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.
DNA Sequence Analysis
To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis
Preparation of Competent Cells and Bacterial Transformation
Growth of Transformant and Plasmid MiniPrep
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 (Basic Local Sequence Alignment Tool)
The BLAST Family
default 11 bp or 3 aa
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
Submit Search by Clicking Here
Execute Search by Clicking Format
NCBI’s ORF FINDER and Open Reading 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
Minimum ORF Length: Can Redraw
with lower cut-off
Submit for BLAST