Can I read Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences on EtoBox?

Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences by Toshitsugu Fujita; Shoko Nagata; Hodaka Fujii is a Chemistry article available to read on EtoBox.

What is Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences about?

Blocking PCR is a method that inhibits amplification of DNA possessing a nucleotide sequence complementary to that of a blocker; the method can be used to suppress amplification of target wild-type DNA while amplifying mutated DNA. Previously, we demonstrated that an oligoribonucleotide (ORN) functions as a cost-effective and sequence-specific blocker. This blocking PCR system, named ORN interference-PCR (ORNi-PCR), is compatible with DNA polymerases lacking 5′-3′ exonuclease activity but not with those possessing the activity (e.g., Taq DNA polymerase), which can remove a hybridized ORN during DNA extension. Here, we demonstrate that under specific experimental conditions, an intact or phosphorothioated ORN strongly suppresses extension of target DNA by Taq DNA polymerases. This method was applied successfully to real-time ORNi-PCR and one-step real-time reverse transcription-ORNi-PCR using a dual-labeled fluorescent probe to detect a single-nucleotide mutation in DNA and RNA in a sequence-specific manner. The results reaffirm the utility of blocking PCR and provide technical hints for its improvement.

Who reads Oligoribonucleotide-Mediated Blockade of DNA Extension by Taq DNA Polymerases Increases Specificity and Sensitivity for Detecting Single-Nucleotide Differences?

Author

Toshitsugu Fujita; Shoko Nagata; Hodaka Fujii

Publisher

American Chemical Society (ACS)

Published

2023

Language

EN

Field

Chemistry (Physical Sciences)