Moleuclar Diagnostics Flashcards
What are the two reasons for molecular techniques, given you know the sequence of the pathogen (1) or gene (2)?
- To rapidly detect/identify disease causing organism
2. To dignose inherited disorders in humans
What is hybridization?
When ssDNA binds to another strand of DNA or RNA (complementary sequence) to form DNA-DNA or DNA-RNA hybrids.
What is hybridization useful for?
For detection and quantification of target DNA or RNA in a sample containg a mix of both
What are single-stranded oligonuclotides called?
probes that are labeled with radioactive or fluorescent tags
What is southern blotting?
DNA+DNA, both probe and target nucleic acid are ssDNA
What is northern blotting?
DNA +RNA, probe is ssDNA and target is mRNA
What is the purpose of southern blotting?
to determine which restriction fragments are associated with a gene
What is the purpose of northern blotting?
measure size and quantities of mRNA molecules (gene expression)
What is the purpose of a western blot?
to measure the amount of protein or antibody
Steps of blotting techniques?
DNA or RNA is electrophoresed in agarose gel to separate and then transfered to membrane so surface exposed. Probe DNA/RNA is added and hydrolyzed to ssDNA
What is polymerase chain reaction (PCR)?
A process to amplify a small amount of dsDNA to however large you would like.
What is the process of PCR?
- Denature DNA with high temps to break H bonds
- Annealing: sample cools, anneals primer to both strands (3’-5’)
- TAQ polymerase (heat stable) & all 4dNTPs added to synthesize DNA
What is the final product of PCR?
DNA doubles in each cycle and can quickly multiple cycle after cycle (in thermocycler)
Disadvantages vs advantages of PCR?
A:Only need small amount of template DNA
Dis: Need to know sequence for flanking, error prone, contamination
Difference between PCR and qPCR (quantitative)?
- Used to quantify copy number
2. Uses probe that fluoresces only in presence of PCR product, by primer
What is qPCR used for?
detect levels of an infectious agent and determine levels of gene expression
What are restriction fragment length polymorphism (RFLP) and variable number tandem repeats (VNTR) used for?
Diagnostics and forensics
- prenatal dianosis
- newborn screening
- genetic carriers
RFLP use?
Restriction enzymes used to match suspect DNA with evidence DNA. If the two are the same DNA, endonucleases will cut the DNA at the same area. Southern blotting then used to trasnfer to membrane, add probe (radioactive)
How many bans will you have if there are 3 restriction sites? (where endonucleases will cut)
2 sites?
3 sites = 2 bans
2 sites = 1 ban
So if disease has 2, normal 3, and you run RFLP+ blotting and have 1 ban, you will have the disease
When running gel electrophoresis, what proteins will be at the top and which will be at the bottom of the gel post electrophoresis?
Large proteins will be at the top of the gel, small at the bottom
What is variable number of tandem repeat (VNTR) and what is it useful for?
Pattern of short repeats, they can be isolated by restriction sites or pcr.
Useful in identification and severity of disease (HD)
Process of VNTR for HD?
Take healthy and HD DNA, do PCR, probe with Alpha32P (phosphorus uses xray), # of repeats will be represented, small at bottom, large at top
Generally, how are medications made?
Using recomibinant proteins to make insulin, growth hormone, clotting factors, vaccines
Example of Recombinant protein steps with insulin?
- Human insulin gene cut from DNA from pancreas
- bacterial plasmid DNA cut with restriction enzymes and insulin added to vector to form recomb. DNA
- REcombinant Bact multiply in fermentation tank, producing insulin
- insulin extracted and purified
