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DNA Sequence Analysis. Broad and Long Term Objective. To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis. Research Plan. Preparation of Competent Cells and Bacterial Transformation. Growth of Transformant and Plasmid MiniPrep. Cycle Sequencing.
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Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis
Research Plan Preparation of Competent Cells and Bacterial Transformation Growth of Transformant and Plasmid MiniPrep Cycle Sequencing Sequence analysis
Today’s Laboratory Objectives To learn how to characterize a DNA sequence using various web based bioinformatics tools including: 1. BLASTN- has this piece of DNA been sequenced before? Does it look like anything already in GeneBank at the nucleotide level? 2. BLASTX- Can we identify the putative function of the transcripts? 3. ORF Finder- What does the open reading frame look like? Do we have a full length clone with an identifiable start and stop codon? 4. ClustalW- How does it compare with other sequences either at the nucleotide or amino acid level? What residues are conserved and thus likely to be important? And what residues are divergent?
BLAST Database Search Tool BLAST (Basic Local Sequence Alignment Tool) • Available on the internet and downloadable • Quick and simple • http://www.ncbi.nlm.nih.gov/
The Blast Algorithm • Identify HSP’s (High Scoring Segment Pairs) default 11 bp or 3 aa Perfect match • Slide query and target sequence across each other until the maximum number of HSP for that target is found
The Blast Algorithm • Score the Alignment a scoring matrx such as BLOSUM62 or PAM is used gaps introduced between GSP’s during sliding get negative score a match gets a positive score total alignment score is subjected to statistical analysis to calculate the significance vs. chance of the score • Repeat for every sequence in the target database • Return total results
PasteSequence here Submit Search by Clicking Here
Interpreting BLAST Results • Length • E-Value • Bit Score • Identities • Positives
NCBI’s ORF FINDER and Open Reading Frames • Begin with “ATG” start codon • End with “TAA”, “TAG”, or “TGA” stop codons • Can occur in any six possible reading frames • Sense Strand: Frame +1 • Frame +2 • Frame +3 • Antisense Strand: Frame -1 • Frame –2 • Frame -3
ORF Finder Algorithm • Iterates over all frames: Iterate to the end of frame Find first/next Start codon Continues to the next Stop codon Records the size and location of ORF • List OFRs sorted by length in descending order
ORF Table Minimum ORF Length: Can Redraw with lower cut-off Graphical View Clickable
Submit for BLAST Selected ORF ORF Length ORF Translation
Multiple Sequence Alignment with Clustal W • Homologous residues in a set of sequences are aligned together in columns • Ideally, homology reflects structural and evolutionary conservation • Evolutionary history of a residue can be deduced from sequence alignments of sequences from different organisms
Alignment Editor Pairwise Scores
Download file Colored Alignment
