Ceq 8800 genetic analysis system

We confirmed the presence of the M_wurzburg allele by sequencing exon 5 of the SERPINA1 gene. We sequenced the intron 4 region to search for the M_whitstable variant.
Polymerase chain reactions (PCR) were performed with the AccuPrimeTM Taq DNA Polymerase System kit (Invitrogen by Thermo Fisher Scientific, Waltham, Massachusetts, USA) and both reaction conditions were as follows: 100 ng of DNA, 2.5 μL of Buffer II, 0.25 μL of MgCl2, 0.5 μM of each primer, and 0.5 μL of Taq DNA Polymerase. Primers used in the amplification and sequence reactions are listed in Table S1. Amplification and sequence reactions were carried out in an I-cycler Thermal Cycler (Bio-Rad Laboratories, Hercules, California, USA). Then, the reaction products were purified with a commercial kit based on magnetic beads (Ampure XP, Inc., Brea CA, USA). Sequence reactions were performed as indicated by the manufacturer, and sequence products were purified by a commercial kit based on magnetic beads (Agencourt CleanSeq, Inc., Brea, CA, USA).
Sequencing was performed by the CEQ 8800 genetic analysis System (Beckman Coulter, Pasadena, California, USA).
The NCBI Reference Sequence which we refer to is the following: NM_001002235.2.

The primers used in this study were designed with Primer3 (http://fokker.wi.mit.edu/primer3, (accessed on 3 September 2014)) based on the sequences flanking microsatellite-like loci determined in the sequence of G. candidum [5 (link)] (Table S1). Multiplex PCRs were performed with primer mixes: (B3/B4, B5/B6, D1/D2, E5/E6, G7/G8), (A3/A4, A7/A8, D3/D4, F7/F8), and (A5/A6, I3/I4, I5/I6, F1/F2). Amplification used the enzyme Ex-Taq (TaKaRa) under the manufacturer’s recommended conditions with a cycle of 94 °C for 4 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 40 s. The program was terminated by 5 min at 72 °C. Fragment lengths were measured with a Beckman Coulter CEQ 8800 Genetic Analysis System.

As previously reported [24] (link), wild-type ERRγ-LBD encoding 222-458 residues was generated by PCR using a human kidney cDNA library (Clontech Laboratories, Mountain View, CA, USA) and cloned into the vector pGEX-6p-1 (GE Healthcare Life Sciences, Piscataway, NJ, USA) using the EcoRI and XhoI restriction enzyme sites. Full-length ERRγ was cloned into the vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA). The resulting plasmids were designated as pGEX-ERRγ-LBD and pcDNA3.1-ERRγ-Full, respectively.
A series of ERRγ mutants were prepared according to the manufacturer's instructions by using PfuTurbo DNA Polymerase (Stratagene, La Jolla, CA, USA) with pGEX-ERRγ-LBD or pcDNA3.1-ERRγ-Full as a template and a set of overlapping sense and antisense primer pairs. The mutations were introduced by PCR mutagenesis in a two-step reaction essentially as reported previously [24] (link), [25] (link). Each mutant LBD or full-length ERRγ was amplified and cloned into the expression vector pGEX-6p-1 or pcDNA3.1(+) at the EcoRI and XhoI sites. The accuracy of all PCR product sequences was confirmed by using a CEQ™ 8800 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA).

Biochemical and genetic tests to diagnose AATD were performed at the Centre for Diagnosis of Inherited Alpha1-Antitrypsin Deficiency in Pavia (Italy) with the understanding and written consent of each subject. All methodologies were in accordance with the Declaration of Helsinki and were approved by the local ethics committee. The plasma levels of AAT and C-reactive protein were determined by a rate immune nephelometric method assay (Immage Immunochemistry System; Beckman-Coulter, Milan, Italy). DNA was isolated from whole peripheral blood using a commercial extraction kit (QIAmp DNA Blood Minikit on QiaCube, Qiagen, Milan, Italy). Genotyping for detection of the S and Z allelic variants was performed by PCR with fluorescently labelled Taq-Man probes (Vic or Fam labels) on a LigthCycler 480 (Roche Diagnostics, Monza, Italy) [57 (link)]. The new mutation was identified by sequencing all coding exons (II-V) of the SERPINA1 gene (RefSeq: NG_008290) as reported previously [58 (link)], using the CEQ 8800 genetic analysis System (Beckman Coulter, Milan, Italy). The clinical data were obtained from direct observation of clinical charts and they are reported in an anonymized form. Clinical data presented here are part of Italian Registry of Alpha 1-antitrypsin Deficiency (RIDA1), that received ethical approval by IRCCS Policlinico S. Matteo, Pavia (Italy) on 14 January 2019 (n°0385).

Genomic DNA was extracted from peripheral venous blood on EDTA by means of the Gentra Puregene Blood kit (QIAGEN S.p.A, Milan, Italy), following the manufacturer’s instructions.
Genomic DNA was extracted from the blood spot paper cards by means of the QIAamp® DNA Micro kit (QIAGEN S.p.A., Milan, Italy), according to the manufacturer’s instructions.
We amplified by PCR all the exons of PAX8, TSHR, FOXE1, NKX2.1, NKX2.5 genes by means of intronic primers. Fragments were first analyzed by Denaturing High Performance Liquid Chromatography on a WAVE DNA Fragment Analysis System (Transgenomic, Omaha, NE), and sequenced on an automated CEQ 8800 Genetic Analysis System (Beckman Coulter GmbH, Germany) whenever a sequence variation was suspected. PCR conditions, partial denaturing temperature (t_pd) for DHPLC analysis and sequencing conditions for TSHR and PAX8 genes have been previously described [28 (link)]. For FOXE1, NKX2.1 and NKX2.5 genes, sequencing conditions are available upon request.

To determine the 23S rRNA gene sequences, all samples were DNA sequenced after melting curve analysis. Amplification prior to sequencing was performed by a T100 thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) with the same reaction mixture as that for the SimpleProbe real-time PCR, excluding the probe at an annealing temperature of 63°C. The higher annealing temperature reduces unspecific amplification in a mixture where there is no probe, as the specific binding of the probe in the SimpleProbe reaction helps to ensure specific detection at a lower annealing temperature. The analysis of the size of the amplicon and DNA concentration was performed using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA), and product cleanup was achieved with a rapid PCR cleanup enzyme set (New England BioLabs Inc., Ipswich, MA, USA). A GenomeLab DTCS quick start kit (Beckman Coulter Inc., Brea, CA, USA) was used for dye terminator cycle sequencing of the 241-bp amplicon from the 23S rRNA gene with the forward primer Mg23S S. The sequence products were analyzed by a CEQ8800 genetic analysis system (Beckman Coulter Inc., Brea, CA, USA).

We used the CEQ 8800 Genetic Analysis System (Beckman Coulter Inc., Fullerton, CA, USA) to analyze the product size resulting from the amplification of VNTR loci. Sequences of all seven VNTR loci primers and characteristics are shown in Additional file 3: Table S3. To measure systematic error for the seven loci that we used, 31 S. Enteritidis strains were selected for PCR amplification and DNA sequencing. The following formula was used in MLVA to calculate the actual size of the repeat: $$\text{Size}\text{of}\text{the}\text{repeat}=\left[\left(\text{A}\pm \text{B}\right)-\text{C}\right]/\text{D}$$ where A is observed PCR product size, B is the systematic error, C is the flank that does not contain repeats, and D is the length of one repeat unit.