Lecturer: David

*Detecting Proteins, RNA, and DNA through Laboratory Techniques

*PCR Review

*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

*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

PCR*Amplification of DNA

RT-PCR*Using reverse

transcriptase to convert RNA cDNA

*Amplification of cDNA

*RT-PCR vs. PCR

*Gel Electrophoresis

*Allows us to know whether the correct DNA fragments were generated.

*Separates fragments by size

*Other lab techniques

*Southern Blot

*Northern Blot

*Western Blot

*ELISA

*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

*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

*Hybridization of the probe to the DNA fragments indicates the fragment contains a complementary sequence to the probe.

*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

*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

*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

*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.

*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

*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

*Antibody is linked to an enzyme

*Substance is added that the enzyme can convert to some detectable signal

*Enzymes in ELISA

*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

*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.