12 Introduction To Molecular Techniques Flashcards
What is a restriction enzyme?
- Enzymes produced by bacteria
- Recognise and cut specific DNA sequences (mostly palindromes) (no lemon, no melon - aattc, cttaa)
- Recognise and degrade foreign DNA
What is DNA gel electrophoresis?
Protein gel electrophoresis?
- Used to separate mixtures of DNA, RNA, proteins according to molecular size
- DNA cut with restriction enzymes
- Samples loaded into wells at one side
- Gel placed in electric field with positive electrode on side furthest away from samples
- DNA negatively charged so will travel to positive electrode
- Fragments separated on basis of size; larger molecules move slowly, smaller move faster
Same with proteins
What is serum protein electrophoresis?
- Serum is blood plasma in blood not including fibrinogens
- Serum run on gel
- Schematic representation made (graph)
- Compared to ‘normal’ serum graph
E.g. Monoclonal gammopathy - More gamma globulins produced causing multiple myeloma (cancer of plasma cells)
What is SDS-PAGE?
Form of gel electrophoresis
Denature proteins with SDS (Sodium Dodecyl Sulphate detergent removes -ve charges breaking down disulphide bonds) so we can have in linear form
Gel electrophoresis
Use for size of unknown protein by running known and comparing unknown
What is isoelectric focusing?
Form of gel electrophoresis
Stable pH gradient est. in gel after application of electric field
Sample of protein added
Electric field reapplied
After staining, proteins migrate until pH = pI (as no net charge at pI so stop moving toward anode)
What is 2D-PAGE?
Form of gel electrophoresis
Seperated by isoelectric point first
Then separate by mass
Use for complex mixtures of protein
Important for diagnosing disease states in different tissues
What is PCR Method of PCR?
DNA segment amplification tool - good to investigate single base mutations, deletions, insertions, variation
Heat DNA to 95, denatures
Forward and reverse primers added to mixture as well as abundant free nucleotides
Cool mixture to 60
DNA denatures with primers and Taq pol attaches free nucleotides to DNA template
We now have two copies of DNA piece
What is Taq polymerase?
Thermostable DNA pol - an enzyme able to withstand protein denaturing conditions required in PCR
How is gene cloning performed?
- Isolate gene of interest with restriction enzymes
- Insert gene into plasmid vector
- Introduce recombinant DNA molecule into suitable host cells (bacteria)
- Cell will replicate, plasmid replicates too!
- Identify and isolate clones containing DNA of interest
What is a plasmid?
- Small circular dsDNA found in bacteria
- Carry genes to replicate independently
- Can transfer to other bacteria (antibiotic resistance!)
Why clone human genes?
To make useful proteins e.g. proinsulin
To find out what genes do
Genetic screening
Gene therapy
If we have an mRNA strand, what must we do to the strand before gene cloning?
Reverse transcriptase used on mRNA to produce dsDNA
What is the method of protein identification (proteomics)?
- Digest protein with trypsin (protease secreted by pancreas the cleaves peptide bonds after lys and arg making protein smaller)
- Perform mass spec
- Generate list of peptide sizes
- Use database to identify
What are the two types of antibodies and what they do?
- Polyclonal antibodies
- produced by many B lymphocytes, multiple different Ab
- 1 antigen but recognise multiple epitopes - Monoclonal antibodies
- produced by 1 B lymphocyte, 1 Ab
- 1 antigen, 1 epitope
- Ab identify and neutralise pathogens
What is western blotting?
- Artificial Ab created that react with specific target protein
- Detect presence in cell extracts
- Use SDS-PAGE to seperate protein sample
- Transfer onto solid support
- Add antibody (which will bind to its specific protein on blot)
- Can detect binding by secondary antibody to see whether protein expressed
