Figure 1.
Amplification of conserved MLV-like regions from mouse tail DNA.
13 different PCRs (Table S2) were conducted with 10 pg or 1 pg mouse tail DNA as indicated. M – GeneRuler 1 kb Plus DNA Ladder, 75–20,000 bp (Fermentas) - selected fragment sizes are as indicated on the 2% agarose gel images. H2O – no template, PCR negative control.
Figure 2.
Alignment of primers with plasmid clone of 419F/1154R amplicon.
Figure 3.
Detection sensitivity of gagO and gagI PCR with various amounts of mouse tail DNA in 500 ng of human whole blood DNA.
(A) gagO amplicons were not visible after 30 cycles but with further cycling to a total of 45 cycles, gagO amplicons were amplified from 10 pg, 1 pg, and 100 fg mouse tail DNA. (B) 40 cycles of gagI PCR following 30 cycles of gagO PCR showed amplicons from 10 pg, 1 pg and 100 fg mouse tail DNA. As a positive control, VAMP2 PCRs resulted in amplicons from all samples with human DNA. M – GeneRuler 1 kb Plus DNA Ladder, 75–20,000 bp (Fermentas, Glen Burnie, Maryland). H2O – no template, PCR negative control.
Figure 4.
Detection sensitivity of gagL PCR with various amounts of mouse tail DNA.
(A) 500 ng and (B) 50 ng of human whole blood DNA. gagL amplicons were detected in 1 pg of mouse tail DNA. M – GeneRuler 1 kb Plus DNA Ladder, 75–20,000 bp (Fermentas). H2O – no template, PCR negative control.
Figure 5.
Sensitivity of PCR in detection of gag and mouse-specific targets in 500 ng of human PBMC DNA with a range of mouse tail DNA concentrations.
The same master DNA dilution series was used as template in each of the PCR amplifications. gagL fragments were amplified from 1 pg mouse tail DNA, as were gagI fragments following gagO PCR. coxI following coxO PCR showed detection limit of 100 fg. IAP PCR was successful from 10 pg through 10 fg and variably sized fragments were observed between 200 and 300 bp; the lower amplicons of approximately 200 bp were sequenced and matched human DNA sequences. VAMP2 PCR was the positive quality control for human DNA. M – GeneRuler 1 kb Plus DNA Ladder, 75–20,000 bp (Fermentas). H2O – no template, PCR negative control.
Figure 6.
PCR detection of gag but not mouse sequences in 500 ng of whole blood gDNA.
Fragments were separated on 2% agarose. (A) gagL fragments at 367 bp observed in 2 samples. (B) Neither mCOX2 nor IAP were amplified from the same 2 DNA samples, by nested coxO followed by coxI PCR, or IAP PCR, respectively. The amplicons of IAP PCR as indicated by the arrows were sequenced and were non-specific human DNA amplification, not of mouse origin. M – GeneRuler 1 kb Plus DNA Ladder, 75–20,000 bp (Fermentas). H2O – no template, PCR negative control. There is an empty lane between coxI and IAP lanes.
Figure 7.
DNA sequence alignment of gag sequences with selected MLV-like sequences and mouse tail DNA sequences.
Nucleotides that are identical to the reference 22Rv1Genome (Genbank FN692043) sequence are shown as dots and gaps are shown as dashes. Pmv9, Mpmv10, Xmv12, Xmv43, Xmv8 are endogenous polytropic, modified polytropic, and xenotropic viral sequences from the C57BL/6J genome that group into different clades according to Jern et al [18]. All other sequences shown were identified in this study. Sequences of nested gagO/gagI and gagL amplicons were distinct from representative MLV-like sequences, especially from the xenotropic sequences. Sequences amplified from blood and from mouse tail DNA were not 100% identical.
Figure 8.
Phylogenetic tree created from alignments of sequences obtained from gagO/gagI PCR.
Phylogenetic tree was created with DNASTAR MegAlign Version 8.1.2. by ClustalW (weighted) method. The LoMuLV sequences [9], XMRV-VP62 [19], and XMRV-WPI1106 [1] sequences were taken from Genbank. All other sequences shown were identified in this study.
Figure 9.
Phylogenetic tree created from alignments of sequences obtained from gagL PCR.
Phylogenetic tree was created with DNASTAR MegAlign Version 8.1.2. by ClustalW (weighted) method. Sequences described in legends to Figure 7 and 8.