Big dye chemistry

To verify NGS genotypes of the second exon of MHC-DRB, we used the unmodified primers JS1 and JS2 to PCR 18 samples following the above Ion Torrent PCR protocol. Following manufacturers’ instructions, we cloned these PCR products using pGEM-T® Easy Vector (Promega, Madison, WI) and Library Efficiency® DH5α Competent Cells (Invitrogen, Life Technologies, Grand Island, NY). Due to the incredible diversity of MHC-DRB, we sequenced between 50 and 90 clones per individual, using ABI 3730xL Analyzer and Big Dye chemistry (Applied Biosystems®, Life Technologies, Grand Island, NY). Using MEGA 5.2 [83 (link)], we aligned and analyze sequences against NGS sequences for these individuals.

BRAF testing focusing on the clinically relevant tyrosine kinase domain mutation status was independently performed in a routine session using conventional Sanger sequencing based on Big Dye chemistry (Applied Biosystems, Foster City, CA, USA) in an ABI 310 genetic analyzer.

The genomic DNA library from V. ammodytes49 (link), prepared in phage λGEM 12, was screened with a 155 bp long fragment of the V. ammodytes trypsin inhibitor. The preparation of ³⁵S-labelled probe was described previously10 (link). The library was screened by the plaque hybridization method50 under the following conditions: 24 h hybridization at 42 °C in hybridization buffer (6 X SSC (NaCl/Na-citrate), 50%(v/v) formamide, 5 X Denhardt’s solution, 0.5% (w/v) SDS and 500 μg denatured herring sperm DNA. The filters were washed in 4 X SSC/0.1% SDS for 15 min and 2 X SSC/0.1% SDS for 5 min at room temperature. Positive clones were purified by repeating the screening procedure. Phage DNA was isolated from plate lysates50 and digested with Eco RI, Hind III, Sac I and Bam HI restriction enzymes. Restriction fragments were separated by 0.7% agarose gel electrophoreses. Positive fragments were determined by Southern blotting using ³⁵S-labelled probe described above and ligated into pUC19 vector. The inserts were sequenced on both strands with an ABI 310 sequence analyzer using BigDye chemistry (Applied Biosystems).

Viral vector construction and AAV production was performed, as previously described [25 (link)]. Briefly, CLU or GFP expression plasmids were cloned into an AAV vector. The constructs were sequence-verified using ABI3730 with Big Dye chemistry (Applied Biosystems, Foster City, CA). AAV vectors expressing GFP and CLU under the control of the glial fibrillary acidic protein (GFAP) promoter to drive expression in astrocytes were co-transfected with AAV2/8 helper plasmids into HEK293T cells. Cells were harvested and lysed in the presence of 0.5% sodium deoxycholate and 50 U/ml Benzonase (Sigma, St. Louis, MO) by freeze-thawing 48 h post-transfection, and the virus was isolated using a discontinuous iodixanol gradient. Quantitative PCR was used to measure the genomic titer of each virus.

Genomic DNA was extracted from fresh leaf samples using Magen Plant DNA Extraction Kit (Magen, Guangzhou, China). Nuclear ribosomal ITS region, Dbr1, two copies of SOS4 (SOS4a, SOS4b), PCRF1 and the chloroplast intergenic spacer (trnV-trnM) were amplified and sequenced using the primers listed in Table 1. We purified the PCR products using the Pearl Gel Extraction Kit (Pearl Bio-tech, Guangzhou, China) and then directly sequenced them on an ABI 3730 DNA automated sequencer with the BigDye chemistry (Applied Biosystems, Foster City, CA, USA). For sequences with more than one polymorphic site and insertion/deletion polymorphisms, PCR products were cloned and sequenced to phase the haplotypes. We conducted ligation reactions with a pMD18-T&A Cloning Kit (Takara, Dalian, China) and selected eight positive colonies for each individual for sequencing. The sequences of all haplotypes were deposited in GenBank under accession numbers MN380832–MN380893.