Calculating the melting temperatures of primers

1. Calculating the melting temperatures of primers

2. What is a primer? A primer is a short piece of nucleic acid strand that serves as a starting point for DNA replication. Reminder: DNA consists of two long chains of nucleotides (aka nucleic acids) twisted into a double helix structure.

3. Nucleotides Adenine Cytosine Guanine Thymine Reminder: In a DNA double helix, nucleotides are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine and cytosine pairs with guanine.

4. DNA

5. What is DNA replication? The copying of double stranded DNA Necessary for cell reproduction prior to cell division Researchers commonly replicate DNA in vitro (i.e. outside the body = in the lab) using the polymerase chain reaction (PCR)

6. DNA Replication Click here for video of DNA replication

7. Polymerase Chain Reaction Click here for a video of PCR

8. Professionals that use PCR Clinical doctors Researchers Geneticists Lawyers Law enforcement Historians Anthropologists Archeologists �PRC is the most important new scientific technology to come along in the last hundred years� - Mark Hughes, deputy director of the National Center for Human Genome Research

9. Back to primers One of the most critical parameters for successful PCR is the design of primers. The choice of length of the primers and their melting temperatures (Tm) must be determined.

10. Melting temperature of primer The temperature at which 50% of a particular DNA double helix will dissociate and become single strand DNA.

11. 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

12. Back to primers