Smai restriction enzyme

Plasmids allowing controlled expression of human p53 proteins pT7-7wtp53 (full length wild type p53, aa 1–393), pGEX-4Tp53CD (GST-p53CD, aa 94–312) and pGEX-2TKp53CT (GST-p53CT, aa 320–393) were described previously [5 (link)]. Nonspecific competitor (pBSK/SmaI) was prepared by SmaI restriction enzyme (Takara, Japan) cleavage of pBluescript SK II- (pBSK, Stratagene). pPGM1 was derived from pBSK by cloning 20bp p53 consensus sequence into HindIII restriction site. Plasmids pBSK/TTC and pPGM1/TTC were prepared by cloning into the EcoRI/SmaI restriction site, establishing a continual 18× GAA·TTC repetition. All plasmids were isolated from E. coli bacterial strain TOP10 (Stratagene) and verified by sequencing.

PFGE was performed according to a previously published protocol12 (link)16 (link). Briefly, strains were grown overnight on 5.0% blood agar plates. The cells were harvested and washed two times with solution buffer (Tris-HCl, 0.01 M; EDTA, 0.1 M; pH 8.0). Streptococcus cells were lysed with 1.0 mg/mL lysozyme and 1.0 mg/mL proteinase K (Sigma, USA). The bacterial suspensions were mixed with an equal volume of 1.0% low-melting-point agarose (Cambrex, USA) and pipetted into a 100 μl plug. A solution in CLB (50 mM Tris, 50 mM EDTA, 1% SDS, 0.1 mg/mL proteinase K) was then added. The plugs were incubated in a solution with 12 U of SmaI restriction enzyme (Takara, China) and its associated buffers and then then sent for PFGE assay using the following program: a switch time of 4–40 s, 20 h, a 120° angle and a voltage gradient of 6 V/cm in a CHEF Mapper XA (Bio-Rad, USA). A lambda ladder PFGE marker (New England Biolabs, USA) was used as a DNA size marker. The gels were stained with ethidium bromide and photographed under UV light. PFGE patterns were then analysed and compared using BioNumerics version 6.5 software (Applied Maths BVBA, Belgium).The unweighted-pair group method was used with arithmetic averages and Dice’s coefficient in the UPGMA Programme to process the data.

Blood samples were obtained from all participants and were stored in tubes containing ethylene diamine tetraacetic acid. The genomic DNA was extracted using a commercial DNA isolation kit (Promega, WI, USA) according to the instruction manuals. The CYP2C19*2 polymorphism was detected by polymerase chain reaction (PCR) – restriction fragment length polymorphism (RFLP) analysis. The forward primer of CYP2C19*2 alleles was 5′‐ ACC AGA GCT TGG CAT ATT GTA TCT‐3′, and the reverse primer of such alleles was 5′‐GAT TCT TGG TGT TCT TTT ACT TTC T‐3′. Both primers were provided by BGI Beijing Corporation. The PCR was performed according to the standard procedures: (1) Initial denaturation at 94°C for 5 min., (2) 35 cycles of denaturation at 94°C for 30 sec., (3) Annealing at 60°C for 30 sec., (4) Elongation at 72°C for 30 sec.; (5) Final extension at 72°C for 10 min. The PCR‐amplified products were digested with SmaI restriction enzyme (Takara biotechnology, Dalian, China) and submitted for direct DNA sequencing.

PFGE was performed using SmaI restriction enzyme (Takara, Japan) digestion according to the protocol described by the Center for Disease Control and Prevention (CDC) with minor modifications (https://www.cdc.gov/pulsenet/pathogens/pfge.html). The chromosomal DNA of Salmonella enterica serovar Braenderup (H9182) was used as the normalization standard and molecular marker. Digested plugs were loaded into the wells of a 1% agarose gel and run in 0.5X TBE using a CHEF (contour-clamped homogeneous electric field)-DR III system (Bio-Rad, Hercules, CA). After completing the electrophoresis process, the gel was stained using a 1.5 μg/mL ethidium bromide solution for 30–40 min on a rocking shaker in a covered container. Destaining was done three times with distilled water on the shaker for 45 min, and then the gel was visualized and photographed. Gel photos were processed and analyzed using GelCompare II software V. 4.0 (Applied Maths, Saint Martens-Latem, Belgium). Similarities between electrophoretic patterns were calculated using the Dice coefficient and set to 80% to determine the pulsed-field type clusters after reviewing the epidemiologic data associated with each of the clusters of isolates. The unweighted pair group method with arithmetic means (UPGMA) was used to construct a dendrogram with 1.5% tolerance and optimization as standard settings.