The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: Application to in vitro combinatorial selection of aptamers

Kang, Jonghoon; Lee, Myung Soog; Gorenstein, David G.

doi:10.1016/j.jbbm.2005.06.003

Cited by 76 publications

(51 citation statements)

References 19 publications

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“…Lipids and proteins have limited solubility in DMSO, which removes proteins through its denaturing and precipitating action and also inhibits the activity of nucleases (Arakawa et al 2007), which helps reduce false DNA fragmentation during sample processing. In addition, DMSO is known to inhibit the formation of folded DNA structure (Kang et al 2005), which results in increased band intensity on the agarose gel following staining with fluorochromes such as ethidium bromide.…”

Section: Resultsmentioning

confidence: 99%

An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

2011

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Conventional DNA ladder assay has certain shortcomings such as loss of DNA fragments during sample processing, involvement of multiple steps and requirement of expensive reagents. The present study demonstrates a rapid, easy-to-perform cost-effective method for detection of apoptotic DNA fragments with considerable improvement in the sensitivity by avoiding loss of DNA fragments. It involves a few minutes of procedure involving direct lysis of cells with dimethyl sulphoxide (DMSO), brief vortexing, addition of 2% SDS-TE buffer, and a single step of centrifugation. This cost-and time-efficient method reduces the assay time considerably and can be used for a large number of samples with excellent sensitivity.

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Section: Resultsmentioning

confidence: 99%

An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

2011

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“…Previous reports have shown that complete denaturation of DNA at 98°C is essential for successful amplification of highly GC-rich sequences [19,20]. Other previous reports have shown that 5% DMSO and 1 M betaine can be used together to reduce stable secondary structures in DNA templates [21]. Thus, we used a denaturation temperature of 98°C and included both 5% DMSO and 1 M betaine in our PCRs to eliminate putative secondary structures and facilitate 5′-RACE of the adaptor-ligated cDNA preparation (Fig.…”

Section: Resultsmentioning

confidence: 99%

A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes

Shi

Jarvis

2006

Analytical Biochemistry

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Rapid amplification of cDNA ends (RACE) is widely utilized to determine the 5′-and 3′-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none address the difficult problems that arise when trying to isolate the ends of extremely GC-rich genes. In this study, we found that we were unable to isolate the correct 5′ or 3′ ends of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first strand cDNA synthesis at 70°C with Thermo-X™ reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98°C denaturation steps and Phusion™ DNA polymerase in the presence of 1M betaine and 5% DMSO. The use of these conditions yielded 5′-and 3′-RACE products that were about 80% GC over 213 and 162 bp, respectively, and included shorter, internal regions of 82-89% GC.

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“…Kang et al. 18 added dimethyl sulfoxide (DMSO) and betaine into the PCR reaction system to reduce the amounts of by‐products. Jie and co‐workers 19 obtained pure products through separating them from by‐products by using capillary electrophoresis.…”

Section: Discussionmentioning

confidence: 99%

Construction and optimization of an efficient amplification method of a random ssDNA library by asymmetric emulsion PCR

Shao

Shi

Zhu

et al. 2016

Biotech and App Biochem

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Construction of a random ssDNA sublibrary is an important step of the aptamer screening process. The available construction methods include asymmetric PCR, biotin–streptavidin separation, and lambda exonuclease digestions, in which PCR amplification is a key step. The main drawback of PCR amplification is overamplification increasing nonspecific hybridization among different products and by‐products, which may cause the loss of potential high‐quality aptamers, inefficient screening, and even screening failure. Cycle number optimization in PCR amplification is the main way to avoid overamplification but does not fundamentally eliminate the nonspecific hybridization, and the decreased cycle number may lead to insufficient product amounts. Here, we developed a new method, “asymmetric emulsion PCR,” which could overcome the shortcomings of conventional PCR. In asymmetric emulsion PCR, different templates were separated by emulsion particles, allowing single‐molecule PCR, in which each template was separately amplified, and the nonspecific hybridization was avoided. Overamplification or formation of by‐products was not observed. The method is so simple that direct amplification of 40 or more cycles can provide a high‐quality ssDNA library. Therefore, the asymmetric emulsion PCR would improve the screening efficiency of systematic evolution of ligands by exponential enrichment.

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The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: Application to in vitro combinatorial selection of aptamers

Cited by 76 publications

References 19 publications

An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes

Construction and optimization of an efficient amplification method of a random ssDNA library by asymmetric emulsion PCR

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