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Single DNA Sequence Analysis Tools. BME 110: CompBio Tools Todd Lowe May 6, 2008. Today’s topics. ORF prediction Restriction enzyme search tools PCR primer design Using on-line tools from ExPASy. Basic ORF Finding. Look for “long” open reading frames

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single dna sequence analysis tools

Single DNA Sequence Analysis Tools

BME 110: CompBio Tools

Todd Lowe

May 6, 2008

today s topics
Today’s topics
  • ORF prediction
  • Restriction enzyme search tools
  • PCR primer design
  • Using on-line tools from ExPASy
basic orf finding
Basic ORF Finding
  • Look for “long” open reading frames
  • Scan sequence at nucleotide #1 (Frame #1), begin ORF at first start codon: ATG
  • Continue scanning to first stop codon: TAA, TGA, or TAG
  • That is your ORF!
  • Repeat, starting at nuc #2 (Frame #2) , then again, starting at nuc #3 (Frame #3)
  • Take reverse complement of sequence ->
  • (i.e. 5’-CGAAC -> 5’-GTTCG)
  • Scan sequence starting at nuc #1 (Frame –1), nuc #2 (Frame –2), nuc #3 (Frame –3)
on line orf finding @ ncbi
On-line ORF Finding @ NCBI

  • Shows all ORFs of min. length 50, 100, or 300 nucleotides long
  • Also shows all start (green) & stop (red) codons
  • Allows “alternate” start codons (TTG, GTG, CTG)
  • When you’ve selected an ORF you like, click on “Accept” button, then can display nucleotide or protein sequence for further analyses
historical note annotating the yeast genome
Historical Note: Annotating the Yeast Genome
  • Yeast is a eukaryote, but does not have many introns
  • A strict cutoff for ORF length was used: minimum of 300 nucleotides ORF required to be considered a gene in original genome annotation (1996)
  • Since then, many smaller ORFs have been found experimentally
more sophisticated methods
More Sophisticated Methods
  • Can analyze entire genome at once, use codon frequencies, not just one gene
  • GeneMark (
  • Also, Glimmer from TIGR


  • Not available on-line, must download and run locally
cutting dna restriction enzymes
Cutting DNA: Restriction enzymes
  • enzymes isolated from prokaryotes that break DNA at very specific sequence-specific positions
  • in nature, act as host-defense against viruses


Nla III: 5’ ... CATG^ ... 3’

BamH I: 5’ ... G^GATCC... 3’

Dra III: 5’ ... CACNNN^GTG... 3’

> 3,000 RE’s found to date with >200 specificities!

many re s create sticky ends
Many RE’s Create “Sticky” Ends

Before cutting:


After cutting, “sticky ends”:



  • Useful to increase efficiency & specificity of re-joining ends
sample agarose gel
Sample Agarose Gel

Same DNA cut by different restriction enzymes give different fragment lengths

new england biolabs tools
New England BioLabs Tools

NEBcutter – Display which restriction enzyme cut in your sequence, and where

REBsites – Display a “virtual” digest of your DNA, showing how it would look on an agarose gel


Key Input:

  • Sequence
  • Targets (region to be amplified)
  • Product Size Ranges
  • Primer size (usually 18-22 bp)
  • Primer Tm (annealing temperature)

(Rest are usually OK as defaults)

expasy proteomic tools
ExPASy Proteomic Tools

All ExPASy Tools Website

  • Basic protein statistics: ProtParam
  • Scan all motif/domain/profile databases: InterProscan
  • Protein topology / location in cell: Psort, TMHMM
  • Many, many others