Biomedical subjects

Maneesh R Pingle

Publications and source records attributed to Maneesh R Pingle.

3 recordsLinked to original sources

PubMed2006

Harnessing asymmetrical substrate recognition by thermostable EndoV to achieve balanced linear amplification in multiplexed SNP typing.

Multiplexed amplification of specific DNA sequences, by PCR or by strand-displacement amplification, is an intrinsically biased process. The relative abundance of amplified DNA can be altered significantly from the original representation and, in extreme cases, allele dropout can occur. In this paper, we present a method of linear amplification of DNA that relies on the cooperative, sequence-dependent functioning of the DNA mismatch-repair enzyme endonuclease V (EndoV) from Thermotoga maritima (Tma) and Bacillus stearothermophilus (Bst) DNA polymerase. Tma EndoV can nick one strand of unmodified duplex DNA, allowing extension by Bst polymerase. By controlling the bases surrounding a mismatch and the mismatch itself, the efficiency of nicking by EndoV and extension by Bst polymerase can be controlled. The method currently allows 100-fold multiplexed amplification of target molecules to be performed isothermally, with an average change of <1.3-fold in their original representation. Because only a single primer is necessary, primer artefacts and nonspecific amplification products are minimized.

Base Pair Mismatch↗

PubMed2004

High sensitivity EndoV mutation scanning through real-time ligase proofreading.

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5' terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.

Artifacts↗

PubMedDate not supplied

Endcaps for stabilizing short DNA duplexes.

The syntheses of endcaps for covalently linking the 3' and 5' hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.

Base Sequence↗