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MCBG > 12. Intro To Molecular Techniques > Flashcards

12. Intro To Molecular Techniques Flashcards

1
Q

Where do restriction enzymes come from?

A

Bacteria-produced endonucleases

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2
Q

Which restriction enzyme does E.coli produce?

A

Eco R1

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3
Q

What does DNA gel electrophoresis separate fragments based on?

A

Size of DNA fragments

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4
Q

What is the positive electrode in gel electrophoresis called?

A

Anode

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5
Q

What would you carry our after restriction enzyme digest?

A

Gel electrophoresis to see the result by analysing fragment sizes

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6
Q

What are the 4 requirements for gel electrophoresis?

A
  1. Gel
  2. Buffer - keeps pH constant
  3. Power supply - generates charge difference
  4. Stain/detection method
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7
Q

How could a nucleotide mutation alter restriction analysis?

A

Could destroy or create a restriction site, which will alter the number and size of fragments in the gel electrophoresis

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8
Q

Outline the steps of gene cloning.

A
  1. Isolate gene of interest following digestion with restriction enzymes
  2. Insert gene of interest into plasmid vector
  3. Introduce recombinant DNA into host cells
  4. Identify and isolate clone of interest
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9
Q

What are reasons for cloning a gene?

A

Make useful proteins - insulin
Find out the function of a particular gene
Genetic screening - BRCA genes

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10
Q

What does a PCR do?

A

amplify a DNA segment of interest/target DNA

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11
Q

What will a PCR mixture contain?

A

Target DNA
Forward and reverse primers
Taq polymerase
Nucleotides

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12
Q

What are the temperatures which are cycled during PCR?

A

95, 50, 72

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13
Q

By how much does the DNA in the PCR mixture increase by in each cycle?

A

Doubles each time

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14
Q

What can PCR be used for?

A

Investigating single base mutations by combining with restriction analysis

Investigate small deletions or insertions

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15
Q

How can proteins be separated using protein gel electrophoresis?

A

Size and charge

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16
Q

What is the negative charged electrode in gel electrophoresis called?

A

Cathode

17
Q

How does SDS-page separate proteins?

A

Size only as SDS detergent adds negative charges and all tertiary structure is removed to leave a linear peptide

18
Q

How does isoelectric focussing separate proteins?

A

On the basis of charge - they will stop migrating at the pH equal to their isoelectric point.

19
Q

How does 2D gel electrophoresis separate proteins?

A

First by isoelectric point, then by size using SDS page.

20
Q

What is 2D gel electrophoresis useful for?

A

Separation of complex mixtures of proteins

Looking at whole cells, organs or tissues

21
Q

How can you use cleavage sites on proteins?

A

Similar to restriction sites, mutations can alter or destroy enzyme cleavage sites and alter fragments produced

22
Q

What are monoclonal antibodies?

A

Antibodies that all recognise the same 1 specific epitope on a protein

23
Q

Describe the process of western blotting.

A

Primary antibody against the protein of interest will bind. A secondary antibody which binds to the primary antibody is enzyme linked and will produce a detectable product to show presence of protein.

24
Q

Before western blotting, how is the protein first separated?

A

By SDS-page

25
Q

What is ELISA used for?

A

Measure the concentration of various proteins e.g. Hormones

26
Q

What are 2 types of enzyme assays?

A

Continuous- detect the production of product

Discontinuous - measure after a certain time

27
Q

Why are enzymes assays useful?

A

Increased or decreased levels are often markers for disease

28
Q

What are aspartate transaminase and alanine transaminase markers for?

A

Liver damage or disease

29
Q

What are 2 enzymes which are elevated after MI?

A

Creatine kinase

Cardiac troponin 1 - ELISA measurement of this is the gold standard MI marker.

30
Q

What clinically important metabolite can enzymes measure?

A

Glucose, by using glucose oxidase enzyme.

31
Q

What method could you use to detect duplication or deletion of exons?

A

Southern blotting - probe for the deleted region would not hybridise in mutated DNA

32
Q

If a mutation alters DNA size, what methods could you use to detect it?

A

PCR and gel electrophoresis

2D gel electrophoresis

33
Q

Which investigative method compares the gene expression in 2 conditions?

A

Microarray

MCBG (17 decks)

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