Abi prism 310 genetic analyser

Sanger sequencing was performed in six children before 2014. All exons of NPHS1, NPHS2, PLCE1, LAMB2, LMXIB, COQ6, and COQ2, as well as exons 8 and 9 of WT1, were amplified by the polymerase chain reaction (PCR) method. The primers for NPHS1, NPHS2, PLCE1, WT1, LAMB2, LMXIB, COQ6, and COQ2 were designed on the basis of previously published information regarding the intron-exon boundaries [3 (link), 5 (link)–8 (link), 19 (link), 20 (link)]. The PCR products were purified with a QIA Quick PCR Purification Kit (Qiagen, Hilden, Germany). The purified products were cycle-sequenced with Big Dye terminators (Applied Biosystems, Foster City, CA, USA), and the cycle sequence products were analysed with an automated sequencer (ABI Prism 310 Genetic Analyser). The novel mutations were investigated in a number of mutation databases on human populations, such as the ExAC Browser (http://exac.broadinstitute.org/), the 1000 Genomes Project (http://www.internationalgenome.org/), HGMD (http://www.hgmd.cf.ac.uk/ac/index.php), and, in the 100 healthy control subjects, by direct sequencing.

As a pair of isoschizomers was used HpaII and MspI which recognise tetranuclotide CCGG, but have differential sensitivity to methylation at the inner or outer cytosine. The first sample containing 250 ng of DNA was digested by EcoRI and MspI restriction enzymes and the second equivalent sample by EcoRI and HpaII restriction enzymes. Subsequent adaptor ligation and pre-amplification reactions, using adaptors and primers designed for EcoRI and HpaII/MspI combinations, were performed as in [37 ]. In the case of selective amplification, three differently labelled EcoRI-derived primers, EcoRI-ACA (FAM), EcoRI-AGC (NED) and EcoRI-ACG (JOE), were combined with five HpaII/MspI-derived primers, HpaII/MspI-TCAA, HpaII/MspI-TCAC, HpaII/MspI-TCGC, HpaII/MspI-GCAT, and HpaII/MspI-TACC. This means altogether 15 primer combinations were used. Amplification products were separated electrophoretically using capillary system of ABI PRISM 310 genetic analyser (Applied Biosystems). GeneScan 500 ROX (Applied Biosystems) was used as a size standard and POP 4 polymer (Applied Biosystems) as a medium for fragment separation.

A 4 μL aliquot of the purified amplicon DNA and the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) was used for cycle sequencing according to the manufacturer’s instructions. Besides primers SIN-Reg-B and cSIN-Reg-B (see above), primers Sin-Reg-B-nf: 5’- ATGACATCAAGATTAGCACC-3’ (position 8872–8891) and Sin-Reg-B-nr: 5’- TGATGCGACGGCTAAG –3’ (position 9734–9749) were used in sequencing reactions to reach full redundancy. The reaction was purified using Centri-Sep columns as recommended (Princeton Separations Inc, Adelphia, USA) and analysed by an ABI PRISM 310 Genetic Analyser (Applied Biosystems).
Sequences were aligned using GramAlign v3.0 [33 (link)]. A Maximum clade credibility (MCC) tree with dated tips and internal nodes was inferred using a MCMC Bayesian approach under the GTR model with gamma-distributed rate variation (Γ) and a proportion of invariable sites (I) using a relaxed (uncorrelated lognormal) molecular clock [34 (link)] in BEAST version 1.8.4 [35 (link)]. Four independent MCMC runs of four chains each were run for 10,000,000 states. The First 1,000,000 were used as burning and the MCC was establish from the remaining states. A median joining network [36 (link)] of the sequences was constructed and edited using a 2,190-character set in SPLITSTREE v4.12.3 [37 (link)]

Germline DNA samples of both patients (HPC311 and P308T) sharing the same HOXB13 germline mutation were genotyped for the 11 microsatellite markers (6 localized in chromosome 17 and one for chromosome 2, 3, 5, 9 and 13) indicated in S2 Table. All 11 markers were assayed by PCR using fluorescently end-labeled primers (S2 Table) and capillary electrophoresis was performed on an ABI PRISM 310 Genetic Analyser (Applied Biosystems, Foster City, CA).

A fragment from the Mitochondrial 16S rDNA (407 bp) was amplified by PCR using primers 16S-F and 16S-R as reported previously [10 (link)] in a 2720 thermal cycler (Applied Biosystems, Foster City, California). The amplicons were examined on 1.5 % agarose gel stained with ethidium bromide for DNA visualization under UV light. The purified PCR products were directly cycle-sequenced from both directions on ABIPRISM 310 Genetic Analyser (Applied Biosystems, Foster City, California) using the BigDye Terminator Cycle Sequencing Kit 1.1 (Applied Biosystems, Foster City, California). Individual mite consensus sequences were manually trimmed of primer sequences, aligned, compared and edited using BioEdit v7.0.9.0 [16 ].