20 pmol of each paired oligo was incubated with 2 U of Taq DNA polymerase (Promega, Madison, USA), 1X reaction buffer, 2.5 mM MgCl2, and 0.4 mM of dNTPs. The reaction mix was incubated at 94°C for 30 seconds followed by 55°C for 30 seconds and 72°C for 30 seconds for 5 cycles. After the reaction, the end product was run on a 2% agarose gel and visualized under UV light with ethidium bromide staining.
During this process, the complementary sequences located at 3′ end of the paired oligo would form a dimer and in the presence of DNA polymerase and dNTP, each oligo would extend its 3′ end using the second oligo as a template.
Cloning of the double-stranded DNA
To ensure the purity of the standard template, the products were ligated into the pGEM-T Easy Vector System II (Promega, Madison, USA) and transformed into competent Escherichia coli JM109 cells, according to the manufacturer’s protocol. Screening for the gene insert was performed by quick lysis at 95°C of the E. coli cells for 5 minutes, followed by PCR using 5 μl of the cell lysate as previously described.
Sequencing of cloned dsDNA and sequence analysis
The plasmid DNA from positive clones was extracted using the QIAprep Spin Miniprep Kit according to the manufacturer’s protocol (Qiagen, Melbourne, Australia). The inserts were sequenced using Applied Biosystems BigDye terminator chemistry version 3.1 (Foster City, CA, USA), on an ABI Prism 373 DNA sequencer. Sequences were identified using the FastA program group accessed through Biomanager.
Ligase Chain Reaction (LCR) in the detection of templates containing resistance mutation
Liner templates containing resistance and wild type were generated by PCR amplification of plasmid DNA, followed by purification using Millipore PCR purification plate (Millipore, Billerica, MA, USA). The linear PCR products were quantitated using spectrophotometer and dsDNA DNA copy number were estimated by DNA calculator. 5 × 1011 copies of standard templates were used for testing the specificity of the LCR system. LCR probes targeting resistance template were designed. Each template was targeted by four LCR probes about 35-45nt with 2 of the probes having 5′ end phosphorylation. Ligation of LCR probes to standard templates was carried out by mixing the standard template with 1pmol of each LCR probes, 2U of pfu DNA ligase (Stratagene, Integrated Sciences, Cedar Creek, TX, USA) in 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl2, 0.1% Igepal, 0.01 mM rATP, 1 mM DTT with total reaction volume of 25 μl. Multiple cycle ligation was conducted to validate the specificity of the probe in recognizing its corresponding template. The reaction condition include one cycle of 5 min at 94°C to denature the dsDNA followed 10 cycles of by 94°C 30 s and 4 min ligation at 65°C. The final product were run on 10% TBE gel (Invitrogen, Mount Waverley VIC Australia) and visualized under UV light with ethidium bromide staining.