Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone*

Eilert, Kenneth D.; Foran, David R.

doi:10.1111/j.1556-4029.2009.01116.x

Cited by 51 publications

(31 citation statements)

References 46 publications

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“…It is well known that different DNA polymerases have different abilities to retain polymerizing activity in various complex reaction environments, explained either by their different origins [7][8][9][10] or Table 1 Improved DNA amplification of crime scene samples using alternative DNA polymerase-buffer systems. Note.…”

Section: Discussionmentioning

confidence: 99%

“…However, AmpliTaq Gold might not be the optimal choice for samples containing PCR inhibitors. In several studies, AmpliTaq Gold has shown lower tolerance to PCR-inhibitory substances compared with other DNA polymerases [7][8][9][10][11]. We have previously shown that short tandem repeat (STR) DNA analysis of complex crime scene samples can be improved by replacing AmpliTaq Gold with either of the alternative DNA polymerase-buffer systems Bio-X-Act Short (Bioline, London, UK), ExTaq Hot Start (TaKaRa Bio, Shiga, Japan), or PicoMaxx High Fidelity (Stratagene, La Jolla, CA, USA) [6].…”

Section: Introductionmentioning

confidence: 99%

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Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

Hedman

Nordgaard

Dufva

et al. 2010

Analytical Biochemistry

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a b s t r a c tThe success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 ''inhibited" crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

Hedman

Nordgaard

Dufva

et al. 2010

Analytical Biochemistry

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“…However, just as iron contributes to reduction of antibody reactivity (figure 3; electronic supplementary material, figure S5), it may also confound efforts to sequence biomolecules, by diminishing signals in mass spectrometry via ion suppression or by inhibiting enzymes required for DNA sequencing [81,82]. Iron chelation in soft tissue analysis is a technical advance in analysing biomaterials from fossil bone because chelation reduces signal inhibition in many fossil analyses, thus broadening the range of specimens from which molecular data may be obtained.…”

Section: Discussionmentioning

confidence: 99%

A role for iron and oxygen chemistry in preserving soft tissues, cells and molecules from deep time

Schweitzer¹,

et al. 2014

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The persistence of original soft tissues in Mesozoic fossil bone is not explained by current chemical degradation models. We identified iron particles (goethite-aFeO(OH)) associated with soft tissues recovered from two Mesozoic dinosaurs, using transmission electron microscopy, electron energy loss spectroscopy, micro-X-ray diffraction and Fe micro-X-ray absorption nearedge structure. Iron chelators increased fossil tissue immunoreactivity to multiple antibodies dramatically, suggesting a role for iron in both preserving and masking proteins in fossil tissues. Haemoglobin (HB) increased tissue stability more than 200-fold, from approximately 3 days to more than two years at room temperature (258C) in an ostrich blood vessel model developed to test post-mortem 'tissue fixation' by cross-linking or peroxidation. HB-induced solution hypoxia coupled with iron chelation enhances preservation as follows: HB þ O 2 . HB 2 O 2 . 2O 2 þO 2 . The well-known O 2 /haeme interactions in the chemistry of life, such as respiration and bioenergetics, are complemented by O 2 /haeme interactions in the preservation of fossil soft tissues.

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“…It was shown to be a N-terminal deletion of 278 amino acids along with some other polymorphisms [24]. Eilert et al [3] has shown that some DNA polymerases from T. aquaticus and other species can have a greater tolerance for inhibitory substances including humic acids.…”

Section: Introductionmentioning

confidence: 99%

“…The affects of inhibitory compounds on PCR is different depending on the inhibitory compound, the amplification conditions and the DNA polymerase being used [1][2][3][4][5]. Some substances will inhibit the enzymatic activity of the DNA polymerase while others will cause template inhibition.…”

Section: Introductionmentioning

confidence: 99%

Assessing PCR Inhibition from Humic Substances

Matheson¹,

Gurney²,

Esau³

et al. 2014

TOEIJ

120

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Inhibition remains the greatest methodological challenge in molecular analysis of buried biological remains. Inhibitory compounds associated with soil environments comprise primarily of humic acids and fulvic acids, collectively referred to as humic substances. We examined the sensitivity of 13 DNA polymerases to both humic acids (11ng-110 g) and fulvic acids (9.4ng-94 g) and the concentration at which successful amplification can be achieved. This research identified that all 13 DNA polymerases tested exhibited inhibition with varying concentrations of humic acids and that 5 out of the 13 DNA polymerase tested exhibited inhibition with varying concentrations of fulvic acid. The most tolerant DNA polymerase to inhibition due to the presence of humic and fulvic acids is pfu DNA polymerase followed by KlenTaq ® LA DNA polymerase and RealTaq DNA polymerase that were both only inhibited by 11 g and 110 g of humic acids. In addition, we present the use of size exclusion chromatography to remove small molecular weight humic substance, dramatically increasing the success of molecular analysis on material associated with burial. This research has implications to the fields of environmental microbiology, soil science, forensic science and archaeological science.

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Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone*

Cited by 51 publications

References 46 publications

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

A role for iron and oxygen chemistry in preserving soft tissues, cells and molecules from deep time

Assessing PCR Inhibition from Humic Substances

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