Abi 3730 automatic sequencer

Genomic DNA was extracted from liver tissue of five individuals of Cnemaspis (Table 1) using the Qiagen DNAeasy tissue kit (Valencia, CA, USA). A 1,251 bp fragment of mitochondrial (mt) DNA consisting of the NADH dehydrogenase subunit 2 (ND2) gene and the flanking tRNAs Trp, Ala, Asn, and Cys was amplified using polymerase chain reaction (PCR) under the following conditions: initial denaturation at 95 °C for 2 min, followed by a second denaturation at 95 °C for 35 sec, annealing at 52 °C for 35 sec, followed by a cycle extension at 72 °C for 35 sec, for 33 cycles using the light strand primer L4437b (5’-AAGCAGTTGGGCCCATACC-3’; Macey et al. 1997 (link)) and heavy strand primer H5934 (5’ AGRGTGCCAATGTCTTTGTGRTT-3’; Macey et al. 1997 (link)). PCR products were purified using the AccuPrep PCR Purification Kit (Bioneer, Daejeon, Korea), and were sequenced using the amplifying primers on an ABI 3730 automatic sequencer (Applied Biosystems, CA, USA). Sequences were edited and aligned using Geneious R11 (Biomatters, Ltd, Auckland, New Zealand). All new sequences were deposited in GenBank under accession numbers MT112890–MT112894 (Table 1).

Sequencing libraries for the quality-checked gDNA were generated using a TrueLib DNA Library Rapid Prep Kit for Illumina sequencing (Illumina, Inc., San Diego, CA, USA). The libraries were subjected to size distribution analysis using an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), followed by real-time PCR quantitative testing. The successfully generated libraries were sequenced using an Illumina NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA). A total of 150-bp paired-end reads with a 300-bp insert library were generated. Three pairs of primers designed to match generally conserved regions of the published target mtDNA were used to amplify short fragments from nad3-nad5, rrnL and the control region (CR) (Supplementary Table S1). The PCR products were cloned into pMD18-T vectors (Takara, Kyoto, Japan) and subsequently sequenced, or they were sequenced directly by the dideoxy nucleotide procedure, using an ABI 3730 automatic sequencer (Applied Biosystems, Foster City, CA, USA) [10 (link)] (Supplementary File S1).

The mtDNA control region was amplified using the forward and reverse primers 5′‐GTT AGA GCG CCA GTC TTG TA‐3′ and 5′‐CCT GAA GTA GGA ACC AAA TGC‐3′, as described by Sekino et al. (2002 (link)). Amplification was performed in a 30 μl reaction volume containing 10 μM of each primer, 10 ng template DNA, 100 μM of each dNTP, 3 μl 10× PCR buffer containing MgCl₂, and 1.25 U of DNA polymerase (Ex Taq™, Takara Co). The cycling conditions were as follows: preliminary denaturation at 94°C for 4 min, followed by strand denaturation at 94°C (45 s), annealing at 58°C (45 s), and primer extension at 72°C (1 min) for 33 cycles; and final elongation at 72°C (10 min). The PCR products were evaluated using 1% agarose gel electrophoresis. Products with a single bright band were selected and purified. The purified fragments were sequenced using an ABI‐3730 Automatic Sequencer (Applied Biosystems).

DNA was extracted using the DNEasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Twelve microsatellite primers labeled with a fluorescent dye (FAM, HEX or NED) and 1μL (~1ng) of purified DNA were used in the PCR reactions, which were performed as described before [41 (link)] in an E6331000025 Eppendorf Thermocycler (Mastercycler Nexus Gradient, Eppendorf, Hamburg, Germany) (S1 Table). Amplified fragments were visualized on a 1% agarose gel stained with GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA) and examined under UV light. PCR products were diluted 1:7 by mixing 3 μL of each product labeled with a different dye with 21 μL of Ultra-Pure Water (Applied Biosystems, Foster City, CA, USA) to a final volume of 30 μL, and 2 μL of the dilution was suspended in 8.925 μL of Hi-Di formamide (Applied Biosystems, Foster City, CA, USA) and 0.075 μL of GeneScan 500 ROX size standard (Applied Biosystems, Foster City, CA, USA) to a final volume of 11 μL. The samples were then sent to the University of São Paulo Center for Human Genome Studies and size-sorted in an ABI 3730 automatic sequencer (Applied Biosystems, Foster City, CA, USA). Fragment analysis was performed using GeneMarker v1.85 (SoftGenetics, State College, PA, USA).

To evaluate an association between changes in the chromosomal carbapenemase gene sequence of isolates and their antimicrobial resistance pattern, a two-step approach was adopted. An initial AFLP assay was carried out on bla_{OXA-23-like⁺} CR-AB, followed by DNA sequence analysis of the bla_OXA-23-like gene of a representative isolate from each AFLP genotype group.
Briefly, we used a high fidelity Pfu DNA polymerase (Fermentas, Lithuania) to generate bla_OXA-23-like specific amplicons, which were purified using a AccuPrep® PCR Purification Kit (Bioneer, Daejeon, Korea) and cloned into pTZ57R (InsT/A Clone PCR product cloning kit, Fermentas, Vilnius, Lithuania). DNA was then transferred into competent E. coli TOP10 cells, which were then isolated using Luria-Bertani (LB) agar supplemented with ampicillin (100 μg/mL). Plasmid DNA was prepared with the AccuPrep Plasmid MiniPrep DNA Extraction Kit, (Bioneer, Daejeon, Korea) and sequenced using an ABI3730 automatic sequencer (Applied Biosystems, CA, USA). The sequences were analyzed using a BLAST algorithm against the NCBI GenBank database [http://www.ncbi.nlm.nih.gov/guide/dna-rna/ (accessed 05.06.11)].

30 µl at 20 ng/µl of genomic DNA (gDNA) was requested, and concentration and purity were checked spectrophotometrically using the Trinean DropSense96 UV/VIS droplet reader for all consortium samples. Gender and DNA fingerprint were determined for all DNA samples using an in-house developed multiplex polymerase-chain reaction (PCR) genomic DNA Fingerprint panel comprising 13 short tandem repeat (STR) markers distributed over multiple autosomal loci—D20S480, D22S1174, D3S1287, D3S1744, D3S1764, D7S672, D7S2426, D8S1746, D14S1005, D20S866, D10S1237, D20S912, D6S965—and two sex chromosome markers—DXS1187, chrom Y: 2655362–2655672—to enable fast and accurate sample identification and gender determination in a single PCR. After selective amplification of 20 ng gDNA under empirically defined reaction conditions, amplification products were size separated on an ABI 3730 automatic sequencer (Applied Biosystems) using GeneScan-600 LIZ (Applied Biosystems) as internal size standard and genotypes were assigned using in-house developed TracI genotyping software (http://www.vibgeneticservicefacility.be). Duplicate samples, gender mismatches and failed samples due to low DNA quality or contamination were excluded, resulting in the final study population of 1,808 FTLD, 395 ALS, and 1,625 control individuals.