Firestormm
Senior Member
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Morning, I can't see that this has been posted before. If it has please remove. The full text is available free is anyone's interested from the same link and I have pasted the 'discussion' beneath this abstract:
Note: this was published in August 2011 and maybe it hasn't been available in full for free before but you might just want to shove it in the library or something.
DNA extraction columns contaminated with murine sequences: http://www.ncbi.nlm.nih.gov/pubmed/21876752
Erlwein O, Robinson MJ, Dustan S, Weber J, Kaye S, McClure MO.
Source: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 nave 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 detections 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.'
Note: this was published in August 2011 and maybe it hasn't been available in full for free before but you might just want to shove it in the library or something.
DNA extraction columns contaminated with murine sequences: http://www.ncbi.nlm.nih.gov/pubmed/21876752
Erlwein O, Robinson MJ, Dustan S, Weber J, Kaye S, McClure MO.
Source: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 nave 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 detections 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.'