RNA-templated DNA ligation for transcript analysis

Ligase-mediated gene detection has proven valuable for detection and precis e distinction of DNA sequence variants, We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA se quences. Here we describe parameters that influence RNA-templated DNA ligat ion by T4 DNA ligase, The reaction proceeds much more slowly, requiring mor e enzyme, compared to ligation of the same oligonucleotides hybridized to t he corresponding DNA sequence, The reaction is inhibited at high concentrat ions of ATP and NaCl and both magnesium and manganese ions can support the reaction, We define reaction conditions where 80% of RNA target molecules c an template a diagnostic ligation reaction, Ligase-mediated RNA detection s hould provide a useful mechanism for sensitive and accurate detection and d istinction of RNA sequence variants.