lecturer: david. * reverse transcription pcr * used to detect rna levels * rna is converted to cdna...
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Lecturer: David
*Detecting Proteins, RNA, and DNA through Laboratory Techniques
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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
*cDNA is reverse transcribed from mRNA, so no introns are found on the DNA sequence only exons
*No splicing is required
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PCR*Amplification of DNA
RT-PCR*Using reverse
transcriptase to convert RNA cDNA
*Amplification of cDNA
*RT-PCR vs. 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
*Southern Blot
*Northern Blot
*Western Blot
*ELISA
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*Southern blot
*Used to detect a specific DNA sequence
*First: Restriction endonucleases are used to cut DNA into fragments
*Then: They are run on agarose electrophoresis to separate by size
*Next: A sheet of nitrocellulose paper is placed over the gel and baked in vacuum or oven
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*Southern blot
*You expose the membrane to a hybridization probe – a specific DNA sequence
*Hybridization probe – a DNA or RNA fragment used to detect a complementary DNA sequence
*DNA binds to the membrane since DNA is negatively charged, and the membrane is positively charged
*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
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*Hybridization of the probe to the DNA fragments indicates the fragment contains a complementary sequence to the probe.
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*Northern blot
*Technique used to study gene expression by detecting RNA
*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
*The nylon membrane is positively charged which binds effectively to the negatively charged RNA
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*Once the RNA is bound to the membrane it is covalently linked to it via UV light or heat
*A tagged probe is then hybridized with the RNA
*This technique allows you to observe gene expression
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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:
*Isoelectric point – the pH when the protein as no charge
*Molecular weight
*charge
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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 molecular weight
*SDS coats the protein with a negative charge and proteins travel to the positive anode.
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*The proteins on the gel are then transferred to nitrocellulose paper by using an electric current
*Primary antibodies are mixed with the membrane and binds to the protein
*Secondary antibodies, which are tagged, are added and bind to the primary antibodies
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*Enzyme-Linked ImmunoSorbent Assay
*A test that uses antibodies and color changes to identify a substance
*An unknown amount of antigen is affixed to a substrate
*Then, a specific antibody is washed over the surface so that it can bind to the antigen
*ELISA
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*Antibody is linked to an enzyme
*Substance is added that the enzyme can convert to some detectable signal
*Enzymes in ELISA
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*Sample with unknown amount of antigens is fixed to medium (plate/well, etc)
*Detection antibody is then added, forming a complex with the antigen
*Detection antibody can be covalently linked to an enzyme, or a secondary antibody attached to an enzyme can be used
*Process of an ELISA
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*Between each step the plate is typically washed with a mild detergent solution
*Detergent removes any proteins or antibodies that are not specifically bound
*The plate is developed by adding an enzymatic substrate to produce a visible signal
* Signal indicates the quantity of antigen in the sample
*Process of an ELISA cont.