Amplification efficiency of thermostable DNA polymerases

Arezi, Bahram; Xing, Weimei; Sorge, Joseph A.; Hogrefe, Holly H.

doi:10.1016/s0003-2697(03)00465-2

Cited by 129 publications

(106 citation statements)

References 22 publications

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“…These values correspond to an increase in the frequency of 5′-CNaM from 18.71% to 24.65%, with the subpopulation having a 5′-GCNaM sequence increasing from 2.30% to 3.48%. These biases are not larger than the biases observed among natural sequences (39), and they are unlikely to interfere with any in vitro application of DNA containing the unnatural base pair, even including those applications requiring massive amplification.…”

Section: Resultsmentioning

confidence: 93%

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Malyshev

Dhami²,

Quach³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

159

130

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The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.expanded genetic alphabet | hydrophobic | artificial DNA | unnatural nucleotides | bioinformatics E xpansion of the genetic alphabet to include an unnatural base pair has emerged as a central goal of chemical and synthetic biology. Success would represent a remarkable integration of orthogonal synthetic components into a fundamental biological system and build the foundation for a semisynthetic organism with increased potential for information storage and retrieval (1). Moreover, the constituent unnatural nucleotides could be used to site-specifically label DNA or RNA with different functionalities of interest (2-4) and potentially revolutionize the already ubiquitous in vitro applications of nucleic acids, such as aptamer and DNA/RNAzyme selections (5, 6), PCR-based diagnostics (7, 8), and DNA-based nanomaterials and devices (9).Although many candidate unnatural base pairs have been reported (10-21), only a few are actually replicable by DNA polymerases (10,11,13,16). Moreover, it is clear that most applications will require that the unnatural base pair not only be replicated with high efficiency and fidelity but also, that replication be at least approximately independent of sequence context. Sequence dependencies would cause biased amplification and effectively preclude many uses of the unnatural base pair. No candidate unnatural base pair has been shown to be replicated without sequence bias, and thus, none can yet claim functional equivalence to a natural base pair.In general, the most promising unnatural base pair candidates currently available have been developed by pursuing one of two different strategies. The first strategy, pioneered in the work by Benner and coworkers (22), relies on the use of nucleotide analogs bearing nucleobases that pair through complementary hydrogen bonding (H-bonding) patterns that are orthogonal to those patterns of the natural pairs. Early ef...

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Section: Resultsmentioning

confidence: 93%

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Malyshev

Dhami²,

Quach³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

159

130

View full text Add to dashboard Cite

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“…As SYBR Green intercalates nonspecifically into all double-stranded DNA molecules during the reaction, the assay must rely entirely on the PCR primers for specificity (21). In addition, the inhibition of the Taq DNA polymerase by SYBR Green could result in lower specificity (2). In our experiments, the annealing temperature was lowered in the reactions using SYBR Green compared with those using internal probes to counter this inhibition.…”

Section: Discussionmentioning

confidence: 99%

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

Bilodeau¹,

Lévesque²,

Cock³

et al. 2007

Phytopathology®

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Bilodeau, G. J., Lévesque, C. A., de Cock, A. W. A. M., Duchaine, C., Brière, S., Uribe, P., Martin, F. N., and Hamelin, R. C. 2007. Molecular detection of Phytophthora ramorum by real-time polymerase chain reaction using TaqMan, SYBR Green, and molecular beacons. Phytopathology 97:632-642.Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), β-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using β-tubulin and seemed to be more sensitive.

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“…19,21,22 It has been noted that the GC content of DNA can significantly affect polymerase chain reaction amplification efficiency, sometimes causing premature chain termination at the beginning of G(C)-rich regions. [23][24][25][26] Although the exact mechanism of how GC content may affect DNA polymerase function is not well understood, it has been hypothesized that amplification of GC-rich templates can be hampered because of the formation of secondary structures such as hairpins and by higher melting temperatures, which can inhibit primer extension by DNA polymerases as well as enzyme processivity. 24,[27][28][29] These variables may also significantly affect the displacement of the complementary DNA strand in the branching reaction.…”

Section: Discussionmentioning

confidence: 99%

Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

Bredel

Juric

et al. 2005

The Journal of Molecular Diagnostics

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Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffinembedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalinfixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fineneedle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. The availability of a simple and reliable method for the amplification of entire genomes from limited sources of DNA would lend itself to high-throughput genetic analyses in virtually all areas of translational research. High-throughput genomic profiling, for example cDNA microarray-based comparative genomic hybridization (array-CGH), 1 requires g quantities of genomic DNA. A particular challenge for translation of array-CGH methodology to clinical application is to link it to a robust up-front technology that allows reliable and unbiased amplification of limited sources of DNA, for example from fine-needle biopsies. Much effort has been invested in developing methods for whole genome amplification, for example polymerase chain reaction-based methods. [2][3][4] In particular, the pitfalls of substantial variation in the extent of amplification occurring between different markers, incomplete representation, and inadequate average DNA size have limited the use of most existing amplification methods, making them particularly unsuitable for genomic applications. 2-4Bacteriophage Phi29 DNA polymerase random-primed DNA amplification 5-7 is based on an isothermal strand displacement amplification reaction in which random hexamer primers anneal to the genomic template at multiple sites and Phi29 initiates replication at these sites on the denatured linear DNA. As synthesis proceeds, strand displacement of complementary DNA generates new single-stranded DNA available to be primed by additional primers. The subsequent strand displacement replication of this DNA leads to the formation of double-stranded DNA. The presence of an associated proofreading activity of Phi29 ensures a high sequence accuracy of the amplified DNA, indicating its suitability f...

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Amplification efficiency of thermostable DNA polymerases

Cited by 129 publications

References 22 publications

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

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