Method to increase sensitivity of detection of low-occurrence mutations
View Patent ↗A method for detecting a low-occurrence mutation in isolated DNA adds a blocking probe to reagents during amplification of the isolated DNA. The blocking probe is an oligonucleotide complementary to wild-type DNA corresponding to the sample. The blocking probe spans a site of a suspected mutation within a region of interest in the isolated DNA. After amplification, fragments of the amplified DNA is sequenced using next generating sequencing and an output is generated to display the sequenced fragments. In some embodiments, the blocking probe is locked nucleic acid (LNA).
1. A method of detecting a low-occurrence mutation in a sample from a patient, the method comprising:
isolating DNA from the sample;
adding a blocking probe to the isolated DNA, the blocking probe comprising an oligonucleotide complementary to wild-type DNA corresponding to the sample, the blocking probe adapted to span a site of a suspected mutation within a region of interest in the isolated DNA, wherein the region of interest is KRAS codon 12 and the blocking probe is CCTACGCCACAGCTCCAA (SEQ ID NO. 1);
amplifying the isolated DNA;
sequencing fragments of the amplified DNA in parallel; and
generating an output corresponding to the sequenced fragments.
2. The method of claim 1 , wherein the blocking probe is locked nucleic acid (LNA).
3. The method of claim 1 , further comprising, prior to adding a blocking probe, fragmenting the isolated DNA into fragments comprising one or more regions of interest.
4. The method of claim 1 , further comprising, prior to amplifying the isolated DNA, fragmenting and tagging the isolated DNA with one or more enrichment probes.
5. The method of claim 1 , wherein the sample is selected from the group consisting of whole blood, stool, biopsied tissue, bone marrow, fine needle aspirate (FNA), and peripheral blood.
6. A method for detecting a low-occurrence mutation in isolated DNA comprising:
adding a blocking probe to reagents during amplification of the isolated DNA, wherein the blocking probe comprises an oligonucleotide complementary to wild-type DNA corresponding to the sample, the blocking probe adapted to span a site of a suspected mutation within a region of interest in the isolated DNA, wherein the region of interest is KRAS codon 12 and the blocking probe is CCTACGCCACAGCTCCAA (SEQ ID NO. 1);
sequencing fragments of the amplified DNA in parallel; and
generating an output corresponding to the sequenced fragments.
7. The method of claim 6 , wherein the blocking probe is locked nucleic acid (LNA).
8. The method of claim 6 , further comprising, prior to adding a blocking probe, fragmenting the isolated DNA into fragment comprising one or more regions of interest.
9. The method of claim 6 , further comprising, prior to amplifying the isolated DNA, fragmenting and tagging the isolated DNA with one or more enrichment probes.
10. The method of claim 6 , wherein the sample is selected from the group consisting of whole blood, stool, biopsied tissue, bone marrow, fine needle aspirate (FNA), and peripheral blood.