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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.

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Broad and long term objective
Broad and Long Term Objective

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


Research plan
Research 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 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 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 algorithm
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 algorithm1
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


PasteSequence here

Submit Search by Clicking Here




Interpreting blast 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: Frame +1

  • Frame +2

  • Frame +3

  • Antisense Strand: Frame -1

  • Frame –2

  • Frame -3


Orf finder algorithm
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



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
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



Alignment Editor

Pairwise Scores


Download file

Colored Alignment


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