PCR Primer Designfrom Edwards and Logan 2009, "Performing Real-time PCR" Chapter 6 in Real-Time PCR: Current Technology and Applications. Good primer design is vital tool for all PCR and real-time qPCR procedures. Many PCR software programs are available for PCR primer design and other important PCR applications.
In any PCR or real-time qPCR the following criteria should be optimised: buffer composition, cycle conditions, magnesium chloride (MgCl2) concentration, primer concentration, probe concentration and template concentration. Commercial master mixes, which are widely available, simplify the optimisation and are convenient. Primers should be designed with the aid of appropriate software and attention should be paid to the optimal size of the amplicon produced in the real-time application. Real-time PCR products are usually <500 bp, this may be much smaller than the amplicon size generated on a block-based thermal cycler. The following guidelines should be observed for optimal and accurate primer design:
Edited by: Jim Huggett and Justin O'GradyI would highly recommend this book (Doodys) read more ...
The application of molecular technology in clinical diagnosis in two key diagnostic areas: cancer and infectious diseases.
Edited by: Nick A. Saunders and Martin A. Lee"an invaluable reference" (Doodys); "wide range of real time PCR technologies" (Food Sci Technol Abs) read more ...
Provides both the novice and experienced user with an invaluable reference to a wide-range of real-time PCR technologies and applications and supplies detailed technical insights into the underlying principles, methods and practice of real-time PCR.
- The primer 3' ends should be free from secondary structure, repetitive sequences, palindromes and highly degenerate sequences.
- Forward and reverse primers should not have significant complementary sequences.
- The forward and reverse primers should have equal GC contents, ideally between 40-70%.
- The binding sites should not have extensive secondary structure.
The CT (cycle threshold) value of a real-time PCR procedure is the number of cycles required for the fluorescent signal to exceed the background level i.e. "cross the threshold". CT values are dependent on various factors including primer design and DNA template concentration. This value is often referred to as crossing point (CP) in LightCycler terminology.
- Bacterial-Plant Interactions
- Metagenomics of the Microbial Nitrogen Cycle
- Pathogenic Neisseria
- Human Pathogenic Fungi
- Applied RNAi
- Molecular Diagnostics
- Phage Therapy
- Bioinformatics and Data Analysis in Microbiology
- The Cell Biology of Cyanobacteria
- Pathogenic Escherichia coli
- Campylobacter Ecology and Evolution
- Next-generation Sequencing
- Omics in Soil Science
- Applications of Molecular Microbiological Methods
- Genome Analysis
- Bacterial Toxins
- Bacterial Membranes
- Cold-Adapted Microorganisms