Amplitaq gold buffer

DNA was amplified using a 1:3 ratio of cpn60 primer pairs (M729/M730 and M1612/M1613) modified to include MiSeq adaptors on the 5′ end (Table 1), as previously described [28 (link), 29 (link)], with 2 μL of extracted amplicon added to 48 μL of PCR mastermix: 1X AmpliTaq Gold Buffer (Applied Biosystems, Foster City, CA), 2.5 mM MgCl₂, 0.2 mM dNTPs, 0.05 U/μL AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA), 0.1 μM each of M729/M730, and 0.3 μM each of M1612/M1613 primers. For mock community DNA (manufacturer-extracted), 1 μL of the ZymoBIOMICS mock community D6305 DNA standard (Zymo Research, Orange County, CA, USA) was added to 49 μL PCR mastermix as above. No template (mastermix-only) controls were also used. For PCR amplification, initial denaturation at 95°C for 2 minutes was followed by 40 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 2 minutes, as previously described [30 (link)]. Products were visualized using the QIAxcel DNA Screening Kit (QIAGEN Inc., Toronto, ON, Canada) to confirm adaptor-ligated cpn60 UT amplicon size (~650 bp).

Up to 20 mg of prostate tissue were digested with proteinase-K and DNA was extracted using standard phenol-chloroform protocol followed by ethanol precipitation. Modification with sodium bisulfite was performed using EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol.
Bisulfite-modified DNA was used as a template for methylation-specific PCR (MSP) with ABCB1 primers [18 (link)] specific for either methylated or unmethylated DNA. The MSP reaction mix (25 μL) contained 1x AmpliTaq Gold Buffer, 2.5 mM MgCl₂, 1.6 mM dNTP mix, 1.25 U AmpliTaq Gold 360 DNA Polymerase (Applied Biosystems), 0.5 μM of each primer, and 1 μL of bisulfite-modified DNA. Amplification conditions were as follows: 95 °C for 10 min, then 37 cycles at 95 °C for 45 s, 60 °C for 45 s, and 72 °C for 45 s, and final extension at 72 °C for 10 min. The MSP products were run on 3 % agarose gel with ethidium bromide staining. Bisulfite-modified leukocyte DNA from healthy donors served as a negative control for methylated DNA and SssI methylase-treated (Thermo Fisher Scientific) bisulfite-modified leukocyte DNA served as a positive control. Non-template controls were included in each PCR run.

PCR amplification and sequencing of rpoB was performed as described elsewhere (71 (link)). Briefly, PCR was performed in 25-µl volumes containing 1× AmpliTaq Gold buffer (Applied Biosystems, Foster City, CA), 3 mM MgCl₂, 2.5 mM each deoxynucleoside triphosphate (Promega, Madison, WI), 0.4 µM rpoB forward primer (5′-GAGCGCATGACCACCCAGGACGTCGAGGC-3′), 0.4 µM rpoB reverse primer (5′-CCTCGTAGTTGTGACCCTCCCACGGCATGA-3′), 10% dimethyl sulfoxide, 1.25 U AmpliTaq Gold (Applied Biosystems, Foster City, CA), and 50 to 200 ng DNA. The following reaction conditions were used: 95°C for 10 min for initial denaturation, followed by 35 cycles of 95°C for 20 s, 65°C for 30 s, and 72°C for 45 s, and final extension at 72°C for 10 min. Sequencing of PCR products was performed at the Cornell Life Sciences Core Laboratories Center. Sequences were assembled manually, and trace files were inspected visually and uniformly trimmed to achieve a final length of 377 bp.
Sequences used in phylogenetic analyses were aligned using MUSCLE (72 (link)). Maximum likelihood trees were constructed with the general time-reversible model of nucleotide substitution (73 (link)), incorporating an estimated proportion of invariant sites and discrete gamma distribution (GTR+I+G) within the RAxML program (74 (link)). Trees were rooted using Mycobacterium smegmatis as the outgroup.

