Calculating Primary Melting Temperature

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					Calculating the melting temperatures
              of primers
              What is a primer?
• A primer is a short       • Reminder: DNA
  piece of nucleic acid       consists of two long
  strand that serves as a     chains of nucleotides
  starting point for DNA      (aka nucleic acids)
  replication.                twisted into a double
                              helix structure.
               Nucleotides
•   Adenine          • Reminder: In a DNA
•   Cytosine           double helix,
•   Guanine            nucleotides are
                       matched between
•   Thymine            strands through
                       hydrogen bonds to form
                       base pairs. Adenine
                       pairs with thymine and
                       cytosine pairs with
                       guanine.
DNA
        What is DNA replication?
• The copying of double   • Researchers commonly
  stranded DNA              replicate DNA in vitro
                            (i.e. outside the body =
• Necessary for cell        in the lab) using the
  reproduction prior to     polymerase chain
  cell division             reaction (PCR)
               DNA Replication
• Click here for video of DNA replication
      Polymerase Chain Reaction
• Click here for a video of PCR
        Professionals that use PCR
•   Clinical doctors   • “PRC is the most
•   Researchers          important new scientific
•   Geneticists          technology to come
                         along in the last
•   Lawyers              hundred years”
•   Law enforcement      - Mark Hughes, deputy
•   Historians           director of the National
•   Anthropologists      Center for Human
•   Archeologists        Genome Research
               Back to primers
• One of the most critical   • The choice of length of
  parameters for               the primers and their
  successful PCR is the        melting temperatures
  design of primers.           (Tm) must be
                               determined.
   Melting temperature of primer
• The temperature at which 50% of a particular DNA
  double helix will dissociate and become single
  strand DNA.
               Back to primers
• Primer Design
  - DNA primers of 18-24 nucleotides are commonly
  used for PCR
  - They may also be degenerate at one or more
  nucleotides to permit amplification of target DNAs
  of more variable sequence
  - Degenerate primers are mixtures of primers with
  different nucleotides occurring at the same primer
  position on different primer molecules
              Back to primers
• Primer Design
  - GC content in the range of 40-60%
  - Relatively high melting temperature (Tm; ideally
  55-65°C)
  - Use primer pairs with Tm not more than 5°C
  different
  - Tm = 4(G+C) + 2(A+T)°C.
  - The annealing temperature for use in PCR can
  further be approximated by subtracting 5°C from
  the Tm
               Back to primers
• Primer Design
  - Long stretches of a single nucleotide
  (as 5’-AAAAAAA-3’) should be avoided
  - Primers should not be self-complementary nor
  complementary to the other primer used in the
  PCR reaction
  - Restriction enzyme cleavage sites may be
  added to the 5’ ends of primers to facilitate post-
  amplification cloning of PCR products
        Calculating Tm of primer
• Tm = 4(G + C) + 2(A + T)   • Tm increases with length
                               and with increased
• Tm is expressed in °C        guanine and cytosine
                               content because the
                               base paired held with
                               three hydrogen bonds
                               instead of two as in
                               adenine and thymine.
Calculating Tm of primer - example
• Primer:                •   Calculation:
  TGACCTGAAAAGAC         •   Tm = 2(6 + 2) + 4 (3 + 3)
• Count nucleic acids:   •   Tm = 24 + 36
• Guanine = 3            •   Tm = 60°C
• Cytosine = 3
• Adenine = 6
• Thymine = 2
                    Summary
• Calculating the melting point of a primer is necessary
  for the design of a successful primer
• One of the most critical parameters for successful
  PCR is the design of primers.
• However, other factors that affect the PCR reaction
  that need to be in consideration are PCR profile, DNA
  template, cofactors such as MgCl2 concentration, and
  DNA polymerase.

				
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