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

Abstract

Mutant detects mutation. A mutant DNA polymerase, which was derived from Pyrococcus furiosus DNA polymerase (M2 in figure), was found to have improved single‐nucleotide discrimination properties, and was used for selective microarrayed‐primer extensions. This approach allowed multiplexed SNP detection.

Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

“…Recognition of the target DNA by formation of a rigid helical double strand leads to separation of the termini which can easily be detected by fluorescence. Besides the genetic analysis (Kostrikis et al 1998; Socher et al 2008) and detection of point mutations (Kranaster et al 2008; Ficht et al 2004; Valis et al 2005), the molecular beacon concept is also useful in real-time PCR (Tyagi and Kramer 1996; Tyagi et al 1998), RNA detection in living cells (Matsuo 1998), and also in the identification of protein–DNA interactions (Li et al 2000). The introduction of single molecule detection for the interaction of a molecular beacon with DNA leads to an increase in sensitivity from about 10 −7  M to 10 −11 to 10 −12  M (Knemeyer and Sauer 2000).…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…The double-headed thymidine (21), n ¼ 1 or 2, has been incorporated into oligonucleotides but showed little effect when incorporated into DNA or RNA three-way junctions. 126 The four diastereoisomers of the double-headed acyclic nucleoside (22), (3R, 2S (D-erythro) isomer shown) were synthesised and incorporated into oligonucleotides where they were found to be destabilising, but the two S isomers were only as destabilising as GNA (9). 127 The presence of a cyclohexenyl nucleoside (CeNA) (23) into DNA enhances the duplex stability and resistance to nuclease digestion.…”

“…21 Using allele-specific primer extension, a microarray-based method has been used with a mutant allele-specific DNA polymerase for the detection of SNPs. 22 Software devised to design imperfectly-matched sequences has been used to detect single base differences in miRNA expression, giving rise to many fewer false positive results than when using a conventionally designed array. 23 Array probes using analogues (e.g., LNA, 2,6-diaminopurine) that give rise to isoenergetic 2 0 -O-methyl oligonucleotides have been used to map secondary structures in a retrotransposon (R2Bm 5 0 RNA).…”

Engineered DNA Polymerases in Biotechnology

“…[58] Furthermore, the obtained Pfu DNA polymerase mutants can be applied as useful tools in genotyping assays such as allele-specific real-time PCR [36] and DNAchip-based allele-specific primer extensions. [59] In both cases, employment of the engineered polymerase leads to a higher showing some amino acids of motif C (QVH) and the DNA primer-template duplex and incoming triphosphate (PDB ID: 3KTQ [53] ). The 3'-primer terminus paired to the template, incoming dNTP and protein backbone are shown.…”