Laboratory Techniques & Theory Flashcards
DNA Cloning
DNA Cloning: Is the propagation of a piece of DNA
Recombinant DNA: DNA molecule formed in the lab by joining together DNA seq. from different biological sources
-is an artificial creation - not found in nature
How to Clone a Fragment
- DNA for cloning purified from cells/tissues
- Restriction enzymes used to produce specific DNA frag. - cuts at specific sequence
- Frag. joined to other DNA molecules that serve as vectors
- Recombinant DNA (frag + vector) transferred to host cell - molecule replicates producing clones
- As host replicates, so does DNA molecule - creates pop. of cell carrying molecule
- Cloned DNA can be recovered from host cell, purified & analysed
- Cloned DNA can be transcribed, mRNA translated & encoded gene product isolated & used for research or sold
Point of a Vector for DNA Cloning
Vector: carrier DNA molecules that transfer and help replicate inserted DNA fragments.
Vectors;
-allows fragment to be propagated (w/out vector it would be dissolved)
-Supplies a Origin of Replication (required for replication - occurs infrequently)
-Provides a selectable resistance gene - only about 3% of cells take insert (DNA frag) - need a way to tell which ones haven’t taken it up
-Selectable resistance gene allows us to select for cells that have taken it up (i.e. ampicillin resistance gene in E. coli - grow bacteria on ampicillin agar & only successful ones grow)
Restriction Enzymes
- Produced by bacteria as a defensive mechanism against infection by viruses (can prevent viral infection by degrading DNA of virus)
- binds to DNA & recognises a specific nucleotide seq (RECOGNITION SEQUENCE)
- usu. 4 to 6 nucleotides long (can be 8+)
- can create a restriction fragment
- Most recognition seq. exhibit a form of symmetry = Palindrome (reads same both ways when read from 5’ to 3’)
- Sticky end: cut where a nucleotide is overhanging
- Blunt end: cut where nucleotides are flush/flat
pUC18
- is a small vector (therefore can carry large DNA inserts)
- has original of replication
- large no. of restriction enzyme recognition seq. - clustered into one region called the polylinker site
- Recombinant pUC18 plasmids easily identifiable
- Has recognition site in middle of lacZ gene (gene produces beta-galactosidase, which when grown on X-gal agar produces a blue product)
- if DNA fragment inserted anywhere into polylinker site, lacZ disrupted and becomes inactive - forms white colonies when grown on X-gal medium.
Electrophoresis
-Separations of molecules on the basis of size and electrical charge in an electric field
-many types but gel electrophoresis used for DNA
How it works;
-P on DNA backbone = negative charge
-when current applied, DNA migrates to positive pole
-DNA moves through a gel matrix (of agarose) that forms a network of long polysaccharide chains w/ gaps between chains
-DNA pulled through gaps by electric field
-DNA mixed w/ substance that makes it heavy and sink to bottom of well
*Migrates according to size (inversely)
What must be included in Gel electrophoresis
- Must have fragments of known sizes running;
- is basically a control - shows DNA has run properly & provides a standard for comparison
- Can work out a standard curve and estimate size of fragments based on how far they’ve travelled.
Applications of Electrophoresis
- Look at extracted DNA quality (if no band on gel = DNA completely degraded)
- Enzyme reaction on DNA - might want to test the success of the reaction
- Test for mutations (e.g. breat cancer
- mutations can change DNA (i.e. may cleave it or make it larger - can tell these things)
- Used to test for different allelic forms of a gene
Gel Concentrations
-Gel can effectively separate 200 - 20 000bp
- 200bp = 3% agarose (over this amount it becomes too brittle)
- 20 000bp = 0.5% agarose (too liquid if under this amount
*Both extremes make it hard to get results
Visualisation of Bands in Gel electrophoresis achieved by: (2)
- Staining gel w/ dye for specific nucleic acids (i.e. ethidium bromide) - fluoresces when exposed to UV light
- Can add label to DNA before placed in gel - i.e. chemical lable that can be dtected by adding antibodies
Southern Blotting
Used when;
- larger DNA has a larger no. of bands that are indistinguishable in gel.
- Used to identify specific gene in large mess
- technique to transfer denatured, s.s, fragments
- membrane placed in a hybridisation soln of a labelled probe - probe binds to only part of DNA fragment
- Membrane then washed to remove any unbound probe
*RNA can be transferred to solid support from gel by Northern blotting (can show its size, its abundance or tissues
Probes (as used to identify a specific gene in gel electrophoresis)
Probe: DNA/RNA molecule w/ base sequence complementary to a sequence in gene of interest
- cut DNA into frag. using 1 or more restriction enzymes, then separates fragments w/ gel electrophoresis
- separated fragments must be denatures & transferred to a permanent solid medium (via Southern blotting)
Polymerase Chain Reaction
- method for amplification of a specific DNA sequence (200-400bp) without having to clone the DNA
- larger region for amplification is - less efficient process will be
How PCR works
-DNA polyermase used
- start out w/ dsDNA
- heat @ 94 degrees for 3 mins to separate strands (denatures DNA)
- reduce temp to allow nucleotide primers to anneal to strands (65-70 degrees C) [anneals by H bonding]
- primers of 15-20 bases used
- add DNA polymerase - shift temp to 72 degrees for 1 min to allow DNA polymerase to synthesis new strands
- ALWAYS syn. in 5’ to 3’ direction
- heat to 94 degrees for 1 min and reapeat 30 - 40 times
-DNA polymerase from Thermus aquitirus (Taq polymerase) - can w/stand hight temps
Primers in PCR
-Primers determine the specificity of PCR (tells you what sequences you’re going to amplify)
HOW?
-determined by the annealing temperatures which is set so that the primers only bind to perfectly complimentary sequence
-sometimes primers can bind to a similar sequence (has a mismatch)
-if use a temperature just below the Tm (melting temp) for PCR, all the mismatched DNA will be denatured and therefore won’t be replicated (as one mismatch in sequence decreases the Tm by about 5 degrees)
*this concept ensures that only one site (the right site) is amplified
