DNA Sequence Analysis

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DNA Sequence Analysis
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Broad and Long Term Objective

• To characterize a single clone from an
  Emiliania huxleyi cDNA library using sequence
  analysis

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Research Plan
Preparation of Competent Cells and Bacterial
Transformation
Growth of Transformant and Plasmid MiniPrep
Cycle Sequencing
Sequence analysis
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Todays 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?
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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/

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The BLAST Family
Program Query Sequence Database Target
BLASTN Nucleotide (both strnds) Optimized for speed not accuracy Not good for distant homologues Dust Option (low complexity) Nucleotide Database
BLASTX Nucleotide translated 6 frames Less sensitive to sequence errors and mismatches Useful for preliminary data/EST Dust Filter Option Protein Database
TBLASTX Nucleotide translated 6 frames Good for ESTs and Single Pass Sequences, Very Slow Nucleotide Database Translated 6 frames
BLASTP Protein Protein Database
TBLASTN Protein Proteins against nucleotides and ESTs Nucleotide Database Translated 6 frames
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The Blast Algorithm

• Identify HSPs (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

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The Blast Algorithm

• Score the Alignment
• a scoring matrx such as BLOSUM62 or PAM
  is used
• gaps introduced between GSPs 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

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Paste Sequence here
Submit Search by Clicking Here
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Execute Search by Clicking Format
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BLASTX Results
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Interpreting BLAST Results

• Length
• E-Value
• Bit Score
• Identities
• Positives

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NCBIs 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

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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

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www.ncbi.nlm.nih.gov/gorf/gorf.html
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ORF Table
Minimum ORF Length Can Redraw with lower
cut-off
Graphical View
Clickable
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Submit for BLAST
Selected ORF
ORF Length
ORF Translation
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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

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http//www.ebi.ac.uk/clustalw/
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Alignment Editor
Pairwise Scores
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Download file
Colored Alignment