seeks to explore if and how contamination might be induced which in
turn informs the scientific process.
PLoS One. 2011;6(8):e23484. Epub 2011 Aug 18.
DNA extraction columns contaminated with murine sequences.
Erlwein O, Robinson MJ, Dustan S, Weber J, Kaye S, McClure MO.
Jefferiss Research Trust Laboratories, Section of Infectious Diseases,
Imperial College London, London, United Kingdom.
Abstract
Sequences of the novel gammaretrovirus, xenotropic murine leukemia
virus-related virus (XMRV) have been described in human prostate
cancer tissue, although the amounts of DNA are low. Furthermore, XMRV
sequences and polytropic (p) murine leukemia viruses (MLVs) have been
reported in patients with chronic fatigue syndrome (CFS).
In assessing the prevalence of XMRV in prostate cancer tissue samples
we discovered that eluates from na=EFve DNA purification columns, when
subjected to PCR with primers designed to detect genomic mouse DNA
contamination, occasionally gave rise to amplification products.
Further PCR analysis, using primers to detect XMRV, revealed sequences
derived from XMRV and pMLVs from mouse and human DNA and DNA of
unspecified origin. Thus, DNA purification columns can present
problems when used to detect minute amounts of DNA targets by highly
sensitive amplification techniques.
Discussion
There are many commercially available kits that rely on DNA binding
columns to extract and purify DNA from tissues or cultured cells. Our
observations by two different laboratory investigators (OE and MJR)
using three different kits and working in separate laboratories,
demonstrate that they can be contaminated with DNA of diverse
provenance. This includes DNA from mice. It cannot be ruled out that
some of the buffers used during the DNA extraction process were
contaminated and, in turn, resulted in contamination of some of the
columns. We tested the elution buffers from several kits and found no
evidence for contamination. A confounding issue was the fact that
tissue lysis buffers and washing buffers in these kits were found to
contain substances that inhibited the PCR and, therefore, the buffers
could not be reliably tested. Therefore, it is possible that these
buffers contain traces of DNA which bind to the columns and are eluted
in later steps, contaminating the sample. However, it is telling that
dismantled column parts soaked in elution buffer resulted in an IAP
signal while the elution buffer control did not, suggesting that the
columns themselves can be contaminated.
Recently, several publications documented that widely used PCR enzymes
and buffers can be contaminated with murine DNA [13], [14], [24].
Taken together with the data presented here, these results may explain
some of the spurious =93detections=94 of XMRV or related pMLV sequences
[2], even in laboratories that use neither mice nor XMRV-infected cell
lines and avoid enzymes known to contain traces of murine DNA. For
those involved in detecting minute amounts of retroviral sequences in
human tissue, these data may serve as a useful reminder to check
reagents to confirm that murine sequences are absent before analysing
tissue samples.
PMID: 21876752 [PubMed - in process] PMCID: PMC3158089
Full text of the paper can be accessed here:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0023484
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