Technical note: Removal of metal ion inhibition encountered during DNA extraction and amplification of copper‐preserved archaeological bone using size exclusion chromatography

Abstract

A novel technique for the removal of metal ions inhibiting DNA extraction and PCR of archaeological bone extracts is presented using size exclusion chromatography. Two case studies, involving copper inhibition, demonstrate the effective removal of metal ion inhibition. Light microscopy, SEM, elemental analysis, and genetic analysis were used to demonstrate the effective removal of metal ions from samples that previously exhibited molecular inhibition. This research identifies that copper can cause inhibition of DNA polymerase during DNA amplification. The use of size exclusion chromatography as an additional purification step before DNA amplification from degraded bone samples successfully removes metal ions and other inhibitors, for the analysis of archaeological bone. The biochemistry of inhibition is explored through chemical and enzymatic extraction methodology on archaeological material. We demonstrate a simple purification technique that provides a high yield of purified DNA (>95%) that can be used to address most types of inhibition commonly associated with the analysis of degraded archaeological and forensic samples. We present a new opportunity for the molecular analysis of archaeological samples preserved in the presence of metal ions, such as copper, which have previously yielded no DNA results.

The effects of metal ion PCR inhibitors on results obtained with the Quantifiler ® Human DNA Quantification Kit

“…Metal ions have the potential to function as inhibitors of PCR-based STR assays and are present in a variety of forensically-relevant sample types, e.g., bones [19]. Furthermore, metal ions can form soluble, high molecular weight complexes with humic substances in soils, have been implicated in adduct-formation with DNA, and can form crosslinks between DNA and proteins [20][21][22][23]. These interactions can reduce the efficiency of DNA extraction techniques and impair access to the DNA template during PCR.…”

Research Article Inexpensive metagenomic DNA extraction protocol with high quality from marine sediments contaminated by petroleum hydrocarbons.

“…Metal ions can form soluble, high-molecular-weight complexes with humic substances in soils. Additionally, they can form adducts with DNA and can form crosslinks between DNA and proteins (Matheson et al, 2009). These chemical interactions can impair access to the DNA template during PCR.…”

“…In particular, marine soils are commonly rich in fulvic and humic acids, which are frequently co-extracted with DNA (Nair et al, 2014) because they possess physical and chemical characteristics similar to DNA molecules and thus are too difficult to remove (Fitzpatrick et al, 2010). Heavy metals are also present in marine sediments and strongly inhibit any reaction in PCR amplification (Matheson et al, 2009). Contaminants other than proteins should be kept to a minimum (Felczykowska et al, 2015).…”

Minimizing inhibition of PCR-STR typing using digital agarose droplet microfluidics

“…These techniques can be generally divided into two categories: (1) novel DNA extraction procedures to purify the target DNA prior to PCR amplification; and (2) modification of the DNA amplification process to minimize the effect of inhibitors that are not removed. The pre-PCR removal methods produce PCR-compatible DNA samples using silica-based extraction [9][10][11][12][13], magnetic separation [14][15][16], column chromatography [17][18][19], and other new protocols [20,21]. Mitigation of inhibition during PCR can also be achieved by extensive dilution of DNA extracts [22,23], utilization of genetically engineered DNA polymerases with better resistance to inhibitors (e.g.…”