A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

Pratyush, Daliparthy D.; Tiwari, Shalbha; Kumar, Ashok; Singh, Surya Kumar

doi:10.1016/j.gene.2012.01.031

Cited by 11 publications

(7 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The use of N 4 me-dCTP results in amplification of the correct target, and reduces non-specific amplification (Figure 3, Lane 1). Using the canonical set of nucleotides with 2.5%10% DMSO, as commonly reported (5, 21, 22), there is a reduction in the number of nonspecific products, but in no case is the 522-bp fragment obtained as the sole amplification product (Figure 3, Lanes 36). DMSO concentrations >10% were not tested, as they are reported to inhibit the DNA polymerase (15).…”

Section: Resultssupporting

confidence: 57%

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

et al. 2016

View full text Add to dashboard Cite

GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

show abstract

Section: Resultssupporting

confidence: 57%

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

et al. 2016

View full text Add to dashboard Cite

show abstract

“…The smart technology, which is considered the most advanced approach to obtain the full length cDNA of a certain gene with a partial known sequence, was chosen for the full length cDNA of the HTA gene (11). At the same time, hot start PCR polymerase was applied to avoid non-specific amplification in lower temperatures (14) and a touch-down PCR program was applied to avoid non-specific amplification in early cycles and to improve the efficiency of specific amplification in later cycles of the program (15).…”

Section: Discussionmentioning

confidence: 99%

Molecular clone and functional study of a novel hepatoma associated gene

Liu

Zhao²,

Ju³

et al. 2013

International Journal of Oncology

View full text Add to dashboard Cite

HTA is a novel hepatoma associated gene screened by bioinformatic strategies in our previous study. In the present investigation, the full-length sequence of the HTA gene was cloned by 3'-rapid amplification of cDNA ends (RACE) and 5'-RACE, which was 1414 bp and the open reading frame (ORF) was constituted with 3 exons and 2 introns. There are two types of splicing of the mRNA of HTA. Northern blot analysis showed that the 1.4 kb HTA mRNA and 1.7 kb HTA mRNA transcripts were present in the hepatocellular carcinoma (HCC) cell lines HepG2 and QGY-7703 but not in the normal cell line L02 and the human umbilical vein endothelial cells (HUVECs). Overexpression of the HTA gene in the hepatic cell line QSG-7701 via stable transfection can promote its proliferation rate and colony forming ability and change the cell cycle distribution of the cell lines. These results showed that the HTA gene is a potential therapeutic target in HCC and the clarification of its gene structure and sequence information provide an essential tool for future research.

show abstract

“…Amplification of templates with a high GC content using PCR is usually difficult compared to low GC targets. PCR amplification of GC-rich nucleotide sequences is often associated with insufficient yield of the target DNA sequence and amplification of non-specific products [1][2][3][4][5]. GC-rich templates are difficult to denature because of their high melting temperature.…”

Section: Introductionmentioning

confidence: 99%

DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

Neffati

Petrounia

Moreira

et al. 2021

Preprint

View full text Add to dashboard Cite

PCR amplification of GC-rich regions often leads to low yield and specificity. Addition of PCR-enhancing compounds is employed in order to overcome these obstacles. PCR-enhancing additives are low molecular polar organic compounds that are included as undisclosed co-solvents in commercial PCR buffers. In the interest of transparency and to permit further optimization by researchers of PCR compositions for challenging amplification problems, we studied eight PCR buffers by GC/MS to identify and quantify their co-solvents. Buffer specificity, both rich in water and salified substances, required a suitable sample preparation before injection into the GC/MS system. The aqueous phase of each buffer was replaced by an organic solvent to remove, by precipitation and filtration, salified substances which are detrimental to the GC/MS analysis. This approach has demonstrated the advantage of eliminating both water and salified substances without any loss of co-solvents. The sensitivity of the developed method was demonstrated as the main co-solvents were easily detected, identified and quantified. The methodology for identifying the co-solvents is mainly based on comparison of both library matching of acquired MS spectra with NIST library and experimental mass spectra obtained from authentic chemical standards. For the quantification of each co-solvent, deuterated Internal standards of similar structure to the co-solvents were used to correct the variable recovery caused by sample preparation, matrix effects, and ion source variability. The recovery ratio of the developed method was verified and found to be in the range 90-120 %. We then characterized the effects of specific organic co-solvents identified during PCR amplification -- using DNA melting, polymerase thermostability, polymerase activity and real-time PCR methods -- in order to elucidate their mechanism of action and to permit further optimization of their effects on amplification efficiency and specificity.

show abstract

A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

Cited by 11 publications

References 20 publications

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

Molecular clone and functional study of a novel hepatoma associated gene

DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

Contact Info

Product

Resources

About

A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

Cited by 11 publications

References 20 publications

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N 4 -Methyl-2′-Deoxycytidine 5′-Triphosphate

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N 4 -Methyl-2′-Deoxycytidine 5′-Triphosphate

Molecular clone and functional study of a novel hepatoma associated gene

DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

Contact Info

Product

Resources

About

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate