Genetics - Gene Analysis Flashcards

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1
Q

Why is DNAP not used in PCR?

A

Derives from mesophilic bacteria having optimal temperature of 37-40C

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2
Q

How does temperature disrupt enzymatic reactions?

A

Disruption of activation energy barrier for activity

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3
Q

Activation Energy

A

This is the energy of the transition state for a given reaction relative to initial state.

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4
Q

How does thermal energy effect catalysis?

A

Increases rate of reaction by overcoming activation barrier however denatures the protein.

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5
Q

Why does denaturing of the protein occur with temperature?

A

Disruption of bonds maintaing 3D structure required for activity, like hydrogen bonds, van der Waals force and hydrophobic interactions.

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6
Q

How does Taq polymerase bypass denaturation?

A

Containing of disulfide bonds and salt bridges.

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7
Q

Disulfide bonds

A

Covalent interactions between sulfur atoms of two cysteine residues

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8
Q

Salt bridges

A

Electrostatic interactions between two oppositely charged groups, being an anionic carboxylate and cationic ammonium

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9
Q

Isoelectric Point

A

This is the point at which the molecule has no net electrical charge.

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10
Q

What is the first step in PCR?

A

94C temperatures increasing systems energy, breaking hydrogen bonds, DNA strands denature with increased entropy.

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11
Q

What is Gibbs Free Energy related to?

A

Enthalpy and Entropy

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12
Q

Entropy

A

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

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13
Q

What does positive enthalpy equate to?

A

Energy is needs to be added to break hydrogen bonds.

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14
Q

What does positive entropy lead to?

A

Indicates increase in disorder dominates the thermodynamics, with an overall negative G, meaning denaturaiton is spontaneous.

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15
Q

What is the second step in PCR?

A

Exothermic annealing of primers

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16
Q

Why is the second PCR step exothermic?

A

H bonds formed are stronger than the ones broken in denaturation.

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17
Q

Why is entropy small in second PCR step?

A

Primer/Template bind is ordered compared to ssDNA, G being determined by enthalpy(negative, menaing reaction spontanoeus.

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18
Q

What is the third step in PCR?

A

Primer elongation with Taq polymerase being exothermic due to negative enthalpy change.

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19
Q

How is the energy of the phosphodiester bonds provided?

A

dNTP hydrolysis to dNMP and pyrophosphate drive polymerisation.

20
Q

Advantages of PCR?

A

Small amount required
Non-invasive methods(saliva, urine, blood)

21
Q

Disadvantages of PCR?

A

Error incorporation by primers
Specialised equipment(Thermal Cycle)

22
Q

What is requried for PCR?

A

Template DNA
Primers
Nucleotides
DNA polymerase

23
Q

How can PCR be visualised?

A

Ethidium Bromide
Fluorphores prior to PCR
GE

24
Q

Where is PCR useful?

A

A
Amplification of sequence for cloning, identifying species/disease alleles, gene expression and forensic applications.

25
Q

Gel Electrophoreosis

A

This is a technique that enables seperation and analysis of charged molecules in an electric field.

26
Q

When is GE used?

A

DNA fragments generated by RE and their seperation.

27
Q

What does GE take advantage of?

A

The fact DNA is negatively charged, an electrical field applied from an anode

28
Q

What is rate of movement dependent on in GE?

A

Strength of electrical field and shape/size of the molecule.

29
Q

How is GE set up?

A

A slab of gel(either Acrylamide or Agarose) with holes where DNA is placed within.

30
Q

What is the function of the gel in GE?

A

Molecular porosity meaning smaller DNA move through faster than large DNA

31
Q

Example of differences in what gel is used?

A

A 0.5 cm slab of 0.5% agarose has larger pores for molecules between 1 to 30kb

32
Q

How is GE centrifgufation visualised?

A

An ethidium bromide soak.

33
Q

What do the bands in GE indicate?

A

DNA fragments of a given size migrate to same band position in gel.

34
Q

How is GE analysed with ETHB?

A

Soak applied intercalating with UV light exposure fluoresence creating DNA bands.

35
Q

How can amount of DNA in band be inferred?

A

Intensity compared to known standards of DNA of a known concentration.

36
Q

DNA Ladder

A

This is used to determine size of DNA fragments by GE.

37
Q

What is the agarose gel composed of?

A

D-Galactose and 3,6-anhydro-L-galactose linked by a1,3 and b-1,4 glycosidic bonds

38
Q

What is southern blotting used for?

A

Detection of specific DNA sequences in a sample.

39
Q

How does southern blotting work?

A

Produce of GE onto nitrocellulose/nylon membrane using capillary transfer
NaOH seperate DNA into ssDNA exposing complementarity
Labelled DNA probe detection follows

40
Q

What detection methods are used in Southern Blotting?

A

Autoradiography, Chemiluminessence or Fluorescence

41
Q

What is the process of southernb lotting?

A

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

42
Q

How is DNA affixed onto nylon membrane?

A

High binding capacity due to nylon being positively charged.

43
Q

What are the functional applications of southern blotting?

A

Specific DNA sequences can be detected, studying gene structure and organization, methlyation patterns of DNA and identification of RE sites.

44
Q

How are specific DNA sequences by hybridization?

A

Probe speicif to gene of interest with fluoresence detected elucidating DNA presence.

45
Q

How can chromosomes be analysed with southern blotting?

A

Autoradiography of theblot visualises dark bands, compared then with samples chromosomes giemsa staining.

46
Q

Restriction Fragment Length Polymorphisms

A

Differences among people in their DNA sequences at sites recognized by Restriction Endonuclease

47
Q

How can the presence of FLP be elucidatied?

A

Restriction enzymes on restirciton sites.