Abi prism 3130xl dna analyzer

Viral RNAs were extracted from 250 μl of plasma with RealBest DeltaMag kit (Vector-Best, Russia) according to the manufacturer's recommendations. The RNA was utilized for reverse transcription PCR and nested PCR. Amplification was performed using a lyophilized ready-to-use Reverse Transcription Master Mix containing all the components for a single-tube reverse transcription and PCR (Vector-Best, Russia) and an in-house set of primers as described previously [15 (link)]. This procedure generates an amplicon of the pol gene encoding the protease-reverse transcriptase (PR-RT, 1400 nt), integrase (IN, 960 nt), and a fragment encoding a part of the major envelope protein, env (732 nt). For several HIV-1 samples, gag (1457 nt) fragment was additionally obtained. After purification, PCR fragments were sequenced by ABI PRISM 3130xl DNA Analyzer (Applied Biosystems, United States).

Peripheral venous blood specimens were drawn from the study subjects. Genomic DNA was isolated from blood leukocytes using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). The genomic DNA sequence of the human HAND2 gene (accession no. NC_000004) was obtained from the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/). Using the online Primer 3 program (http://primer3.ut.ee/), primers to amplify the coding exons and splicing boundaries of HAND2 by polymerase chain reaction (PCR) were designed as shown in Table 1. PCR was performed with HotStar Taq DNA Polymerase (Qiagen, Hilden, Germany) on a Veriti Thermal Cycler (Applied Biosystems, Foster, CA). The amplicons were sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), under an ABI PRISM 3130 XL DNA Analyzer (Applied Biosystems). For an identified sequence variation, the Single Nucleotide Polymorphism (SNP; http://www.ncbi.nlm.nicch.gov/SNP), the 1000 Genomes Project (1000 GP; http://www.1000genomes.org), and the Exome Variant Server (EVS; http://evs.gs.washington.edu/EVS) databases were consulted to verify its novelty.

Genomic DNA was extracted from peripheral blood using the Gentra Puregene Blood kit (Gentra Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. All coding exons and flanking intronic regions were amplified by PCR using primers specific for VHL exons 1–3. The amplified product was directly sequenced with an ABI PRISM 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems).
For screening exon deletions and duplications, multiplex ligation-dependent probe amplification (MLPA) P016-C1/P16-C2 kits (MRC-Holland, Amsterdam, Netherlands) were used. DNA denaturation, probe-target sequence hybridization, probe ligation, and PCR amplification of ligated probes were performed according to the manufacturer’s instructions. The products were loaded onto ABI PRISM 3130xl DNA Analyzer (Applied Biosystems) and analyzed by GeneMarker software version 1.51 (SoftGenetics, LLC, State College, PA, USA).

We tested 24 SSR markers that we observed successful amplification in V. exilis in our previous study (Chankaew et al., 2014b (link)). All primers were labeled with 6-FAM, HEX, or NED (Applied Biosystems). We performed PCR with Multiplex PCR Master Mix (Qiagen) following the manufacturer's protocol, and fragment analysis using ABI PRISM 3130xl DNA Analyzer (Applied Biosystems). Genotypes were determined by GeneMapper 4.0 (Applied Biosystems). We calculated genetic distance using Populations 1.2.30 (http://bioinformatics.org/~tryphon/populations/), and constructed phylogenetic tree by neighbor-joining method (Saitou and Nei, 1987 (link)). We also performed bootstrap test with 1000 repeats.

Genomic DNA extracted from strain 10Alg 79^T with the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany) was used to amplify 16S rRNA genes with 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) primers [25 ] and sequences using an ABI Prism 3130xL DNA analyzer (Applied Biosystems, Hitachi, Japan).
The analysis of 16S rRNA gene sequences was performed on the EzBioCloud server [26 (link)], and the phylogenetic analysis was conducted using MEGA X software, version 10.2.1 [27 (link)], with the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) methods. Genetic distances were calculated according to the Kimura two-parameter model [28 (link)], and bootstrap values were obtained from 500–1000 alternative trees.
The maximum-likelihood phylogeny of RpoC sequences translated from genome sequences was calculated using the IQ-TREE web server, version 1.6.12 [29 (link)], with 100 non-parametric bootstrap replicates and the LG+I+G4 substitution model determined using ModelFinder [30 (link)].

Twenty-four variants detected through NGS were confirmed by Sanger sequencing. Fifteen variants were previously observed in our diagnostic routine panel either by Sanger sequencing performed previously or through restriction fragment length polymorphism (RFLP). As a result, all variants observed through NGS were confirmed by at least one additional method. Sanger sequencing was performed using a Big Dye Terminator V3.1 kit (Applied Biosystems, Austin, TX, USA) in an ABI PRISM 3130xl DNA analyzer (Applied Biosystems). The CFTR DNA amplification was achieved with the set of primers listed in Supplementary Table 1. PCR products were visualized on 1.5% agarose gels and purified using a Sweep Clean up kit (Applied Biosystems, Vilnius, Lithuania). The obtained sequences were aligned with the reference sequence of CFTR in Ensembl (ENST00000003084.10). Sequence analysis was performed with Chromas Lite 2.0 software (Technelysium) and BioEdit Sequence Alignment Editor v6.0.6 (Ibis Therapeutics).

Genomic DNA was extracted from peripheral leukocytes using the Flexigene DNA kit (Qiagen). The coding exons and exon-flanking sequences were analyzed by PCR (Taq polymerase, Qiagen) followed by direct sequencing using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). Capillary electrophoresis was performed on an ABI PRISM 3130xl DNA Analyzer (Applied Biosystems). Primers and PCR conditions for amplification of the coding exons (Exons 3–18) and splice sites have been described previously [7 (link)]. Sequences were aligned to the NCBI reference sequence NM_001014796, using ClustalW2 algorithm and mutations were designated with reference to the translation start site.