GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects

Moreau, Alain; Wang, Da Shen; Forget, Steve; Duez, Colette; Dusart, Jean

doi:10.1385/1-59259-177-9:075

Cited by 2 publications

(1 citation statement)

References 14 publications

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“…This region proved recalcitrant to standard PCR analysis. After trying several combinations of thermostable polymerases and denaturing agents, success was achieved by using Vent polymerase (New England Biolabs) in conjunction with 10% formamide and using a commensurately lowered annealing temperature of 55°C, a similar combination of conditions has been reported previously for amplification of problematic templates (24). Use of further upstream primers, including Rm4x18s, Rm4x810s, and Rm4x10s in concert with Rm4x4a resulted in amplified products of appropriate size when using plasmid DNA (279, 139, and 72 bp, respectively) but yielded no amplified products when using cDNA as template.…”

Section: Figmentioning

confidence: 99%

Structure of the m4 Cholinergic Muscarinic Receptor Gene and Its Promoter

Wood

Roopra

Harrington

et al. 1995

Journal of Biological Chemistry

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Cholinergic muscarinic receptor genes are members of the G-protein receptor gene superfamily. In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene. A rat cosmid clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated. This clone directed expression of the rat m4 gene when introduced in IMR32 cells, a human neuroblastoma that expresses m4, but did not drive expression when introduced into Chinese hamster ovary cells, a line that does not express the m4 gene. S1 nuclease, modified 5-rapid amplification of cDNA ends and polymerase chain reaction analysis of rat cosmid DNA and cDNA showed that the gene consists of a 2.6-kilobase coding exon, extending 34 base pairs ( Signal transduction in the nervous system is largely conducted by activation of G-protein coupled receptors (GPRs).

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Section: Figmentioning

confidence: 99%

Structure of the m4 Cholinergic Muscarinic Receptor Gene and Its Promoter

Wood

Roopra

Harrington

et al. 1995

Journal of Biological Chemistry

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Molecular Cloning and Expression of High GC-Rich Novel Tumor Suppressor Gene HIC-1

Kumar

2014

Mol Biotechnol

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Hypermethylated in Cancer-1 (HIC-1) is a novel tumor suppressor plays crucial role in tumor formation through loss of function by hypermethylation. HIC-1 is known as transcriptional factor whereas little known about its structure and function. Requirement felt to clone and express full coding protein and reveal various domains and binding pattern onto promoters conducting biophysical studies which lack in current scenario. Production of sufficient amounts of protein is frequent bottleneck in structural biology. Cloning full-length HIC-1 with >73 % GC content poses a daunting task with sequencing and expression adds more to the challenge. We describe the methodology for specific amplification, cloning, sequencing, and expression of HIC-1 in E. coli. Standardization using 1.5 U pfu polymerase in (NH4)2SO4 containing buffer gave specific amplification with 10 % DMSO and 1.5 mM MgCl2. Sequencing achieved using base analog 7-de aza dGTP (0.2 mM) or denaturant like DMSO (10 %) or betaine (1 M). Expression using strains of E. coli induced by different concentrations of IPTG (0.5-5.0 mM) for time points of 4, 8, 16, 20, and 24 h at different temperatures 25, 30, and 37 °C. Full-length clone successfully expressed in BL21-Codon Plus-RP using 1 mM concentration of IPTG for 8 h at 37 °C gave prominent band of 74 kDa.

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GC-Rich Template Amplification by Inverse PCR: DNA Polymerase and Solvent Effects

Cited by 2 publications

References 14 publications

Structure of the m4 Cholinergic Muscarinic Receptor Gene and Its Promoter

Structure of the m4 Cholinergic Muscarinic Receptor Gene and Its Promoter

Molecular Cloning and Expression of High GC-Rich Novel Tumor Suppressor Gene HIC-1

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