Sesh 12 And 14: Molecular Techniques Flashcards
What is a restriction enzyme?
An endonuclease produced by bacteria that cleaves phosphodiester bonds of double-stranded DNA at a specific, palindromic recognition site.
What does DNA gel electrophoresis separate on the basis of?
Size, as all DNA fragments are -ve due to phosphate.
Restriction analysis combines what 2 molecular techniques?
Restriction digest and DNA gel electrophoresis.
In gene cloning, why is mRNA used to produce cDNA?
To exclude introns from cDNA.
Which enzyme makes cDNA, using RNA as a template?
Reverse transcriptase.
What 4 things are needed for PCR?
- Target DNA
- DNA Taq polymerase
- Nucleotides
- Forward and reverse primers
What are the 3 stages in a cycle of PCR?
- Denature DNA by heating to 95 C
- Allow nucleotides to anneal at 50 C
- Heat to 72 C for Taq Polymerase to form phosphodiester bonds
Is PCR used to investigate small or large mutations?
Small
Serum protein electrophoresis separates proteins on the basis of what?
Their native properties e.g. Size, shape, charge
What does SDS-PAGE separate proteins based on, and how?
Size.
SD adds negative charge to all proteins, and beta-mercaptoethanol breaks disulphide bonds to make all proteins linear.
Which protein gel electrophoresis method separates proteins only based on their charge?
Isoelectric focussing.
What does 2D-PAGE separate proteins based on?
Size and charge.
Outline the steps in Western blotting.
- SDS-PAGE
- Transferred to solid nitrocellulose blot
- Primary antibody against protein of interest added
- Secondary antibody added (usually enzyme-linked)
- Transfer to get an immunoblot, to visualise and identify protein
What is ELISA used for?
To detect concentration of proteins in solution, using antibodies.
What do enzyme assays measure?
Enzyme activity by measuring rate of product formation over time (continuous), or after a specific time (discontinuous).
Southern blotting is a combination of which 2 molecular techniques?
DNA gel electrophoresis and DNA hybridisation.
During Southern blotting, before DNA can be hybridised, what needs to happen?
DNA needs to be made single-stranded, so is transferred to a nitrocellulose filter soaked in alkaline.
What are the main stages of Southern hybridisation?
- DNA restriction digest
- Separate DNA fragments with gel electrophoresis
- Transfer gel to solid nitrocellulose filter soaked in alkaline
- Hybridise DNA with labelled probe
- Visualise probe by exposure to X-ray film
What can Southern hybridisation be used for?
- Detect large deletions/duplications in a gene
- Investigate gene expansions or triplet repeats
- Use allele-specific probes to investigate disease
- DNA fingerprinting
What does Northern blotting detect?
RNA
What can the Sanger chain termination method be used for?
To work out DNA nucleotide sequence.
What is reverse transcriptase PCR?
A type of gene analysis that indirectly amplifies RNA using reverse transcriptase to form cDNA. Used to look at levels of gene expression.
What is microarray used for?
Genome-wide analysis, and comparing 2 conditions e.g. Normal and diseased state.
What type of mutation can microarray not detect?
Balanced re-arrangements.
What type of DNA is analysed in DNA fingerprinting/profiling?
Non-coding regions of the genome. Analyses patterns of minisatellite repeats.
Which 3 methods are used to analyse DNA at the chromosome level?
- Karyotyping
- Fluorescent in situ hybridisation (FISH)
- Chromosome painting
What stage are the cells in that are used for karyotyping, and why?
Metaphase, as the chromosomes are condensed so can be visualised.
