(L17) Molecular Diagnostics Flashcards
What are southern and northern? L17 S11
Southern blot: -hybridization to detect specific DNA sequence
Northern blot: -hybridization to detect specific RNA sequence DNA/RNA sequence must be known in order to detect
What is PCR and what is it used for? L17 S12
- dsDNA is taken and denatured
- Primer (required sequence must be know) binds to original DNA and flank each end of DNA in 3’ to 5’ direction
- allow it to bind to DNA (anneal)
- add all 4 dNTPs
- Tap polymerase is used to replicate new DNA from primer using original DNA as a template Amplifies DNA
What is qPCR and what is it used for? L17 S15
Quantitative PCR Same as normal PCR but includes a probe that fluoresce in presence of PCR product
- Quantifies copy number
- will only light up when primers extend all the way to the end of the probe
- used to detect levels of gene expression or detect levels of an infectious agent
What is RFLP and what is it used for? L17 S21-22
- Restriction fragment length polymorphism
- Uses PCR to amplify sample followed by restriction enzymes to cleave DNA in a very precise manner
- Due to differences everybody has in SNPs in restriction sites, every individual will have a unique way that the restriction enzymes will cleave their DNA Used as a genetic finger print.
- Can be used in paternity testing or disease detection (ie. sickle cell has 2 restriction sites where normal has 3).
DNA fingerprinting
What is VNTR and how is it used? L16 S23-24
Variable number of tandem repeats
- There are several locations in the genome where there are short tandem repeats (STR).
- find primer that flank the repeats and run a PCR, run what you got in a gel to see the number of repeats
- Several diseases occur due to increased numbers of the STRs (Huntington disease, fragile X, etc.)
- The number of STRs can be used to determine likelihood of developing one of these diseases (technical term is anticipation)
(used in huntingtons dz, useful in identification of herdietary disesaes and the severity of them)
How are recombinant proteins produced? L16 S27-29
Gene for desired protein is isolated and introduced into a plasmid which is then introduced to bacteria. Plasmid is designed so that the bacteria will produce the gene product which can then be isolated and used.
-take a plasmid vector and add an antibitoic resiteance gene, cDNA of protein in the vectors, and restriction sites
How are antibodies produced? L16 S33
Mouse is immunized with desired Ab target. B cells that produce desired Abs are isolated B cells are fused with immortalized tumor cells Hybrid cell is now immortal and produced desired Ab. Cell is cultured and Ab is isolated
What does -mab at the end of a medication name mean? L17 S38
Indicates that the drug product is a monoclonal antibody
What is ELISA? What types are there and what is each used for L17 S38-40
Enzyme-linked immunosorbent assay: indicates how much is in a sample
indirect: -immobilized Ag is on plate -sample is added to plate and if Ab specific fo Ag is present it binds -labeled anti-Ab Ab is added which will bind to and indicate if Ag specific Ab bound
Sandwich: -immobilized Ab is on plate -sample is added to plate and if Ag that the Ab is specific for is present it binds -Ab specific for Ag is added which will bind Ag if it was present -labeled anti-Ab Ab is added which will bind to and indicate if Ag-specific Ab bound
What is Western blotting? L16 S45
Protein mixture is isolated via SDS-PAGE and transferred to nitro cellulose membrane Ab specific for desired protein is added and if protein is present it will bind. Labeled anti-Ab. Ab is added which will bind to and indicate if Ag-specific Ab bound.
Used to confirm HIV
Steps for Hybridization
- get a single stranded DNA and immbolize it it on a membrane (blotting)
- generate single stranded oligonucleotides (probes)
- Hyberdize the probe: will light up when you make the sample
How to blot
1) material seperated by gel eletrophroesis
2) transfer to a membrane
3) add probe
4) visualize probe by florucense or autoradiography
Eastern blot
detects post translational mods on proteins
Insulin normal
-normal insulin has a proline at 28 and lysine at 29 of b chain
lispro
has lysine on 28 and proline at 29 of b chain
