Genetic Engineering Flashcards
Are the three types of blotting done in situ or homogenate?
Homogenate
What is southern blotting used for visualising?
DNA
What is western blotting used for visualising?
Proteins
What is northern blotting used for visualising?
RNA
What is an in situ method for visualising RNA?
In situ hybridisation
What is a method to visualise protein in homogenate?
Western blotting
What is used to visualise DNA in situ?
Chromosome painting/Chromosomal spreads
What is used to visualise protein in situ?
Immunocytochemistry/Immunohistochemistry
What are the advantages of visualising in homogenate?
- Quantification
- Size
- Isolation
What are the disadvantages of visualising in homogenate?
- Requires more tissue
What are the advantages of visualising in situ?
- Tissue distribution
- Function
- Changes in above
What are the disadvantages of visualising in situ?
- Requires tissue processing
- Limited and compromised by reagents and resolution
3 Steps of visualisation and detection of DNA, RNA and protein in homogenates
- Gel electrophoresis
- Blotting (transfer out of gel into a nylon membrane)
- Detection by probing
What is a gel?
A gel is a porous matrix that acts like a sieve. Pores are present between the gel particles
Gel electrophoresis
DNA is loaded into a well on negative side
Electric current through gel and due to negatively charged phosphate moves towards positive electrode
Smaller molecules move further in the set time
Compare bands with DNA standard
Is there a proportion between the distance between the bands and the size of the fragment?
No, distance between bands is not proportional the size of fragments. Based on a logarithmic scale
Four factors affecting DNA migration
- Gel concentration
- DNA shape
- DNA size
- Gel type: agarose, polyacrylamide
Which gel type is most common?
Polyacrylamide
Which gel would be more appropriate for DNA with 15,000 base pairs?
Agarose
What are the optimum base pair ranges for each gel type?
Agarose: 100-20,000bp
Polyacrylamide:10-700bp
Which gel has higher resolution?
Polyacrylamide
What are the two ways of carrying out blotting?
- Blotting by capillary action
- Blotting by an electric field
How would you detect DNA/RNA after gel electrophoresis?
Hybridisation
Labelled with marker
How would you detect protein after gel electrophoresis?
Antibody staining
Where is the DNA polymerase that we study originally from?
T7 in E. coli
When does exonucleolytic proofreading take place?
When an incorrect base pair is added
Which way does the proofreading exonuclease proofread?
3’–>5’
Does DNA polymerase need a primer?
Yes
What is the chain termination an example of?
DNA sequencing
What is PCR used for?
To amplify DNA. Proceeds on an exponential scale 2^#of cycles
What do you need for a PCR?
- RNA primers
- Free DNA nucleotides
- 1 copy of DNA sequence to be amplified (template)
- DNA polymerase
- Buffer solution
3 steps in PCR
- Heat to 95ºC to denature dsDNA to ssDNA
- Cool to ⁓60ºC for annealing of primers to template DNA
- Heat to 72ºC so that DNA polymerase can extend the primers to the end of the template
Cycle repeats but with the products as new templates as well
How can PCR be used to manipulate DNA sequences?
- Primers can be used to insert single point mutations by lowering temperature for annealing to allow imperfect binding between primer and DNA sequence
- Tailed primers can be used to insert restriction enzyme sites to produce ‘sticky ends’ for DNA engineering
