DNA extraction columns contaminated with murine sequences

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 naïve 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.

Figure 1. Amplification of contaminating DNA from empty columns of the QiaAmp FFPE Tissue Kit.

Lanes 1–10, naïve DNA extraction columns; lanes 11–14, PCR water controls; lane 15, positive control; upper panel, McCoy cellular DNA; middle and lower panel, XMRV VP62 infectious clone; lane 16, 100 bp DNA ladder (Invitrogen, Paisley, UK). Upper panel, detection of contaminating sequences using IAP-specific primers IAP for and IAP rev. All columns apart from column no 7 produce amplicons. Size differences reflect the fact that IAP sequences form a class of slightly different retrotransposons. Middle panel, PCR products using primers XTP1 and MLV reverse outer under relaxed annealing conditions. Lower panel, multiplex PCR using the four primers XMRV-R, XMRV Forward outer, XMRV Reverse outer and 1154R under less stringent annealing conditions.

Figure 2. Alignment of the infectious molecular clone of XMRV, VP 62, and sequences obtained from mock eluted columns.

(A) Alignment of the leader/gag region, displaying the XMRV specific deletion. (B) Alignment of the env region.