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Real Time PCR
Transcript of Real Time PCR
Phillip Callihan Real Time PCR History As the sample amplifies: the time it takes a fluorescent signal to reach threshold level correlates with amount of target genes in original sequence (enabling quantification).
The final product can be further described by heating it to determine when the double-stranded product “melts.” (The point at which the strands separate depends on product length and nucleotide composition.) How does it work? Molecular Principle Cancer Diagnostics: Mutations in genes can be determined by melting curve analysis by observing a shift in the melting point of the PCR product.
helpful in understanding patient specific disease process.
Useful in drug development studies. Example of Use Advantages:
- Quantitative, sensitive, very small quantities required
- susceptible to contamination
- lots of room for human error
- In RT real-time PCR RNA is involved = FRAGILE Real Time PCR
Pros & Cons Based on PCR: process of amplifying specific pieces of DNA more than a billion-fold
Addition of fluorescent dyes (Ethidium bromide & SYBR green) led to visualization and recording of nucleic acids
Factors that Led to Need for Real Time PCR
o Need to quantify levels of nucleotides
o The availability of only small amounts of sample in some procedures such as:
cells obtained by laser capture micro-dissection
small amounts of tissue
Hunt, M. Real time PCR. Microbiology and Immunology On-line. University of South Carolina. 2010. <http://pathmicro.med.sc.edu/pcr/realtime-home.htm>
Valasek, M. Repa, J. The power of real-time PCR. American Physiological Society. Vol 29:3. pg. 151-159. Sept 2005. Sources