Synthesis of cDNA was performed using Superscript® III Reverse Tr

Synthesis of cDNA was performed using Superscript® III Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. IgE and IgG heavy chain gene rearrangements were then amplified using an isotype-specific PCR. PCR amplification was performed with 100–200 ng cDNA or aliquots of the PCR1 product as templates, 0.2 μm of each primer, 200 μm of each dNTP, 1.25 units PFU polymerase (Promega, Madison, WI, USA) and a buffer supplied by the manufacturer. Details of the primers used are shown in Table 1. Specific primers

for the three large IGHV gene families (VH1F, VH3F and VH4F) were used as forward primers in separate reactions. IgG1 and IgG2 were amplified by standard PCR using appropriate isotype specific primers (G1 and G2/G4IN) as reverse Bioactive Compound Library manufacturer primers. Reactions times for this PCR were 95 °C for 3 min, followed by 35 cycles of

95 °C for 30 s, 61 °C https://www.selleckchem.com/products/AP24534.html for 30 s, 72 °C for 4 min and then a final extension at 72 °C for 5 min. Semi-nested PCR were used for IgG3 (reverse primers: G3OUT and G3IN), IgG4 (G4OUT and G2/G4IN) and IgE (IGEOUT and IGEIN) sequence amplifications. PCR1 conditions used were initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 4 min and a final extension at 72 °C for 5 min. For PCR2, the only changed condition from those of PCR1 was the annealing temperature, which was 65 °C for IgE and IgG4, and 61.7 °C for IgG3. PCR2 was run for 25 Morin Hydrate cycles. All PCR were run on a Tpersonal 48 cycler (Biometra, Gottingen, Germany). PCR products were then cloned and sequenced at the Ramaciotti Centre for Gene Function Analysis, University of New South Wales, as previously described [13]. Bioinformatic analysis.  Rearranged VDJ sequences were aligned against the germline repertoire using the iHMMune-align program [19] and the UNSWIg repertoire of germline genes [20] (http://www.ihmmune.unsw.edu.au/unswig.html). This repertoire was updated with a number of IGHV polymorphisms that we have identified

in the PNG population and have submitted to GenBank (accession numbers HM855272–HM855948), as well as putative polymorphisms that have been identified in previous studies [20, 21]. Evidence in support of the existence of these putative polymorphisms within rearranged VDJ genes can be found at http://cgi.cse.unsw.edu.au/~ihmmune/IgPdb/. The number of mismatches between the germline IGHV genes and each rearranged sequence was noted. Sequences with more than 45 mismatches were removed from the data set because of the likelihood they included sequencing errors. Clonally related sequences were identified on the basis of shared IGHV, IGHD and IGHJ genes, as well as shared N regions and shared point mutations.

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