Improved efficacy of whole genome amplification from bacterial cells

Figures & data

Figure 1.  Multiple displacement amplification (MDA) products of Salmonella enteritidis LK5.

Whole genomic DNA was amplified from approximately 600 (lane 2) and 60 (lane 3) colony-forming units (cfus) after releasing genomic DNA by heating at 98°C for 10 min. S. enteritidis-specific fragments (294 bp) were amplified from the respective MDA products (lanes 4 and 5). Purified S. enteritidis LK5 genomic DNA was used as positive control to amplify sdf I fragment indicated by the arrow (lane 6). A 1-kb DNA ladder (Invitrogen, Carlsbad, CA, USA) was used as a standard marker (M).

Table 1.  PCR Primers and Sequences

Primer	Sequence
sdf I-F	5′-TGTGTTTTATCTGATGCAAGAGG-3′
sdf I-R	5′-CGTTCTTCTGGTACTTACGATGAC-3′
63F	5′-CAGGCCTAACACATGCAAGTC-3′
1389R	5′-ACGGGCGGTGTGTACAAG-3′
rpoS-F	5′-AGATCTTCTTCTTCAGCCAGGTCGTT-3′
rpoS-R	5′-AGATCTCATCCGGTACGCCAATTACT-3′

All of the oligonucleotides were obtained from Integrated DNA Technologies, Coralville, IA, USA.

Figure 2.  Effect of PCR buffer on multiple displacement amplification (MDA) reaction.

Whole genomic DNA was amplified from approximately 600 (lanes 1 and 2), 60 (lane 3), and 6 (lane 4) colony-forming units (cfus). All samples were heated at 98°C for 10 min in the presence of 2× PCR buffer before the MDA reactions except lane 1, which contained double-distilled water instead. Whole genomic DNA was also amplified using the genomic DNA purified from Salmonella enteritidis LK5 as a template (lane 5). A 1-kb DNA ladder (Invitrogen) was used as a standard marker (M).

Figure 3.  Multiplex PCRs from multiple displacement amplification (MDA) products of Salmonella enteritidis LK5.

MDA products amplified from approximately 600 (lanes 1 and 2), 60 (lane 3), and 6 (lane 4) colony-forming units (cfus) were used as templates to amplify three loci in multiplex PCR (294 bp for sdf I; 800 bp for rpoS promoter; 1300 bp for 16S rRNA gene). The MDA reactions were performed using genomic DNA released by heating at 98°C for 10 min in the presence of 2× PCR buffer except lane 1, which contained double-distilled water instead. S. enteritidis LK5 genomic DNA was used as a positive control for the mutiplex PCR (lane 5). A 100-bp DNA ladder (Promega, Madison, WI, USA) was used as a standard marker (M).