Improved PCR Method for Amplification of GC-Rich DNA Sequences

Hubé, Florent; Reverdiau, Pascale; Iochmann, Sophie; Gruel, Yves

doi:10.1385/mb:31:1:081

Cited by 54 publications

(34 citation statements)

References 11 publications

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“…12) Hubé et al found that 5% formamide increased the efficiency of PCR amplification of a GþC rich sequence (70%) more potently than did 5% DMSO. 19) This agrees with our finding that formamide had a more potent effect than DMSO. Taken together, for PCR and the reverse transcription reaction, formamide appears to be particularly effective when the target DNA or RNA has a high GþC rich sequence.…”

Section: Discussionsupporting

confidence: 92%

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Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

Yasukawa

Konishi

Inouye

2010

Bioscience, Biotechnology, and Biochemistry

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The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). At 42 C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.Key words: dimethyl sulfoxide; formamide; glycerol; organic solvent; reverse transcriptaseReverse transcriptase (RT) [EC 2.7.7.49] is the enzyme responsible for viral genome replication. It possesses RNA-and DNA-dependent DNA polymerase and RNase H activities.1,2) RTs from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV) are the most extensively used in cDNA synthesis 3) and RNA amplification reactions 4,5) due to their high catalytic activity and fidelity.6) The AMV RT is a heterodimer consisting of a 63-kDa subunit and a 95-kDa subunit, while MMLV RT is a 75-kDa monomer. The subunit of AMV RT comprises the fingers, palm, thumb, connection, and RNase H domains, and the subunit comprises these five domains and the C-terminal integrase domain.7) MMLV RT also contains these five domains.8) The active site of the DNA polymerase reaction resides in the fingers/palm/thumb domain, while that of the RNase H reaction lies in the RNase H domain.Enzymes, in general, are inactivated in the presence of organic solvents. However, enzymatic reactions in media containing organic additives can sometimes make previously problematic processes feasible through increased substrate solubility or diminished side reactions, for example. In polymerase chain reaction (PCR), various organic additives have been reported to improve yields and specificity.9,10) Of these, dimethyl sulfoxide (DMSO) and formamide are frequently used for the reaction with a GþC-rich DNA to improve specificity, 11,12) but very little is known about the effects of organic solvents on the reverse transcription reaction, although DMSO has been used empirically in the RNA amplification reaction. 4,5) One of the difficult points in this evaluation is that from a practical viewpoint, the efficiency of the reverse transcription reaction is evaluated by the yields and specificity of the subsequent PCR process. Another difficult point is that organic solvents might act on the primer and the RNA as well as on the RT itself.The purpose of this study was to show that organic solvents can improve the efficiency of the RT reaction under certain conditions. We expected that organic solvents ...

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Section: Discussionsupporting

confidence: 92%

“…Formamide at 10% inhibited PCR amplification by non-engineered Taq DNA polymerase, but increased the activity of the genetically engineered thermostable variant AmpliTaq DNA polymerase. 19,21) This suggests that genetically engineered thermostable MMLV RT variants [22][23][24] might also be highly resistant to DMSO and formamide effects.…”

Section: Discussionmentioning

confidence: 99%

Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

Yasukawa

Konishi

Inouye

2010

Bioscience, Biotechnology, and Biochemistry

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“…Mammalian promoter sequences often contain highly GC-rich regions, which are difficult to amplify under standard reaction conditions [15]. In this study, we tested the efficacy of concentration-dependent combinations of different PCR additives for a reliable amplification of genomic DNA corresponding to a set of human promoter sequences and generated a novel, cheap, and flexible PCR enhancer.…”

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confidence: 99%

An efficient and economic enhancer mix for PCR

Ralser

Querfurth

Warnatz

et al. 2006

Biochemical and Biophysical Research Communications

179

109

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Polymerase chain reaction (PCR) has become a fundamental technique in molecular biology. Nonetheless, further improvements of the existing protocols are required to broaden the applicability of PCR for routine diagnostic purposes, to enhance the specificity and the yield of PCRs as well as to reduce the costs for high-throughput applications. One known problem typically reported in PCR experiments is the poor amplification of GC-rich DNA sequences. Here we designed and tested a novel effective and low-cost PCR enhancer, a concentration-dependent combination of betaine, dithiothreitol, and dimethyl sulfoxide that broadly enhanced the quantitative and/or qualitative output of PCRs. Additionally, we showed that the performances of this enhancer mix are comparable to those of commercially available PCR additives and highly effective with different DNA polymerases. Thus, we propose the routine application of this PCR enhancer mix for low-and high-throughput experiments.

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“…will be partially open inhibiting the ability of the Taq DNA polymerase to act in those regions [22]. McDowell et al [23] demonstrated that GC-rich sequences, in conjunct with high Tm, leads to the formation of stable secondary structures in primers, which can reduce the PCR efficiency by serving as termination or stop sites.…”

Section: Resultsmentioning

confidence: 99%

Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphism of the KLF14 Gene

Zabala¹,

Gomez²,

Alvarez³

et al. 2017

OALib

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GWAS (genome-wide association studies) have associated Type 2 DiabetesMellitus with the single nucleotide polymorphism (SNPs) rs972283 (A/G) of KLF14 gene with in a global population. The most common techniques used to analyze SNPs are time consuming, multi-step process and require expensive instruments. Therefore, in order to overcome these problems, we have developed a new, rapid and cost effective T-ARMS PCR assay to genotype rs972283. However, the optimization step can be hardworking and laborious. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. In a first step, we design and validate two specific primer pair for T-ARMS PCR. Later, the amplification conditions were optimized for DNA concentration, annealing temperature, Taq DNA polymerase units and primers concentration. The last one was considered the main interference factor for a correct amplification and appropriate band intensity. Finally, the results obtained by T-ARMS PCR were concordant with sequencing. T-ARMS PCR assay developed in our laboratory for genotyping rs972283 (A/G) of KLF14 gene is time saving and cost-effective compared to the available methods used for SNP studies.

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Improved PCR Method for Amplification of GC-Rich DNA Sequences

Cited by 54 publications

References 11 publications

Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

An efficient and economic enhancer mix for PCR

Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphism of the KLF14 Gene

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