A Method for Isolation Bacteriophage Particles-Free Genomic DNA, Exemplified by TP-84, Infecting Thermophilic Geobacillus

Abstract

DNA purification methods are indispensable tools of molecular biology, used for many decades. Nevertheless, for certain specialized applications, the currently employed techniques are not sufficiently effective. While examining a number of the existing methods to purify the genomic DNA of the thermophilic bacteriophage TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus), we have found out that the obtained DNA is contaminated with trace amounts of infectious TP-84 particles. This was detrimental for the bacteriophage genetic manipulation purposes, as finding the recombinant TP-84 clones was essentially impossible due to the appearance of a high background of native bacteriophage plaques. Thus, we have developed a method, which enables the fast and efficient isolation of a bacteriophage genomic DNA from concentrated phage preparations, obtained using CsCl gradient ultracentrifugation, without the need to remove concentrated CsCl solutions. The method employs silica columns and mini-scale isolation of microgram amounts of high quality DNA. It is universal-the silica mini-columns from various manufacturers can be used to conduct the procedure. The purified DNA, free from infectious bacteriophage particles, is ready for further manipulations. This is particularly important for such thermophilic bacteriophages that may partially survive standard isolation procedures and contaminate the final DNA product.

Keywords: DNA purification; TP-84; bacteriophage; genomics; phage display; thermophile.

Figure 1

Scheme illustrating the method for isolation of bacteriophage genomic DNA, free of bacteriophage particles.

Figure 2

Digestion of E. coli genomic DNA with DNase I (new batch, glycerol stock) in the presence of various CsCl concentrations. A total of 0.4 µg of E. coli genomic DNA was digested with 4 µg DNase I at 37 °C for 30 min and inactivated at 80 °C for 15 min. Lane M, GeneRuler 1 kb DNA Ladder; lane K, undigested E. coli DNA (0.4 µg); lanes 1–6, samples with CsCl diluted 1:10, 1:9, 1:8, 1:7, 1:6 and 1:5; lane K1, sample without CsCl; lane K2, sample without DNase I, CsCl diluted 1:20; lane K3, sample without DNase I, CsCl diluted 1:5.

Figure 3

Digestion of E. coli RNA with RNase A in different concentration of CsCl. A total of 7 µg of E. coli RNA was digested with 3.5 µg RNase A at 37 °C for 30 min. RNase-digested RNA was purified using a RNA Clean-Up kit. A total of 1.2% agarose gel electrophoresis: Lane M, GeneRuler 1 kb DNA Ladder; lane K, undigested E. coli RNA (7 µg); lanes 1–6, samples with CsCl diluted 1:10, 1:9, 1:8, 1:7, 1:6 and 1:5; lane K1, a sample without CsCl; lane K2, sample without RNase A and CsCl; lane K3, sample without RNase A and CsCl diluted 1:5.

Figure 4

Effect of the presence of various concentrations of CsCl on the activity of proteinase K. Lysate E. coli pPR B5 (0.25 mL culture, OD₆₀₀ = 3.6) was digested with 48 µg of proteinase K in various concentrations of CsCl at 55 °C for 30 min. SDS-PAGE/glycine analysis, 12% gel: Lane M, protein marker (PageRuler, Prestained Protein Ladder, 10 to 180 kDa); lanes 1–3 samples with CsCl diluted 1:10, 1:7, 1:5; lane K1, sample without CsCl; lane K2, sample without proteinase K, CsCl diluted 1:5; lane K3, sample without CsCl and proteinase K; lane P, proteinase K (80 µg); lane L, lysozyme (50 µg).

Figure 5

DNA of bacteriophage TP-84 extracted with four different commercially available lysis buffers together with dedicated spin-columns. Samples were electrophoresed on 1% agarose/TBE gel. Lane M1, GeneRuler 1 kb DNA Ladder; lane 1, Supplier S1; lane 2, Supplier S2; lane 3, Supplier S3; lane 4, Supplier S4.

Figure 6

Bacteriophage TP-84 DNA isolated using spin-columns obtained from the Supplier S1 and here developed lysis buffers. Samples were electrophoresed on 1% agarose/TBE gel. Lane M, GeneRuler 1 kb DNA Ladder; lane 1, Supplier S1 buffer; lane 2, lysis buffer LB1; lane 3, lysis buffer LB2; lane 4, lysis buffer LB3; lane 5, lysis buffer LB4.

Figure 7

Bacteriophage TP-84 DNA isolated using spin-columns obtained from the Supplier S1 and developed lysis buffers. The quality of the obtained DNA was tested by cleavage with BglII restriction endonuclease at 37 °C for 20 min (Bgl) and control incubation at 37 °C for 1 h (37) in the BglII digestion buffer. Samples were electrophoresed in 1% agarose/TBE gel. Lane K—undigested DNA; lane M, GeneRuler 1 kb DNA Ladder; lanes 1, Supplier S1 buffer; lanes 2, lysis buffer LB1; lanes 3, lysis buffer LB2; lanes 4, lysis buffer LB3; lanes 5, lysis buffer LB4.

Figure 8

The effect of mixing of the reaction mixture at the stage of lysis and incubation in 80 °C on the viability of contaminating TP-84 bacteriophage. The G. stearothermophilus 10 (StrR) was mixed with 5 µL of the purified TP-84 genomic DNA. The mixture was embedded in 0.65% agar TYM broth and incubated on solid agar plates at 55 °C: 1, with host bacterium only; 2, TP-84 DNA isolated with thorough mixing, CsCl diluted 1:5; 3, TP-84 DNA isolated without mixing and CsCl diluted 1:5.