Genetics - Gene Analysis Flashcards
Why is DNAP not used in PCR?
Derives from mesophilic bacteria having optimal temperature of 37-40C
How does temperature disrupt enzymatic reactions?
Disruption of activation energy barrier for activity
Activation Energy
This is the energy of the transition state for a given reaction relative to initial state.
How does thermal energy effect catalysis?
Increases rate of reaction by overcoming activation barrier however denatures the protein.
Why does denaturing of the protein occur with temperature?
Disruption of bonds maintaing 3D structure required for activity, like hydrogen bonds, van der Waals force and hydrophobic interactions.
How does Taq polymerase bypass denaturation?
Containing of disulfide bonds and salt bridges.
Disulfide bonds
Covalent interactions between sulfur atoms of two cysteine residues
Salt bridges
Electrostatic interactions between two oppositely charged groups, being an anionic carboxylate and cationic ammonium
Isoelectric Point
This is the point at which the molecule has no net electrical charge.
What is the first step in PCR?
94C temperatures increasing systems energy, breaking hydrogen bonds, DNA strands denature with increased entropy.
What is Gibbs Free Energy related to?
Enthalpy and Entropy
Entropy
This is the measure of the number of ways in which energy can be distributed among the systems particles, or their number of possible arrangements
What does positive enthalpy equate to?
Energy is needs to be added to break hydrogen bonds.
What does positive entropy lead to?
Indicates increase in disorder dominates the thermodynamics, with an overall negative G, meaning denaturaiton is spontaneous.
What is the second step in PCR?
Exothermic annealing of primers
Why is the second PCR step exothermic?
H bonds formed are stronger than the ones broken in denaturation.
Why is entropy small in second PCR step?
Primer/Template bind is ordered compared to ssDNA, G being determined by enthalpy(negative, menaing reaction spontanoeus.
What is the third step in PCR?
Primer elongation with Taq polymerase being exothermic due to negative enthalpy change.
How is the energy of the phosphodiester bonds provided?
dNTP hydrolysis to dNMP and pyrophosphate drive polymerisation.
Advantages of PCR?
Small amount required
Non-invasive methods(saliva, urine, blood)
Disadvantages of PCR?
Error incorporation by primers
Specialised equipment(Thermal Cycle)
What is requried for PCR?
Template DNA
Primers
Nucleotides
DNA polymerase
How can PCR be visualised?
Ethidium Bromide
Fluorphores prior to PCR
GE
Where is PCR useful?
A
Amplification of sequence for cloning, identifying species/disease alleles, gene expression and forensic applications.
Gel Electrophoreosis
This is a technique that enables seperation and analysis of charged molecules in an electric field.
When is GE used?
DNA fragments generated by RE and their seperation.
What does GE take advantage of?
The fact DNA is negatively charged, an electrical field applied from an anode
What is rate of movement dependent on in GE?
Strength of electrical field and shape/size of the molecule.
How is GE set up?
A slab of gel(either Acrylamide or Agarose) with holes where DNA is placed within.
What is the function of the gel in GE?
Molecular porosity meaning smaller DNA move through faster than large DNA
Example of differences in what gel is used?
A 0.5 cm slab of 0.5% agarose has larger pores for molecules between 1 to 30kb
How is GE centrifgufation visualised?
An ethidium bromide soak.
What do the bands in GE indicate?
DNA fragments of a given size migrate to same band position in gel.
How is GE analysed with ETHB?
Soak applied intercalating with UV light exposure fluoresence creating DNA bands.
How can amount of DNA in band be inferred?
Intensity compared to known standards of DNA of a known concentration.
DNA Ladder
This is used to determine size of DNA fragments by GE.
What is the agarose gel composed of?
D-Galactose and 3,6-anhydro-L-galactose linked by a1,3 and b-1,4 glycosidic bonds
What is southern blotting used for?
Detection of specific DNA sequences in a sample.
How does southern blotting work?
Produce of GE onto nitrocellulose/nylon membrane using capillary transfer
NaOH seperate DNA into ssDNA exposing complementarity
Labelled DNA probe detection follows
What detection methods are used in Southern Blotting?
Autoradiography, Chemiluminessence or Fluorescence
What is the process of southernb lotting?
Gel transferred to membrane.
Soaked in buffer
Sandwhiched between absorbent material like filter paper.
Capillary action of buffer carries NA through gel and membrane, dissolving gel matrix
How is DNA affixed onto nylon membrane?
High binding capacity due to nylon being positively charged.
What are the functional applications of southern blotting?
Specific DNA sequences can be detected, studying gene structure and organization, methlyation patterns of DNA and identification of RE sites.
How are specific DNA sequences by hybridization?
Probe speicif to gene of interest with fluoresence detected elucidating DNA presence.
How can chromosomes be analysed with southern blotting?
Autoradiography of theblot visualises dark bands, compared then with samples chromosomes giemsa staining.
Restriction Fragment Length Polymorphisms
Differences among people in their DNA sequences at sites recognized by Restriction Endonuclease
How can the presence of FLP be elucidatied?
Restriction enzymes on restirciton sites.
