Transcript DNA Sequence Analysis

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
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 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
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
Paste Sequence here
Submit Search by Clicking Here
Execute Search by Clicking Format
BLASTX Results
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:
Antisense Strand:
Frame +1
Frame +2
Frame +3
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
www.ncbi.nlm.nih.gov/gorf/gorf.html
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
http://www.ebi.ac.uk/clustalw/
Alignment Editor
Pairwise Scores
Download file
Colored Alignment