- Course Introduction
- 01: Evidence & DNA
- 02: Forensic Biology
- 03: DNA Extraction & Quantitation
- 04: DNA Amplification
- Overview
- Introduction
- Amplification
- Primer Design
- Primer Length
- Primer Melting Temperature
- Primer Annealing Temperature
- Values for Tm
- Maximum 3′ Stability
- GC Content Percentage
- No Secondary Structures
- No Self-complementarity
- No Complementarity To Other Primers
- No Long Runs With The Same Base
- Distance Between Two Primers On Target Sequence
- Plateau Effect
- Thermal Cyclers
- Thermal Cycling Parameter and Optimization
- Works Cited & Online Links
- Locus Selection
- Multiplexing
- Contamination
- Overview
- 05: Amplified DNA Product Separation
- 06: STR Data Analysis & Interpretation
- 07: Population Genetics & Statistics
- 08: Communicating Results
- 09: Other DNA Markers & Technologies
- Course Resources
- Laboratory Training Manual
President's DNA Initiative
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Advancing Justice Through DNA Technology
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Advancing Justice Through DNA Technology
DNA Analyst Training
No Secondary Structures
Home > DNA Amplification > Overview > Primer Design > No Secondary Structures
A successful PCR reaction requires efficient amplification of the product. At normal temperatures nucleic acids fold into conformations (secondary structures) which have high negative free energy (∆G). The stability of these template secondary structures depends largely on their free energy and melting temperatures (Tm), and is extremely important for designing primers. If these secondary structures in the nucleic acids are stable even above the annealing temperatures, then the primers are unable to bind to the template DNA, and the yield of PCR product is significantly reduced. It is desirable to design specific primer pairs which do not assume secondary structures during the reaction, and this can be determined from a program called mfold server.
Click here to visit a website that uses the mfold server.
