Methods for enriching a target polynucleotide in a sample
AssigneeNexgen Cancer Detection LLC
InventorMatthew Nelson
Life of this patent
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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 composing a mixture of the target poly nucleotide and the reference poly nucleotide. 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.
Claims27
A method for enriching a double-stranded target polynucleotide in a sample by performing a polymerase chain reaction (PCR) amplification to obtain an amplification product, the method comprising the steps of:
- (a) preparing a reaction mixture comprising:
- (i) the sample;
- (ii) a DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity;
- (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide and not to the target polynucleotide;
- (iv) a pair of primers that flank the genetic alteration and do not interfere with the hybridization of the blocking oligonucleotide, wherein the sample comprises the double-stranded target polynucleotide and a double-stranded reference polynucleotide, wherein the target polynucleotide has a genetic alteration relative to the reference polynucleotide;
- (b) increasing the temperature of the reaction mixture above the melting temperatures of the double stranded reference polynucleotide and the double stranded target polynucleotide;
- (c) reducing the temperature of the reaction mixture, wherein the primer pair hybridizes to unblocked reference polynucleotide, if present, and the target polynucleotide;
- (d) extending the primers hybridized to the unblocked reference polynucleotide, if present, or target polynucleotide;
- and
- (e) repeating steps (b) through (d) for two or more cycles so as to enrich the target polynucleotide.
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A method for enriching a double-stranded target polynucleotide in a sample by performing a polymerase chain reaction (PCR) amplification to obtain an amplification product, the method comprising the steps of:
- (a) preparing a reaction mixture comprising:
- (i) the sample;
- (ii) a DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity;
- (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide and not to the target polynucleotide;
- (iv) a pair of primers that flank the genetic alteration and do not interfere with the hybridization of the blocking oligonucleotide, wherein the sample comprises the double-stranded target polynucleotide and a double-stranded reference polynucleotide, wherein the target polynucleotide has a genetic alteration relative to the reference polynucleotide;
- (b) increasing the temperature of the reaction mixture above the melting temperatures of the double stranded reference polynucleotide and the double stranded target polynucleotide;
- (c) reducing the temperature of the reaction mixture to a temperature that is below the melting temperature of the blocking oligonucleotide to the reference polynucleotide but is above the melting temperature of the primer oligonucleotide, wherein the blocking oligonucleotide and the reference polynucleotide form a blocking duplex;
- (d) reducing the temperature of the reaction mixture, wherein the primer pair hybridizes to the target polynucleotide;
- (e) extending the primers hybridized to the target polynucleotide;
- and
- (f) repeating steps (b) through (e) for two or more cycles so as to enrich the target polynucleotide.
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Dependencies are inferred from the claim text.