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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