Design of stem-loop probes and utilization in snp genotyping

A method of detecting single nucleotide polymorphism (SNP) in nucleic acid containing samples, utilizing a pair of stem-loop probes for SNP genotyping of two individual SNP nucleic acid target sequences of a sample;

• the stem-loop probes (100) comprising first (3), second (2), and third (3) single stranded nucleic acid portions;
• the second (2) single stranded nucleic acid portion being located between the first (1) and the third (3) single stranded nucleic acid portions;
• the first (1) and the third (3) single stranded nucleic acid portions building a double stranded, intramolecular stem (10);
• and the second (2) single stranded nucleic acid portion forming a single stranded oligonucleotide loop (20) with a nucleotide sequence that is complementary to one of the individual SNP nucleic acid target sequences,
• wherein a ratio of perfect match probe/target hybrids to mismatched probe/target hybrids is detected at a certain temperature, and wherein the nucleotide sequence of the stem-loop probe is chosen such that the perfect match probe/target hybrids having a melting point Tm that is at least 5° C. higher than the Tm of mismatched probe/target hybrids.