Primer Design and Restriction Mapping

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Primer Design and Restriction Mapping
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Primers

• Synthetic oligonucleotide (oligo)
• 15 to 28 bases
• Single stranded
• Used in PCR and sequencing
• Orientation is 5 to 3
• Must be reverse compliment of nucleic acid you
  are attempting to sequence or PCR amplify
  (template)
• Primer must anneal

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Using Primers

• PCR requires primer pairs
• 2 primers that can amplify the double stranded
  DNA
• Sequencing requires 1 primer
• Melting Temperature and annealing temperature
• Annealing temperature chosen for a PCR depends
  directly on length and composition of the
  primer(s).
• IDT DNA
• 5 TAT TGT TGG CTT CCG GTA CAT 3

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Calculating Ta

• Primer annealing temperature (Ta) should be about
  5 degrees below the lowest melting temperature
  (Tm) for the primer pair
• Ta too low?nonspecific binding multiple PCR
  products
• Ta too high?primer annealing low low yield
• Tmmelting temperature (temperature at which
  double stranded DNA template separates).
• Tm 4(G C) 2(A T)oC.
• What is the Tm for these primer pairs?
• AGAGTTTGATCCTGGCTCAG
• GGTTACCTTGTTACGACTT

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Primer Design Rules

• Primers should be at least 15 base pairs long
  (typically 17-28 bases)
• Have at least 50 G/C content (not greater than
  60)
• Anneal at a temperature in the range of 50-65
  degrees C maximum of 80
• For PCR forward and reverse primer should
  anneal at approximately the same temperature
• 3'-ends of primer pairs should not be
  complementaryprimer dimers
• primer self-complementation should be
  avoidedhairpins
• runs of three or more Cs or Gs at the 3'-ends of
  primers should be avoided

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Primer Design

• The design of PCR (and sequencing) primers is
  relatively simple from a computational point of
  view just search along a sequence and find short
  sub-sequences that fit certain criteria.
• The nature of these criteria is not at all
  obvious
• All primers design software uses approximately
  the same criteria and computing algorithms.
• The rules for choosing PCR primers are a rough
  combination of educated guesses and old fashioned
  trial-and-error.

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Primer Problems

• primers should flank the sequence of interest
• primer sequences should be unique
• primers that match multiple sequences will give
  multiple products
• primers should not have self-annealing regions
  within each primer (i.e. hairpin and foldback
  loops)
• pairs of primers should not anneal to each other
  to form the dreaded "primer dimers"

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PCR Primers

• Flanking the region of interest in the DNA and
  annealing to DNA.

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3
3
5
DNA of interest
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Problems
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Software for Designing Primers

• Primer 3
• Lets try
• Go to Biology WB and use NDJINN to search for in
  the GBBCT database for the following accession
  number (15183169) and import this sequence.
• Now select the sequence and choose Primer 3
• Find a pair of primers that will PCR amplify most
  of this sequence.
• Now click here http//www.bioquest.org/bioinform
  atics/module/cooper/Primer_Design/primer_design.ht
  m
• Choose primers at least 18 nucleotides long.
  Forward and reverse primers.
• http//www.idtdna.com/scitools/scitools.aspx

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Restriction Mapping

• Can use molecular biology software to predict
  restriction enzyme cut sites for a DNA sequence
• Restriction maps for a DNA sequence can be
  obtained
• Necessary to know where your restriction sites
  are to clone in a cloning vector (plasmid)
• Mapping software
• TACG

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Restriction map
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TACG

• Used for restriction enzyme mapping
• A nucleic acid tool
• Allows you to cut a nucleic acid sequence with
  all possible restriction endonucleases or it
  allows you to select specific restriction enzymes
• Steps
• Upload sequence in Biology workbench nucleic acid
  tools.
• Select sequence and select TACG
• You will be allowed to select parameters (see
  next slide for description)

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All5 and/or 3 overhang or blunt Blunt no
overhang
Recognition site influences frequency of cut.
Cut specific sized fragment between two
enzymes?cloning
Linearshows the cuts on the actual sequence and
gives translation Ladder shows a coneptual view
of size fragments (larger than 300)
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What TACG can do

• Total number of hits per sequence
• Lists cut sites for each sequence
• Gives pseudo gel map for fragments produced
• Gives a linear map of sequence

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Lets Try.

• Obtain pBluescript file
• Open it select the sequence copy
• Open the Biology Workbench start a new session
  nucleic acid tools add new nucleic acid sequence
• Past sequence into box and give it a name
  (p_Bluescript) update sequence
• Nucleic acid tools select your sequence choose
  TACG accept defaults submit

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Practice your Knowledge

• If we cut this plasmid with EcoRI and ran the DNA
  on a gel
• How many fragments would we see on the gel?
• What would the gel look like?
• What if we cut with BamHI?
• What if we cut with both BamHI and EcoRI?

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Example exercise

• If we had a circular plasmid (3.4 kb) and we cut
  it with ApoI and EcoRI, ran the DNA on a gel, and
  the gel looked like this

With both enzymes 4 fragments resulted 1386,
1323, 667, 24 With ApoI 2 fragments
resulted 3376, 24 With EcoRI 2 fragments
resulted 2733, 667
3,400 b
2,000 b
1,500 b
1,000 b
500 b
Draw the plasmid map.
100 b
50 b
Gel
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Example exercise

• If we had this 1kb of linear DNA and we cut it
  with ApoI and EcoRI, ran the DNA on a gel

With both enzymes 3 fragments resulted 700, 200,
100 With ApoI 2 fragments resulted 800, 200 With
EcoRI 2 fragments resulted 900, 100
1,000 800 600 400 200
Draw a map