Ultraspecific nucleic acid sensors for low-cost liquid biopsies

A method for preferential amplification of a target DNA molecule comprising a single nucleotide polymorphism (SNP) mutation, wherein the method comprises:

• (a) contacting a SNP-specific remote toehold primer to a sample comprising a target DNA molecule, wherein the SNP-specific remote toehold primer comprises (i) a 5′ docking site complementary to the target DNA molecule, (ii) a spacer domain not complementary to the target DNA molecule, and (iii) a 3′ mutant targeting remote toehold domain which is three to four nucleotides in length and complementary to a region in the target DNA molecule comprising the SNP mutation, and whereby the SNP-specific remote toehold primer associates with the target DNA molecule;
• (b) extending from the 3′ end of the mutant targeting remote toehold domain of the SNP-specific remote toehold primer along the target DNA molecule with a DNA polymerase and deoxynucleotides using the target DNA molecule as a template,
• wherein, in the presence of the target DNA molecule comprising the SNP mutation, the mutant targeting remote toehold domain binds to the region in the target DNA molecule comprising the SNP mutation, thereby positioning the 3′ end of the mutant targeting remote toehold domain to prime polymerization along the target DNA molecule as a template by the DNA polymerase, and
• wherein, in the presence of the target DNA molecule not containing the SNP mutation, the mutant targeting remote toehold domain does not bind to the DNA template and extending from the 3′ end of the mutant targeting remote toehold domain by the DNA polymerase using the target DNA molecule as a template does not occur;
• and
• (c) detecting a product, which comprises the extended mutant targeting remote toehold domain of step (b), indicating the presence of said target DNA molecule comprising the SNP mutation in the sample.