PCR-amplification of GC-rich regions: 'slowdown PCR'

  title={PCR-amplification of GC-rich regions: 'slowdown PCR'},
  author={Ulrich H. Frey and Hagen Sjard Bachmann and J{\"u}rgen Peters and Winfried Siffert},
  journal={Nature Protocols},
The polymerase chain reaction (PCR) technique has become an indispensable method in molecular research. [] Key Method The protocol relies on the addition of 7-deaza-2′-deoxyguanosine, a dGTP analog to the PCR mixture and a novel standardized cycling protocol with varying temperatures. The latter consists of a generally lowered ramp rate of 2.5 °C s−1 and a low cooling rate of 1.5 °C s−1 for reaching an annealing temperature and is run for 48 cycles. We established this protocol as a versatile method not only…

Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates.

This protocol uses a mixture of four additives-betaine, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA)-for use with Taq DNA polymerase.

Improved PCR Amplification of Broad Spectrum GC DNA Templates

Subcycling during the amplification process significantly improved amplification of short template pools, particularly when the template contained a low percent of GC, and 7-deaza-dGTP improved the amplification of longer products.

Amplification of GC-rich DNA for High-Throughput Family-Based Genetic Studies

A method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents and these amplicons have GC contents ranging from 65–85 % and sizes up to 870 bp.

Enhancement of PCR Amplification of Moderate GC-Containing and Highly GC-Rich DNA Sequences

Conditions for optimized PCR to help resolve problems amplifying moderate to high GC-rich templates with moderate and extremely high CG-content are introduced.

Multiple heat pulses during PCR extension enabling amplification of GC-rich sequences and reducing amplification bias.

With this novel type of protocol, all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes, are amplified without addition of cosolvents.

PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.

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 that have been reported to resolve or alleviate problems caused by secondary structures in the DNA.

PCR procedures to amplify GC-rich DNA sequences of Mycobacterium bovis

The result demonstrated the superiority of the 2-step PCR protocol over other protocols in PCR amplification of Mb0129 when specific high fidelity DNA polymerases were used in the presence of an enhancer.



Successful amplification of extremely GC-rich promoter regions using a novel 'slowdown PCR' technique.

OBJECTIVES PCR has become a routine technique in DNA genotyping for diagnostic or pharmacogenetics purposes. Promoter regions of genes are in the main focus for detecting novel regulatory single

Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR.

The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites.

Optimization of the annealing temperature for DNA amplification in vitro.

The optimal annealing temperature (TaOPT) values for several primer-template pairs are experimentally determined and a method for its calculation is developed and found to be a function of the melting temperatures of the less stable primer- template pair and of the product.

Formamide can dramatically improve the specificity of PCR.

PCR of genomic segments from mammals typically generates acceptable specificity, however, specificity is much more difficult to achieve for segments of high GC content, and poor specificity was observed in five GC-rich segments of the human dopamine D2 receptor gene.

7-Deaza-2`-deoxyguanosine allows PCR and sequencing reactions from CpG islands

The use of 7-deaza-2`-deoxyguanosine for PCR amplification of the human p16INK4A promoter and sequencing of HUMARA exon 1 PCR products significantly improves results, particularly when small amounts of poor quality DNA are available as starting material.

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.

Improved PCR method for amplification of GC-rich DNA sequences

An improved polymerase chain reaction (PCR) method is proposed to be used for successful amplification of the tissue factor pathway inhibitor (TFPI)-2 gene promoter region that exhibit >70% G+C content in a sequence of approx 300 bp and a complete CpG island region spanning exon 1, the three transcription initiation sites, and the translation start site.

Optimization and troubleshooting in PCR.

  • K. Roux
  • Medicine, Chemistry
    Cold Spring Harbor protocols
  • 2009
Various optimization strategies are discussed, including touchdown PCR and hot-start PCR, which aim to vary one or more of the many parameters that are known to contribute to primer-template fidelity and primer extension.