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DNA Sequence Analysis

DNA Sequence Analysis

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DNA Sequence Analysis

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  1. DNA Sequence Analysis

  2. Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis

  3. Research Plan Preparation of Competent Cells and Bacterial Transformation Growth of Transformant and Plasmid MiniPrep Cycle Sequencing Sequence analysis

  4. 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?

  5. 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/

  6. The BLAST Family

  7. 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

  8. 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

  9. PasteSequence here Submit Search by Clicking Here

  10. Execute Search by Clicking Format

  11. BLASTX Results

  12. Interpreting BLAST Results • Length • E-Value • Bit Score • Identities • Positives

  13. 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

  14. 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

  15. www.ncbi.nlm.nih.gov/gorf/gorf.html

  16. ORF Table Minimum ORF Length: Can Redraw with lower cut-off Graphical View Clickable

  17. Submit for BLAST Selected ORF ORF Length ORF Translation

  18. 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

  19. http://www.ebi.ac.uk/clustalw/

  20. Alignment Editor Pairwise Scores

  21. Download file Colored Alignment