* Detecting Proteins, RNA, and DNA through
Laboratory Techniques Handout
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PCR Review
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RT-PCR
Reverse transcription PCR
Used to detect RNA levels
RNA is converted to cDNA by reverse transcriptase
Then it is amplified similar to PCR
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Uses of RT-PCR
This technique can detect very low numbers of RNA
Useful in the insertion of genes into prokaryotes
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cDNA is reverse transcribed from mRNA, so no introns are
found on the DNA sequence only exons
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No splicing is required
RT-PCR vs. PCR
PCR
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RT-PCR
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Gel Electrophoresis
Allows us to know whether the correct DNA fragments were generated.
Separates fragments by size
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Other lab techniques
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Southern blot
Used to detect a specific DNA sequence
First:
Then:
Next: You expose the membrane to a hybridization probe – a specific DNA
sequence
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Hybridization probe
DNA
The probe is labeled radioactively or with a fluorescent dye
Any excess probe is washed off and the pattern is then visualized via x-ray
Hybridization of the probe to the DNA fragments indicates the fragment contains
a complementary sequence to the probe.
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Northern blot
RNA is extracted from a tissue or cells
The RNA sample is then separated by size through gel electrophoresis
A nylon membrane is placed over the gel and RNA is transferred from gel to
membrane
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The nylon membrane is
charged which binds
effectively to the negatively charged RNA
Once the RNA is bound to the membrane it is covalently linked to it via UV light
or heat
This technique allows you to observe
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Western blot
This technique is used to detect protein samples in cells
Cell samples are broken down mechanically by a blender or lysed then
precipitated.
The samples are run on gel electrophoresis and may be separated by:
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The most common gel electrophoresis used for proteins is polyacrylamide gel
with sodium dodecyl sulfate
SDS-PAGE for short
SDS-PAGE denatures the proteins and allows for separation based solely on
SDS coats the protein with a
charge and proteins travel to the
positive anode.
antibodies are mixed with the membrane and binds to the protein
antibodies, which are tagged, are added and bind to the primary
antibodies
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ELISA
Enzyme-Linked ImmunoSorbent Assay
A test that uses antibodies and color changes to identify a substance
Then, a specific antibody is washed over the surface so that it can bind to the
antigen
Enzymes in ELISA
Antibody is linked to an enzyme
Substance is added that the enzyme can convert to some detectable signal
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Process of an ELISA
Sample with unknown amount of antigens is fixed to medium (plate/well, etc)
Detection antibody can be covalently linked to an enzyme, or a secondary
antibody attached to an enzyme can be used
Between each step the plate is typically washed with a mild detergent solution
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The plate is developed by adding an enzymatic substrate to
produce a visible signal
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Signal indicates the quantity of antigen in the sample