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22 Cards in this Set
- Front
- Back
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What are some methodologies to study the genome? (4)
What do all of these techniques employ? |
Southern blot (DNA target)
Northern blot (RNA target) PCR Microarrays - base-pairing of nucleic acids, DNA sequencing |
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How are proteins studied?
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mass spectrometry
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What must a sample contain for genomics to be useful? What are examples of substances that would NOT be useful?
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nucleic acids
-saliva, urine, CSF, plasma... |
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What is usually used to study DNA/RNA
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Microarray
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What is used to study proteins?
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Proteomics
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Give 4 examples of uses of genomics/proteomics:
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To analyze expression differences in:
- normal vs. healthy tissue - drug-treated cells vs. control - normal cell cycle - tissue specific genes/proteins |
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Explain how microarray analysis works.
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2 bacterial cultures are grown under diff conditions. RNA is purified, then converted to cDNA (complimentary DNA).
cDNA is labeled with diff fluorescent dyes. Mix equal masses, then hybridize against microarray. cDNA base pairs with genes tethered to microarray (hybridization of target and probe). Visualize with scanner. |
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How does SDS-PAGE work? What can you do with it?
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Polymerize a gel of polyacrylamide. Apply proteins at top, apply electric current. Proteins are separated by mass.
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How does Western blot work?
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Transfer proteins to membrane.
Detect proteins with specific antibodies (labeled secondary antibody attached to primary antibody) |
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What is a limitation of SDS-PAGE?
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Not good for comparative analysis - for looking at expression differences
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What is 2-dimensional SDS PAGE? What can you do with it?
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Resolve individual proteins
Isoelectric focusing (ampholytes establish pH gradient, proteins migrate to their isoelectric point) Then, SDS PAGE |
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What is a limitation of proteomics?
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You're only looking at the most abundant proteins
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Where does trypsin cleave?
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carboxyl side of lysine/argenine residues
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Describe simple mass spectometry.
Matrix assisted laser desorption ionization - time of flight MALDI TOF |
Digest protein with Trypsin - you get a lot of fragments, each with its unique Lys/Arg fingerprint.
Do MaldiTOF mass spectometry: trypsin digested peptides are put onto a metal slide, mixed with UV-light absorbing rganic compound. Bombarded with UV laser, which excites the peptides. UV light turns peptides into gas, ionizes them, repelled from metal plate. Fly to detector - measure time it takes to get from origin to detector (small proteins fly faster) TOF = time of flight |
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What is Tandem Mass spectometry (MS-MS)
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adding a second dimension of mass spectrometry
highly automated, very fast top part = simple mass spec put target slide in, separate on 1st time of flight. then, electronic gate opens to allow indiv peaks in. SO peak gets let through to another flight tube. This flight tube is not a vacuum, has a gas. proteins are flying out of a vacuum and into an env where they will collide with other molecules. WHen the peptides hitt other molecules, they will fragment. Protein can only be fragmented in certain ways. based on the way it fragments, computer can predict the amino acid sequence. Then can understand what protein it is with very high confidence. so after mass spec, it goes thru another tube where it collides with other particles -- fragments -- computer can predict AA seq based on how it fragments. |
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What is a limitation of 2D gels, mass spec?
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takes a few days
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what is HPLC (High pressure liquid chromatography)?
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variety of ways of separating proteins based on physical characteristics
cation/anion exchange, reverse phase |
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What is PKU?
phenylketonuria |
genetic disease where conversion of phenylalanine to tyrosine is absent/greatly decreased. --> elevated Phe/toxic metabolites in blood
retardation, brain damage, seizures Tx: early diagnosis, dietary limitation of Phe MS/MS test - to measure blood Phe levels |
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What are noroviruses?
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highly contagious RNA viruses
MS can be used to detect the norovirus capsid protein in human stool. |
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How can MS/MS be used as a cancer detection tool?
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detect proteins at earliest stages of cancer
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Describe the use of MS for drug testing
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take saliva sample, test for 23 drugs
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What is liquid chromatography (LC) - MS?
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Protein digested by trypsin, separated on HPLC columns.
The individual peptides are identified. The proteins are then "reassembled" using bioinformatics, computing |
