Real Time PCR
as a Diagnostic
Tool
PCR = Polymerase Chain
Reaction
• A way to make lots of copies of DNA
Setting Up!
See real people doing PCR at
http://www.youtube.com/watch?v=sGP
56W0CSV8
What Happens in the Machine?
Do the PCR virtual lab at
http://learn.genetics.utah.edu/content/
labs/pcr/
Real Time PCR
• Amplifies AND quantifies a targeted segment
of DNA
– Regular PCR: DNA detected at end
– Real time PCR: DNA detected as the reaction
progresses
• Fluorescent dyes or probes are added and
fluorescence is measured during the reaction
• Can be used for both diagnostic & basic
research
The Lightcycler (Real-time PCR)
The Lightcycler
consists of a
Computer
attached to a
PCR machine
with a
florescence
detector.
• Used to detect SNPs- single nucleotide
polymorphisms- based on melting curve
analysis
• Quick, inexpensive way to investigate genetic
differences; works as a diagnostic tool.
What’s a Single Nucleotide
Polymorphism (SNP)?
Do the tutorial at
http://learn.genetics.utah.edu/content/health/pharma/snips/
Melting Curve Analysis
• The temperature at which
50% of the DNA is denatured
is called the melting point.
– G-C pairs have 3 hydrogen
bonds.
– A-T pairs have 2 hydrogen
bonds.
– G-C bonds will “melt” at a
higher temperature.
Lightcycler data