Full COLD-PCR enrichment with reference blocking sequence

A kit for enriching a target nucleic acid sequence suspected to be in a nucleic acid sample which sample also contains a concentration of a reference nucleic acid sequence, said target nucleic acid sequence is at least 50% homologous to the reference nucleic acid sequence and differs from the reference nucleic acid sequence by at least one deletion, insertion or substitution and is amplifiable by a same primer pair as the reference nucleic acid sequence, the kit comprising:

• a buffer;
• a DNA polymerase;
• deoxyribonucleoside triphosphates;
• a first primer pair comprising a first forward primer and a first reverse primer that anneal to respective binding sites on complementary strands of the reference nucleic acid sequence in the nucleic acid sample and the target nucleic acid sequence suspected to be in the nucleic acid sample, the first forward primer and the first reverse primer capable of annealing to the respective strands of the reference nucleic acid sequence and the target nucleic acid sequence at or below a first temperature;
• and
• an engineered reference blocking sequence oligonucleotide that is fully complementary with at least a portion of one of the strands of the reference nucleic acid sequence to which one of the first forward primer or the first reverse primer binds, and is not fully complementary with the strand of the target nucleic acid sequence to which said first forward primer or the first reverse primer binds;
• wherein the engineered reference blocking sequence oligonucleotide is selected so that at a second temperature that is higher than the first temperature, the reference blocking sequence oligonucleotide is capable of annealing to the respective strands of target nucleic acid sequence and the reference nucleic acid sequence to which one of the first forward primer or the first reverse primer anneals to form heteroduplexes of the engineered reference blocking sequence oligonucleotide and the target nucleic acid sequence and duplexes of the engineered reference blocking sequence oligonucleotide and the reference nucleic acid sequence,
• wherein the melting temperature of heteroduplexes of the engineered reference blocking sequence oligonucleotide and the target nucleic acid sequence is lower than the melting temperature of duplexes of the reference blocking sequence oligonucleotide and the reference nucleic acid sequence but higher than the first temperature, and wherein the engineered reference blocking sequence oligonucleotide does not include peptide nucleic acid;
• and
• instructions that include cycling parameters including the first temperature, the second temperature and the melting temperature of the heteroduplexes.