Real Time PCRReal Time PCRas a Diagnostic as a Diagnostic

ToolTool

PCR = Polymerase Chain Reaction• A way to make lots of copies of DNA

What Happens in the Machine?Do the PCR virtual lab at

http://learn.genetics.utah.edu/content/labs/pcr/

Setting Up!See real people doing PCR at

http://www.youtube.com/watch?v=sGP56W0CSV8

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)

• Used to detect SNPs- single nucleotide polymorphisms- based on melting curve analysis

• Quick, inexpensive way to investigate genetic differences; works as a diagnostic tool.

The Lightcycler consists of a Computer attached to a PCR machine with a florescence detector.

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