reaction (PCR)
Experiment Goals
Understand how PCR technique works
Perform PCR experiment
Analyze PCR products
What is PCR?
Definition
The polymerase chain reaction (PCR) is a
technique to amplify a piece of DNA very
rapidly outside a living cell
Development of PCR
1971: Khorana described basic principle
of DNA replication using DNA primers
1983: Dr. Karry Mullis developed PCR
technique, for which he received the
Nobel Prize in Chemistry in 1993.
PCR Applications
PCR is now a common and often indispensable
technique used in medical and biological research labs
for a variety of applications.
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Analyze PCR products
Check a sample by gel electrophoresis.
Is the product the size that you
expected?
Is there more than one band?
Is any band the correct size?
May need to optimize the reaction
conditions.
PCR Modifications
Nested PCR
increases the specificity of DNA amplification, by reducing
background due to non-specific amplification of DNA. Two sets
of primers are being used in two successive PCRs.
Multiplex PCR
The use of multiple, unique primer sets within a single PCR
mixture to produce amplicons of varying sizes specific to
different DNA sequences.
Reverse-transcriptase PCR
(Reverse Transcription PCR) is a method used to amplify,
isolate or identify a known sequence from a cellular or tissue
RNA. The PCR is preceded by a reaction using reverse
transcriptase to convert RNA to cDNA.
Polymerase Chain Reaction
Controls for PCR
Blank reaction (Negative control reaction)
Controls for contamination
Contains all reagents except DNA template
Analyse products on 2%
-ve +ve Sample
agarose gel containing
ethidium bromide
Visualize the PCR product
on UV transilluminator