RNA-templated DNA ligation for transcript analysis.
Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.
Nilsson
M
M
Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. m.nilsson@lumc.nl
Antson
D O
DO
Barbany
G
G
Landegren
U
U
D017122:Bacteriophage T4 / Q000201:enzymology; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry*; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D008345:Manganese; D012313:RNA; D004247:DNA; D011088:DNA Ligases; D008274:Magnesium