The six STRs markers (#022, #108, #138, #189, #278, and #279) were amplified separately as previously described (Gits-Muselli et al., 2015 (link)). Briefly, the forward primers were tagged with FAM, HEX or ATTO565 fluorophores. PCR reactions were performed on a GeneAmp PCR System 9700 Thermocycler (Applied Biosystems) in a final volume of 20 μL. The reaction mixture was composed of 1 × AmpliTaq Gold buffer (Life technologies) with 0.25 μM of each primer, 2.5 mM of MgCl2, 0.8 μM of dNTPs, 0.25 UI of AmpliTaq Gold polymerase (Life technologies) and 2 μL of DNA. The PCR protocol was 10 min at 95°, followed by 35 cycles of 30 s at 95 °C (denaturation), 30 s at 56° (primer annealing) and 60 s at 72 °C (extension) followed by a final extension of 10 min at 72 °C. A calibration sample was used in each PCR run as an internal control. Then, 2 μL of the PCR product was prepared for fragment analysis by the addition of 18 μL of formamide (Life Technologies) and 1 μL of Genescan-500 LYZ Size Standard (Life Technologies). The lengths of the PCR fragments were determined by capillary electrophoresis (50 mm) on an ABI 3500 genetic analyser using denaturing polymer POP-7 (Life Technologies) at 60 °C. Analysis was performed with the ABI Gene mapper v4.1 software (Life Technologies).

Ten previously described STRs markers VNTR2bis, VNTR3, VNTR4, VNTR5, VNTR6, VNTR8, VNTR9 (Brisse et al., 2009 (link)), GLM5, GLM6 (Abbes et al., 2012 (link)), and Cg6 (Grenouillet et al., 2007 (link)) were amplified. The forward primers were tagged with fluorophores (FAM, HEX, or ATTO565). All PCR reactions were performed on a GeneAmp PCR System 9700 Thermocycler (Applied Biosystems) in a final volume of 20 μL containing 1X Ampli Taq Gold buffer (Life technologies) with 0.25 μM of each primer, 2.5 mM of MgCl2, 0.8 μM of dNTPs, 0.25 UI of Ampli Taq Gold polymerase (Life technologies), and 2 μL of DNA. The reaction consisted of 10 min at 95°C; 35 cycles of 30 s at 95°C, 30 s at 55°C, and 60 s at 72°C; and a final extension at 72°C for 10 min. After amplification, 2 μL of PCR product was prepared for fragment analysis by adding 18.5 μL of formamide (3700 formamide, Life technologies) and 0.5 μL of Genescan-500 LYZ Size Standard (Life technologies). Capillary electrophoresis was performed with the denaturing polymer POP-7 (Life technologies) in a 50-mm capillary tube at 60°C. The lengths of the PCR fragments were determined on an ABI 3500 genetic analyzer with ABI Gene mapper v4.1 software (Applied Biosystems). Rare genotypes were those for which fewer than 3 isolates/patient were found in our collection. Singletons are genotypes found in only one isolate of our cohort.

The six selected STRs were amplified separately by PCR since multiplexing failed to be as sensitive as single PCR (data not shown). The forward primers were tagged with fluorophores (FAM, HEX or ATTO565). All PCR reactions were performed on a GeneAmp PCR System 9700 Thermocycler (Applied Biosystems) in a final volume of 20 μL containing 1X Ampli Taq Gold buffer (Life technologies) with 0.25μM of each primer, 2.5mM of MgCl2, 0.8μM of dNTPs, 0.25 UI of Ampli Taq Gold polymerase (Life technologies) and 2 μL of DNA. The reaction consisted of 10 minutes at 95°, followed by 35 cycles of 30 s at 95°C (denaturation), 30 s at 56° (primer annealing) and 60 s at 72°C (extension) followed by a final extension of 10 min at 72°C. A sample with a mixed genotype at one locus (sample181) was used in each PCR run as an internal control and to measure reproducibility.