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GENETIC ENGINEERING LECTURES > LEC 3 > Flashcards

LEC 3 Flashcards

1
Q

Agarose gel structure?

A

Tiny pores= mesh. When a potential difference is applied across the gel, the mesh impedes the progression of molecules (smaller molecules impede less)

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2
Q

Loading dye purpose in electrophoresis

A

Physically track the molecules

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3
Q

How to produce agarose gel?

A

Heat up agarose to at least 60 degrees (liquid form) pour into mould (with comb in place) to form the wells

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4
Q

Comb used to form?

A

wells

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5
Q

DNA is charged?

A

NEGATIVELY therefore moves towards the positive anode

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6
Q

Agarose gel is used to analyse what types of genomes?

A

medium sized gels

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7
Q

Polyacrylamide

A

gel used on smaller genomes (as can detect small differences in fragment size)

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8
Q

Production of polyacrylamide? (chemically)

A

Acrylamide+ methylene biacrylamide= polyacrylamide (cross linked).

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9
Q

In forming the gel on a plate (polyacrylamide)

A

Pour onto a plate (with a spacer between to create a gap) overlay with water to stop oxidation of polyacrylamide.

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10
Q

Two types of gel stain’s (DNA)

A

ETHIDIUM bromide (not as safe, cheaper) and SYBR GOLD (safer, more expensive, and detects small volumes of DNA)

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11
Q

Molecular marker use?

A

as a comparison: what fragments are present

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12
Q

gel stains (proteins)

A

coomassie blue (safer) and silver (not as safe but more sensitive to smaller volumes of protein)

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13
Q

Denaturing form?

A

chemical agents to break interactions within the conformation

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14
Q

Native form?

A

natural form

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15
Q

Foramide/ SDS

A

a chemical agent which breaks hydrogen bonds to linearise the protein (denatured)

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16
Q

when is the denaturing form relevant to use

A

when we are only interested in its size rather than conformation.

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17
Q

southern blotting? (not method, background)

A

ED southern 1975.
previous use: DNA
now: RNA

18
Q

Method: southern blotting?

A
  1. Agarose gel
  2. Load DNA samples
  3. Lane for molecular marker
  4. Run gel
  5. Blotting: nitrocellulose membrane, paper towel and a weight.
  6. nitrocellulose has a high affinity for proteins
  7. Gel> membrane
  8. Remove nitrocellulose membrane, mix with a price which is complementary to the fragment of interest
  9. wash to remove the unbound probe
  10. Expose to xray film and if the fragment of interest is present then it will show up on the audio radiogram
19
Q

Northern blotting used with?

A

RNA

20
Q

characteristics of Northern blotting ?

A

quick and easy

To find the vol of protein expressed in particular places

21
Q

method of Northern blotting

A
  1. Extract RNA, isolate, purify.
  2. isolation: magnetic bead s in which have a series of Thymine nucleotide bases attached- oligodT dynabead
  3. these attach themselves to poly a tails of mRNA = dimer beads
  4. run mRNA like blotting
  5. Apply complementary RNA probe again to isolate
  6. Purified mRNA is formamide denatured (linearised)
  7. run on gel
  8. Radioactively labelled with a probe
  9. We can now see by gel electrophoresis by the sizes of the bands how well a particular gene is expressed in tissues
22
Q

Western blotting- antibodies method?

A

-Antibodies are raised against a protein of interest.
- Proteins are electrophoretically transferred to the nitrocellulose layer from the gel
- SDS mutant used to denature protein
- place in a tank with a buffer and apply a potential difference
- Antibody has a great affinity for the nitrocellulose membrane.
- Chemlucinet enzyme flashes light when reaction takes place (s>p)
- bands show up if light emitted
- Expression of genes detected
fc 8 to continue

23
Q

difference between western blotting antibodies and eastern blotting method?

A

In eastern blotting- proteins migrating to nitrocelluose by capillary action

in western blotting- proteins are electrophoretically transferred to the nitrocellulose layer

24
Q

what type of enzyme is used in western blotting?

A

chemilucent

25
Q

structure of complex involved in western blotting

A

protein of interest which is bound to a primary enzyme, which is bound to a secondary antibody, which is bound to substrate which is formed into a product giving out light

26
Q

How can we tell there is a gene being expressed, detection method

A

Light given off when conformational change occurs in the enzyme. S>P (light). This is detected and shows up on a graph . Can also detect the volume of protein present.

27
Q

Antibody is complementary to what within western blotting?

A

substrate is complementary to

28
Q

Modifications to blotting can do what?

A

Widen their practical scope, e.g large DNA molecules and analysing DNA- protein interactions

29
Q

Pulse Field electrophoresis use? How does PFE allow this.

A

For larger DNA molecules. MEGABASE. Alternating the electric filed, allows for larger DNA molecules

30
Q

how many different directions is the electric filed held in PFE?

A

3

31
Q

How does PFE allow for even large DNA molecules to be detected?

A

Larger molecules will take longer to change direction when the electric field changes orientation.

32
Q

At what degrees is the electric field alternated at?

A

120

33
Q

what does ESMA stand for?

A

electrophoretic mobility shift assay

34
Q

what is ESMA used for?

A

Studying DNA-protein interactions (e.g transcription factors) without denaturing anything.

35
Q

Method for ESMA

A
  • Non denaturing gel used
  • Electrophoresis of DNA strands of interest
  • One which has a transcription factor bound is larger and therefore takes longer to migrate
36
Q

SSCP stands for?

A

Single strand conformational polymorphism

37
Q

SSCP use?

A

Detect the presence of mutations

38
Q

Characteristics of SSCP?

A

Quick, robust, gives information on the structure of a sequence not the size (base substitutions will have the same sequence size)

39
Q

Method of SSCP?

A
  • Denature into single strands
  • Snap cool (slow cooling)
  • Each DNA strand will fold into a particular shape depending on its sequence (can tell the difference in structure of a DNA molecule not just its size)
  • Use of a native gel, and will migrate at different rates
40
Q

Snap cooling?

A

cool slowly (on ice) heat to 98 degrees

GENETIC ENGINEERING LECTURES (4 decks)

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