising:
- (a) performing a polymerase chain reaction (PCR) amplification to obtain an amplification product in a reaction mixture comprising:
- (i) the sample;
- (ii) a high fidelity DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity;
- (iii) a pair of primers that flank the genetic alteration;
- and
- (iv) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex;
- and
- (b) sequencing the amplification product and detecting the genetic alteration in the amplification product, thereby detecting the target polynucleotide comprising the genetic alteration in the sample.
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A kit for performing a method for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide, wherein the kit comprises:
- (i) a DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity;
- (ii) a pair of primers that flank the genetic alteration;
- and (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex and obtaining an amplified fragment sample.
Dependencies are inferred from the claim text.