DNA Sequence AnalysisDNA Sequence Analysis

Broad and Long Term ObjectiveBroad and Long Term Objective

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

Research PlanResearch Plan

Preparation of Competent Cells and Bacterial Transformation

Growth of Transformant and Plasmid MiniPrep

Cycle Sequencing

Sequence analysis

Today’s Laboratory Objectives Today’s Laboratory Objectives

To learn how to characterize a DNA sequence using various web To learn how to characterize a DNA sequence using various web based bioinformatics tools including:based bioinformatics tools including:

1. BLASTN- has this piece of DNA been sequenced 1. BLASTN- has this piece of DNA been sequenced before? Does it look like anything before? Does it look like anything

already in already in GeneBank at the nucleotide GeneBank at the nucleotide level?level?

2. BLASTX- Can we identify the putative function of 2. BLASTX- Can we identify the putative function of the the transcripts? transcripts?

3. ORF Finder- What does the open reading frame 3. ORF Finder- What does the open reading frame look look like? Do we have a full length clone like? Do we have a full length clone with with an identifiable start and stop codon? an identifiable start and stop codon?

4. ClustalW- How does it compare with other 4. ClustalW- How does it compare with other sequences sequences either at the nucleotide or amino either at the nucleotide or amino acid level? acid level? What residues are conserved What residues are conserved and thus likely and thus likely to be important? And what to be important? And what residues are residues are divergent? divergent?

BLAST Database Search ToolBLAST Database Search Tool

BLAST (Basic Local Sequence Alignment Tool)BLAST (Basic Local Sequence Alignment Tool) Available on the internet and downloadableAvailable on the internet and downloadable Quick and simpleQuick and simple http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/

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

The BLAST FamilyThe BLAST Family

The Blast AlgorithmThe Blast Algorithm

Identify HSP’s (High Scoring Segment Pairs)Identify HSP’s (High Scoring Segment Pairs)

default 11 bp or 3 aadefault 11 bp or 3 aa

Perfect matchPerfect match

Slide query and target sequence across each other until the maximum Slide query and target sequence across each other until the maximum number of HSP for that target is foundnumber of HSP for that target is found

The Blast AlgorithmThe Blast Algorithm

Score the AlignmentScore the Alignment a scoring matrx such as BLOSUM62 or PAM is useda scoring matrx such as BLOSUM62 or PAM is used

gaps introduced between GSP’s during sliding get gaps introduced between GSP’s during sliding get negative score negative score

a match gets a positive scorea match gets a positive scoretotal alignment score is subjected to statistical total alignment score is subjected to statistical

analysis to calculate the significance vs. chance of the analysis to calculate the significance vs. chance of the scorescore

Repeat for every sequence in the target databaseRepeat for every sequence in the target database Return total resultsReturn total results

Paste Sequence here

Submit Search by Clicking Here

Execute Search by Clicking Format

BLASTX ResultsBLASTX Results

Interpreting BLAST ResultsInterpreting BLAST Results

•Length

•E-Value

•Bit Score

•Identities

•Positives

Begin with “ATG” start codonEnd with “TAA”, “TAG”, or “TGA” stop codonsCan occur in any six possible reading frames

Sense Strand: Frame +1 Frame +2 Frame +3

Antisense Strand: Frame -1 Frame –2Frame -3

NCBI’s ORF FINDER and Open Reading Frames

ORF Finder AlgorithmORF Finder Algorithm

Iterates over all frames:Iterates over all frames:

Iterate to the end of frameIterate to the end of frame

Find first/next Start codonFind first/next Start codon

Continues to the next Stop codonContinues to the next Stop codon

Records the size and location of ORFRecords the size and location of ORF

List OFRs sorted by length in descending orderList OFRs sorted by length in descending order

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

Graphical View

ORF Table

Minimum ORF Length: Can Redraw with lower cut-off

Clickable

Submit for BLAST

Selected ORF

ORF Length

ORF Translation

Multiple Sequence Alignment with Clustal WMultiple Sequence Alignment with Clustal W

HomologousHomologous residues in a set of residues in a set of sequences are aligned together in sequences are aligned together in columnscolumns

Ideally, homology reflects Ideally, homology reflects structuralstructural and and evolutionaryevolutionary conservation conservation

Evolutionary history of a residue can Evolutionary history of a residue can be deduced from be deduced from sequence sequence alignmentsalignments of sequences from of sequences from different organismsdifferent organisms

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

Alignment Editor

Pairwise Scores

Download file

Colored Alignment