Abi 377

Polymerase chain reaction (PCR) primers were designed from the cloned fragments of the HvGA20ox2 gene [13 (link)] and barley genome sequencing information (Additional file 2: Table S2). The relative positions of each primer to the HvGA20ox2 gene are shown in Additional file 1: Figure S1. All primers were synthesized by Gene Works Pty. Ltd. (Australia). The PCR reactions consisted of 50 ng genomic DNA as template, 0.1 μM of each primer, in a final volume of 10 μl containing 1 × PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTP, and 0.5 U Taq polymerase (Bioline, Australia). The PCR reactions were performed using the following program: denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, annealing for 45 s and extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min. The optimal annealing temperature of each pair of primer combination was determined by gradient PCR. The PCR products were cloned into pGEM-T Easy Vector (Promega), and at least two independent clones from each PCR product were sequenced using an automated sequencing system (ABI 377, Applied Biosystems).

For further identification of the non-tuberculous mycobacteria (NTMs), sequencing of the 16S was carried out. Amplification of the DNA using 264 primers A and B was performed. The complete PCR products were sequenced on an automated DNA sequencer (ABI 377; Applied Biosystems, Waltham, MA, USA) by cycle sequencing using the Big Dye RR Terminator cycle sequencing kit (Applied Biosystems). The resulting sequences were aligned and compared to the sequences of the International Nucleotide Sequence Database Collaboration.

Genomic DNA was extracted from the leukocytes of peripheral blood from the 36 patients according to the manufacturer's recommendations (Genomic DNA Extraction Kit, RBC Bioscience, Taiwan). PCR was performed to amplify the promoter, all 13 exons and intron-exon boundaries of the GLP1R gene (GenBank accession number AL035690) using specific primer sets and PCR conditions as described in Supplementary Table  1, in Supplementary Material available online at http://dx.doi.org/10.1155/2015/176949 [20 (link)]. All of PCR products were confirmed by electrophoresis on 1.5% agarose gels and directly sequenced using an automated sequencer ABI 377 (Applied Biosystems, Foster City, CA) to determine the DNA sequences.

Total RNA was extracted from MCL cell lines, following previously reported protocols [44 (link)]. Exon II of the PSMB5 gene was amplified by PCR using the following primer. Forward, 5′-TTCCGCCATGGAGTCATA-3′ and reverse 5′-GTTGGCAAGCAGTTTGGA-3′. The PCR product was sequenced by the ABI377 (Applied Biosystems, Grand Island, NY, USA).

Polymerase chain reaction (PCR) was applied for HPV DNA detection on CSCC patients. The GP6 + /MY11 PCR primer pair was adopted to generate an approximately 192 bp fragment within the L1 region of HPV genome^{27 (link),28 (link)}. HPV genotype was then defined by sequencing the PCR product on ABI 377 (Applied Biosystems, Foster City, CA). HPV testing did not perform for the controls.

Genomic DNA was extracted from peripheral blood leukocytes after obtaining informed consent from the patients. The direct sequencing of polymerase chain reaction (PCR) products was performed for the coding regions of PROC (exons 1–9), PS (PROS1 exon 1–15) and AT (SERPINC1 exon 1–6) genes as described previously [18 (link), 22 (link)]. The exon and exon–intron boundary regions of each gene, including the promoter region, were amplified by PCR, and the products were then subjected to direct sequencing using an ABI 377 (Applied Biosystems, Foster City, CA, USA).

Representative samples of each defined haplotype were typed for LEI0258 using the Beckman CEQ 8800 instrument (Beckman-Coulter, Fullerton, CA, USA), according to the protocol in [46 (link)]. Since the size of the observed allele can vary slightly depending on the detection platform used, all allele sizes were converted to their equivalent when using the ABI 377 (Applied Biosystems, Foster City, CA, USA) platform as reported in [46 (link)].