Sequencing and Bioinformatics: As part of the Cloning and Sequencing Explorer Series

Sequence and analyze your cloned DNA. Students combine plasmid DNA containing PCR products with sequencing primers on a 96-well plate and send them to a sequencing service.

The portion of the GAPDH gene to be cloned using the Cloning and Sequencing Explorer Series can vary from 0.6 to over 2 kb in length. To ensure complete coverage of the gene, four sequencing primers are provided: one forward and one reverse primer specific to either side of the pJET1.2 cloning site and one forward and one reverse primer homologous to different regions in the middle of the GAPDH gene. Thus four independent sequences will be generated for each plasmid — these sequences can be assembled into a contig using the bioinformatics module. We recommend each student team submit two plasmids for sequencing with all four primers. The ultimate goal of the Cloning and Sequencing Explorer Series is to generate sequence data that can be uploaded into NCBI's GenBank database to be used by other scientists. To ensure the data is as accurate as possible, it is important to guard against sequencing errors by having as many sequence reads as possible. Fortunately, since this is a class project, it is probable that multiple groups will independently clone the same GAPDH gene, resulting in the desired repetition needed to confirm the sequence. Additionally, sequencing primers are provided to allow for sequence reads in both forward and reverse directions, providing another method of sequence confirmation.

Once the plasmid has been cloned and sequenced, the real work begins — interpreting the sequence data. This final portion of the Cloning and Sequencing Explorer Series teaches students essential bioinformatics skills needed to link biology with computers. This capstone activity was developed in collaboration with Geospiza, Inc. and the sequencing and bioinformatics module includes a two month subscription to your class's personal iFinch account. The Bio-Rad Bioinformatics Portal gives students access to the latest tools in bioinformatics data mining. Raw sequence files are uploaded and hosted by the web portal. Students are guided through a series of activities. They will view and edit the original chromatograms of their sequences, screen out vector sequences, confirm their sequences are GAPDH, perform NCBI BLAST searches on their data to identify and find related sequences direct from iFinch, assemble their forward and reverse sequences into contigs, predict intron and exon structure, transcribe and translate the GAPDH sequence and compare it to known gene, mRNA, and protein sequences.