Analysis of DNA Sequences: Bioinformatics

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Analysis of DNA SequencesBioinformatics

• Julia Saxonov
• Tom Chen
• Montville High School

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Biological Databases

• The simplest tasks used in bioinformatics concern
  the creation and maintenance of databases of
  biological information.
• Nucleic acid sequences (and the protein
  sequences derived from them) comprise the
  majority of such databases.

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Most Pressing Tasks of Bioinformatics

• Finding the genes in the DNA sequences of various
  organism
• Developing methods to predict the structure
  and/or function of newly discovered proteins and
  structural RNA sequences.
• Clustering protein sequences into families of
  related sequences and the development of protein
  models.
• Aligning similar proteins and generating
  phylogenetic trees to examine evolutionary
  relationships.

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So how do we go about doing this?
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Sequence Analysis Flow Chart
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Use a Waveform program to briefly analyze the
quality of your DNA sequence

• We will be using the 4Peaks program
• Briefly scan through the sequence using the arrow
  keys
• Make sure the sequence is fairly clean with no
  long runs of Ns in the middle
• Long runs of Ns are not good and should be edited
  from the sequence

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The linker used

• AATTCGCGGCCGCT- cDNA insert AGCGGCCGCG
• GCGCCGGCGA- cDNA insert -
  TCGCCGGCGCTTA

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Remove vector and bad sequences from your 5-EX
DNA sequence

• Look for the EcoRI site GAATTC
• If the site was defective, it would be useful to
  look for a different restriction site, such as
  the SmaI site CCCGGG

• This is a starting point. Now in order to
  isolate the cDNA, you must find the linker site.
  Remove it and the bases before.

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Bad Sequences

• Examine the sequence until you are not confident
  with the base-calling, which could be lots of Ns
  or problems in the sequence itself

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Just as you did for the 5-EX DNA sequence,
remove vector and bad sequences from the 3-EX
sequence

• The sequence using the 3-EX end will be read
  from right to left
• This means that although on the DNA, the 3
  sequence begins on the right, your sequences 3
  end is on the left.

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GCTAGCTA
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Sequence will appear
ATCGATCG
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So now repeat the steps the same way as with the
5 sequence

• Again, look for the EcoRI site
• If this is defective, you can search for either
  the PstI site, or the BamHI site
• Remember, since it is being read from right to
  left, these restriction sites will be at the
  beginning of the sequence.

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BLAST for Beginners

• A step-by-step tutorial to searching for DNA
  sequences

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NEB Cutter
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Size of the open reading frame.
These are the different restriction sites that
are found in this section of DNA.
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Blasting Sanger
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This range from 59536 to 62018 is important to
remember
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The range of our match found from before is
within this range of cosmid Y50D7A.
There is no specific gene shown so you must click
the name column to find it yourself.
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These are the four sections where the query
matched the DNA in the library.
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Performing Structural Analysis

• How does it fold?
• Is it composed of one or multiple domains?
• If it is an enzyme, how does it bind substrate?
• Where is the active site?
• Are the regions that are conserved between your
  protein and other homologs on the interior or
  exterior of the protein structure?
• If there are mutants, where would the structure
  be affected?
• Why is this important
• Understanding how structure relates to function
  benefits in the successful design of drugs to
  activate or deactivate proteins. Understanding
  that a structure is related to proteins in
  another homologous organisms, such as humans,
  also allows us to test drugs on lower life forms.

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The End

• Disclaimer
• Presentation was the original work of Tom Chen
  and Julia Saxonov.
• NCBI blast slides courtesy of http//www.geospiza.
  com/outreach/BLAST/