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Organisation & Control of Eukaryotic Genome (3) - Coggle Diagram
Organisation & Control of Eukaryotic Genome (3)
1) Molecular techniques
(a) polymerase chain reaction (PCR)
process used to amplify a specific segment of DNA in vitro -> results in synthesis of large amt. of DNA from a minute amt. of starting material
(i) process of PCR
denaturation
95 degree celsius
denaturation of dsDNA into ssDNA by breaking hydrogen bonds btw nitrogenous base of the 2 strands
annealing
50-60 degree celsius
DNA primers anneal via hydrogen bonding to the flanking sequence of the target DNA sequence due to complementary base pairing
elongation
72 degree celsius
Taq polym. adds deoxyrn. to the 3' OH end of the primers using DNA molecule as a template
called a chain rxn bcus products of the previous rxn are used as reactants in the next cycle
n cycles will produce 2^n molecules of target DNA sequence
(ii) advantages of PCR
sensitivity
large amt. of DNA can be produced from very minute amt. of starting materials
speed & accuracy
large amt. of DNA can be produced in a short period of time with relatively high accuracy of replication
specificity
specific sequence of DNA can be amplified using specific primers
(iii) limitations of PCR
knowledge of DNA / a.a sequence of target gene / protein is req. to synthesise flanking nucleotide primers
there may be non-target DNA sequences which are complementary to the DNA primers
non-target DNA sequence may be amplified alongside the target DNA sequence
Taq polym. (bacteria origin) does not perform proofreading
there may be mistakes in complementarity of the nucleotides added -> mistakes may be amplified
limits on the length of target DNA sequence (3kb)
efficiency of amplification decreases with increase in length of target DNA sequence
(b) gel electrophoresis
a technique of separating nucleic acids / proteins based on the size, electrical charge & other physical properties of the molecule by passing them through a gel which functions as a molecular sieve in an electric field
the larger the molecule, the slower it moves through the gel
agarose gel electrophoresis
agarose is purified from agar
agarose melts at high temp. & cools to form a gel-like matrix -> porous structure allows it to act as a sieve
since DNA is negatively charged due to its sugar-phosphate backbone, this technique separates DNA based on size only
intensity of the band is used to estimate the conc. of DNA sample
process
prior to gel elect., DNA samples are purified & cut with restriction enzymes
A. casting of gel
Agarose gel is prepared by heating agar powder with buffer solution to dissolve it. Conc. of gel can be adjusted to alter the resolution of gel (more agarose -> less porous gel)
Agarose gel solution is poured into a gel tray & cooled. A comb is added at 1 end of the gel to create wells for loading of DNA.
After agarose has cooled & hardened, the comb is removed to reveal the wells. The gel is then placed within an electrophoresis chamber filled with buffer solution.
DNA samples are loaded into the wells at the -ve electrode. As DNA is invisible to the naked eye, a small amt. of loading dye is mixed with the sample -> the blue dye will move along the gel & give an indication of the progress of electrophoresis. Glycerol is added to loading dye as it is dense & allows DNA to sink into the wells. A standard DNA ladder / kb ladder is loaded for estimation of the size of DNA fragments.
B. application of electric field
Direct current is turned on. -> DNA runs from cathode to anode when electric field is applied. -> After DNA has run to abt 2/3 of the gel length, current is stopped. (to prevent 'overrun' such that DNA samples move off the gel into the buffer solution)
After separation in the gel, analysis is not possible as the bands are not visible. -> Ethidium bromide can be added in the buffer / agarose gel before cooling. -> When viewed under UV radiation, DNA bands are fluoresce -> a polaroid photo can be taken to record the position of the bands.
(c) nucleic acid hybridisation