RNA-templated DNA ligation for transcript analysis | Zendy

Mats Nilsson | Zendy; DanOscar Antson | Zendy; Gisela Barbany | Zendy; Ulf Landegren | Zendy

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RNA-templated DNA ligation for transcript analysis

Author(s) -

Mats Nilsson,

DanOscar Antson,

Gisela Barbany,

Ulf Landegren

Publication year - 2001

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/29.2.578

Subject(s) - dna ligase , biology , rna , oligonucleotide , ligation , sequencing by ligation , dna , rna ligase , ligase chain reaction , microbiology and biotechnology , dna ligases , biochemistry , gene , polymerase chain reaction , base sequence , genomic library , multiplex polymerase chain reaction

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.

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