Computational protocol: Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase

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Protocol publication

[…] In order to characterize the novel CHD7 transcript, its coding sequence was re-amplified from a cDNA preparation synthesized using the Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), according to manufacturer's instructions, and 3 μg of total RNA from DU145 prostate carcinoma cell line. PCR reactions were carried out in 50 μl and the reactions mixtures contained 1 μl of a 1/20 dilution of the cDNA preparation, 1× High Fidelity Buffer® (Eppendorf, Westbury, NY), dNTPs (0.4 mM each), 0.4 μM of each primer and 4 U TripleMaster Taq polymerase (Eppendorf, Westbury, NY). The following primers annealing to sequences corresponding to exons 2 and 38 of CHD7 were used: CHD7F forward: 5'-ACCTCAGTGAAGTGAAGCACAGG-3' and CHD7R reverse: 5'-CACACTAGCGTGGAGATTGTCAG-3'. The cycling protocol employed was as follows: 3 min at 94°C (initial denaturation); 1 cycle of 30 sec at 94°C and 12 min at 72°C; 3 cycles of 30 sec at 94°C, 40 sec at 68°C and 12 min at 72°C; 3 cycles of 30 sec at 94°C, 40 sec at 65°C and 12 min at 72°C; 35 cycles of 30 sec at 94°C, 40 sec at 62°C and 12 min at 72°C; and a 15 min at 72°C (final extension).The approximately 3.3 kpb DNA band was gel purified and subcloned into the pGEM-T Easy vector (Promega, Madison, WI) using the TA cloning system, according to manufacturer's instruction. Three bacterial (Escherichia coli XL1 Blue) clones (pGEM-M1, pGEM-M2 and pGEM-M3) were picked and individually grown in liquid LB medium containing 100 μg/ml of ampicilin overnight at 37°C under agitation (250 rpm). Plasmid DNA was extracted from bacterial cultures using the GFX TM Micro Plasmid Prep (GE HealthCare, Piscataway, NJ), according to the manufacturer's instructions. The three clones containing the novel CHD7 transcript cDNA were subjected to sequencing using the ABI 3700 sequencer and the BigDye 3.1 sequencing kit (Applied Biosystems, Foster City, CA) at the GaTE (Genomic and Transposable Elements) lab, Biological Institute, University of Sao Paulo. Sequencing was carried out using sequencing primers annealing to CHD7 exons 2, 37 and 38. The novel CHD7 transcript cDNA sequences (clones M1, M2 and M3) were individually clustered into contigs using the SeqMan II software (DNASTAR, Inc., Madison, WI). The original sequencing files were evaluated for quality using the Trace Quality Evaluation algorithm within SeqMan II. Poor-quality sequences were trimmed and the trimmed cDNA sequences of each clone were assembled into contigs using the SeqMan II assembly process. The contig cDNA sequences of each clone were individually compared to the canonical CHD7 transcript reference sequence (NM_017780.2) using the BLASTN program [] at NCBI []. Determination of sequence overlap between the contig cDNA sequences described above and the CHD7 canonical transcript reference sequence and CHD7 mRNAs and spliced ESTs sequences from the Genbank was performed using the UCSC Genome Browser [,]. The contig cDNA sequences were aligned to the February 2009 version of the human genome sequence assembly using the BLAT alignment tool provided by UCSC. The contigs cDNA sequences open reading frames (ORFs) were determined using the ORFinder tool at []. Translations of the detected ORFs were submitted to alignment to CHD7 reference protein sequence (Genbank: NP_060250.2) using the BLASTP alignment tool []. […]

Pipeline specifications

Software tools BLASTN, BLAT, BLASTP
Databases UCSC Genome Browser
Application Genome data visualization
Organisms Homo sapiens
Diseases Prostatic Neoplasms, CHARGE Syndrome
Chemicals Adenosine Triphosphate