Premix ex taq dna polymerase

Genomic DNA of A. fumigatus was extracted from frozen mycelia as described [113 ]. One ng of DNA was used for real-time qPCR analysis using Premix Ex Taq DNA polymerase (Takara, cat. no. RR039W) on ABI Fast 7500 (Applied Biosystems) Real-Time PCR machine using oligos listed in S12 Table. The ΔΔCt method was used for quantification using the tubulin gene tubA as an internal reference.

The bacterial number of ETEC K88 shed in the feces was determined by quantitative real-time PCR targeting a 70-bp fragment of the K88 fimbrial gene as described by West et al. [14 (link)]. Briefly, the genomic DNAs in the feces were extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The PCR mixture contained 2 μL of DNA template, 100 pmol of the forward primer (5′-GGTTCAGTGAAAGTCAATGCATCT-3′), 100 pmol of the reverse primer (5′-CCCCGTCCGCAGAAGTAAC-3′), 0.5 μL of the probe (Cy5-5′-CCACCTCTCCCTAACACACCGGCAT-3′-BHQ2; GenoTech, Daejeon, Korea), and 12.5 μL of Premix EX Taq DNA polymerase (Takara Bio, Shiga, Japan) in a total volume of 25 μL. Thermal cycling was performed with an initial denaturation at 95°C for 10 min, followed by 45 cycles of 95°C for 20 sec, 62°C for 30 sec, and 72°C for 30 sec. The cfu of ETEC K88 in each fecal sample was assessed from the standard curve for the ETEC K88 standard solution plotted against the threshold (Ct) value using the Smartcycler software (Cepheid, Sunnyvale, CA).

Rs10507391 and rs4769874 polymorphisms in the ALOX5AP gene were investigated. Genomic DNA was extracted from peripheral blood leukocytes using a standard phenol-chloroform method. Primers used for detecting polymorphisms were synthesized by Sangon Biotech (Shanghai) and were as follows: 1) rs10507391 (forward) 5′ GTG TTC AGG AAG GGA GTT TCT GT 3′ and (reverse) 5′ GTC TAT GGT TGC AAC ATT GAG ATT A 3′; and 2) rs4769874 (forward) 5′ CCC ACT TTC CTC GCT GTG CT 3′ and (reverse) 5′ CCG AAA GGG GAC CAA AAG TA 3′. Polymerase chain reaction (PCR) was performed in a 25 µl reaction volume containing 1 µl (0.1 µg) of genomic DNA, 12.5 µl of Premix Ex Taq DNA polymerase (Takara Biotechnology, Dalian), 1 ul of each primer and 9.5 µl of sterile water. PCR conditions were as follows: 95°C for 5 min; (95°C for 30 s; 60°C for 30 s; 72°C for 40 s) ×30 cycles; 72°C for 10 min. PCR products were subsequently restriction-digested at 37°C overnight for restriction fragment length polymorphism (RFLP) analysis. rs10507391 products were digested with VspI (Takara Biotechnology, Dalian), and rs4769874 products were digested with BstuI (New England Biolabs). The digested products were electrophoresed on 3% (rs10507391) and 2% (rs4769874) agarose gels and genotypes were determined using a gel imaging and analysis system. Several PCR products were sequenced to verify RFLP data.

Bisulfite genomic sequencing is regarded as a gold-standard for detection of DNA methylation, because it provides a qualitative and quantitative method to apply to a limit number of loci [64 ]. Twenty-five DMRs were selected randomly to validate the reliability of the sequencing data using a nest PCR (nPCR). All the primers (Additional file 10) were designed using the website of Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl), and synthesized commercially (Invitrogen, Shanghai, China). Briefly, 1 μg of genomic DNA was treated by bisulfite according to the protocol of the EZ DNA Methylation-Gold Kit (Zymo Research, CA, USA), and used as the template for the following nPCR amplification. Then, the bisulfite treatment PCR (BS-PCR) was performed in a 50 μl reaction mixture, containing 25 μl premix EX Taq DNA polymerase (TaKaRa, Osaka, Japan), 25 μg bisulfite-treated DNA and 0.2 μM of each pair of primers with a nest PCR. Next, products were purified using Gel Extraction Kit (Axygen, CA, USA), and cloned into the pMD19-T Simple Vector (TaKaRa, Osaka, Japan). Each DMR amplified with 10–14 clones was sequenced by Invitrogen Trading Shanghai Co.Ltd. (Shanghai, China), and the sequencing results were spliced and edited by BioEdit and vector NTI8 software, then analyzed in the Kismeth website.