Target-dependent transcription using deletion mutants of N4 RNA polymerase
View Patent ↗The present invention comprises novel methods, compositions and kits that use N4 vRNAP deletion mutants to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide or non-nucleic acid analytes, by detecting a target sequence tag that is joined to an analyte-binding substance. The method consists of an annealing process, a DNA ligation process, an optional DNA polymerase extension process, a transcription process, and, optionally, a detection process.
1. A method of making RNA comprising:
(a) obtaining a N4 virion RNA polymerase consisting of a transcriptionally active mini-vRNAP, wherein said mini-vRNAP consists of a sequence at least 95% identical to SEQ ID NOS: 4, 6, or 8;
(b) obtaining a single-stranded DNA oligonucleotide wherein said single-stranded DNA oligonucleotide contains a N4 virion RNA polymerase promoter sequence;
(c) admixing said N4 virion RNA polymerase and said single-stranded DNA oligonucleotide; and
(d) culturing said N4 virion RNA polymerase and said single-stranded DNA oligonucleotide under conditions effective to allow RNA synthesis.