Increased single nucleotide discrimination in arrayed primer elongation by 4′C-modified primer probes

Abstract

Herein we describe the beneficial impact of immobilized 4'C-modified primer probes on detecting single nucleotide variations in arrayed primer extension by a DNA polymerase.

Efficient enzymatic synthesis of LNA-modified DNA duplexes using KOD DNA polymerase

“…DNA and RNA polymerases incorporate their natural substrates with high specificity and fidelity, and different research groups have shown that various polymerases accept a range of sugar-modified nucleotides as substrates. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] We have recently reported the enzymatic incorporation of LNA nucleotides by primer extension, PCR amplification and transcription reactions in vitro using Phusion TM High Fidelity DNA polymerase, 31,32 9 • N m TM DNA polymerase 33 and T7 RNA polymerase. 33 Kuwahara et al have in another recent report described the use of two genetically engineered KOD DNA polymerases, KOD (exo-) and KOD Dash, with the main focus on the incorporation of DNA nucleotides complementary to the LNA monomers of an LNA-modified DNA template strand.…”

Mutant DNA Polymerase for Improved Detection of Single‐Nucleotide Variations in Microarrayed Primer Extension

“…In the next step, we transferred the concept of fluorophor‐incorporation based primer extension to a solid support. We immobilised primer probes on 1,4‐phenylene diisothiocyanate (PDITC)‐activated glass slides by using an aminoalkyl linkage at their 5′ termini,6f and performed comparative primer‐extension reactions with wild‐type Pfu DNA polymerase as well as M2 (Scheme ). Primer probes were found to be covalently attached to aminopropyl PDITC activated glass substrate.…”

A universal locked nucleic acid-integrated X-shaped DNA probe design for amplified fluorescence detection of single-nucleotide variant

“…Up to now, many methods, such as allele-specific hybridization methods, have been developed for SNV detection, but complicated optimization and rigorous control of the assays made direct sequencing sub-optimal [6,7]. On the other side, allele-specific enzymatic methods, including ligase-based ligation [8][9][10], endonuclease-based cleavage [11], primer extension [12][13][14], polymerase based nucleotide incorporation [15] and proof-reading [16,17], have become a prevailing option for SNV detection because of its specificity and robustness. However, the inherent shortcomings of enzymes, such as the activity sensitive to reaction conditions and high cost, limit their application [18].…”