Abi prism bigdye terminator v1.1 cycle sequencing kit

Parasite DNA from both clinical samples were subject to PCR amplification of Leishmania-specific ITS regions. In brief, total genomic DNA was isolated from blood buffy coat using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), as per manufacturer’s instructions. The complete ribosomal ITS region (~1.1 kb) was then amplified using Leishmania-specific primers (forward, 5'-CTG GAT CAT TTT CCG ATG-3'; and reverse, 5'-ACA CTC AGG TCT GTA AAC-3') [28 (link)]. The amplification was performed in the thermal cycler (Eppendorf, Hamburg, Germany) and initially heated to 95 °C for 2 min followed by 34 cycles of 95 °C for 20 s, 53 °C for 30 s and 72 °C for 1 min. The final extension was carried out at 72 °C for 1 min. The PCR amplicon was excised for purification using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) from a 1.8% agarose gel prepared in 1× TAE buffer, The purified PCR amplicon was then sequenced using the ABI Prism Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA).

Clonal HIV-1 variants or bulk isolates were obtained as described previously [18 (link)] and total DNA was isolated using the L6 isolation method. Nef was amplified using a nested PCR with the outer primers Nef-1-fw (5′-AGCCATAGCAGTAGCTGAGG-3′) and Nef-1-rev (5′-GCTTATATGCAGGATCTGAGG-3′) and the inner primers Nef-2-fw (5′-AGCTTGTAGAGCTATTCGCCACA-3′) and Nef-2-rev (5′-AGCAAGCTCGATGTCAGCAG-3′). Nef PCRs were performed using Taq polymerase (Promega, Madison, WI, USA) in the presence of 2 mM MgCl2 using the following amplification cycles: 2 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 55 °C, 2 min at 72 °C, followed by a 10-min extension at 72 °C and subsequent cooling to 4 °C. PCR products were purified using EXOSAP-IT reagent (USB, Cleveland, OH, USA) and sequenced using an ABI prism Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) using the nested PCR primers. Sequences were analyzed on the Applied Biosystems/Hitachi 3130 xl Genetic Analyzer (Foster City, CA, USA). DNA sequences were translated and analyzed with the BioEdit program (BioEdit v 7.0.5, Tom Hall, Ibis Therapeutics, Carlsbad, CA, USA).

A systematic analysis of the complete exon 5 of CHRNA4 gene was performed in three overlapping fragments by polymerase chain reaction (PCR) and direct sequencing. The PCR primers were designed with the free internet tool Primer3 v.0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/). PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye^® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. All primer sequences and PCR conditions can be obtained on request from the authors. All sequences were analyzed using Mutation Surveyor software v3.2.
Remark: In an earlier paper, we have discussed the possibly of population stratification in our sample [16 (link)]. We did not genotype a standard panel of ancestry-informative SNPs to control for genetic stratification since we investigated a genetically highly homogeneous sample of subjects with Germany ancestry (or from adjacent countries) and because participants were randomly selected from the general population. Also, the European autosomal gene pool is quite small, in particular in northern and middle Europe populations [26 (link)].

Selected target fragments (prioritized based on phenotypic information and NGS data) were amplified from genomic DNA and cDNA, when available, using a standard PCR protocol. Primer sequences are available upon request. Amplicons were examined by applying 2.5 µl of each PCR reaction on a 1.3% agarose gel. PCR products were purified using ExoSAP-IT (USB, Cleveland, Ohio, USA) according to the protocol and then sequenced with the ABI PRISM BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) using the PCR primers as sequencing primers. The sequencing was performed on an ABI 3730 or ABI 3130xl Prism DNA Analyzers, and data analyzed with DNA Sequencing Analysis software, version 5.2 (Applied Biosystems) and Sequencher version 4.7 (Gene Codes Corporation, Ann Arbor, USA).