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Semester 1 - MCBG > 17. Intro to Molecular techniques > Flashcards

17. Intro to Molecular techniques Flashcards

1
Q

What can be used for analysis of DNA at the gene level?

A
  • restriction enzymes
  • DNA gel electrophoresis
  • PCR
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2
Q

What can be used for analysis of proteins?

A
  • protein electrophoresis
  • immunoassays
  • enzyme assays
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3
Q

What are restriction enzymes?

A

specific endonucleases that cut phosphodiester bonds in both strands within SPECIFIC DNA sequences (restriction sites)

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4
Q

Where are restriction enzymes made?

A

Bacteria produce endonucleases

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5
Q

What do restriction enzymes commonly work on?

A

Palindromes of 4,5,6,8 bp

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6
Q

What type of ends do restriction enzymes leave?

A

Sticky ends

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7
Q

Define overhang.

A

Single stranded DNA produced at the site of cut by restriction enzymes.

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8
Q

How does gel electrophoresis work?

A
  • A solution of DNA molecules is placed in gel with electric field across it
  • DNA is negatively charged due to phosphate groups so moves towards the positive electrode
  • Small DNA molecules move more quickly through the gel than larger DNA molecules.
  • DNA fragments can be separated on the basis of size (or shape)
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9
Q

What are molecular weight size markers?

A

set of standards that are used to identify the approximate size of a molecule run on a gel.

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10
Q

What is used to stain DNA in gel electrophoresis?

A 
Ethidium bromide (flat, hydrophobic: sits between bases)
- made visible by shining UV light.
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11
Q

What are the requirements for gel electrophoresis?

A
  1. Gel: A matrix that allows separation of DNA fragments
  2. Buffer: Allows charge on the DNA samples across the gel
  3. Power supply: Generates charge difference across the gel
  4. Stain/detection: To identify the presence of the separated DNA
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12
Q

Why use restriction analysis?

A
  • To investigate the size of DNA fragments
    e. g. small deletions
  • To investigate mutations - affects restriction sites and so alters size of fragments
    e. g. Sickle Cell disease
  • To investigate DNA variation
    e. g. DNA fingerprinting
  • To clone DNA
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13
Q

What are plasmids?

A
  • small circular dsDNA
  • found in bacteria
  • carry genes to replicate independently
  • can transfer to other bacteria
  • often carry antibiotic resistance genes
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14
Q

What are four basic steps in gene cloning?

A
  • Isolate relevant gene of interest following digestion with restriction enzymes
  • Insert gene of interest into plasmid vector (recombinant DNA molecule)
  • Introduce recombinant DNA molecule into suitable host cells e.g. E. coli
  • Identify and isolate the clone containing the DNA of interest
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15
Q

What must be done before inserting DNA into plasmid?

A

Bacteria have no mechanism to remove introns:

  • mature mRNA converted back into DNA
  • using reverse transcriptase from viruses
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16
Q

Why clone human genes?

A
  • To make useful proteins
    e. g. insulin
  • To find out what genes do
    e. g. HTT
  • Genetic screening
    e. g. Huntington’s, BRCA1/2, Cystic Fibrosis
  • Gene therapy ?
    e. g. Cystic Fibrosis
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17
Q

What does PCR stand for?

A

polymerase chain reaction

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18
Q

How does PCR work?

A

Denaturation:
- DNA is heated to 95C to denature and separate the strands by breaking hydrogen bonds.

Annealing:
- Temperature cooled to 50-65C to allow primers (that are complementary to the strands) to anneal

Elongation:
- Temperature raised to 75-80C to allow polymerisation and add to the primer (free nucleotides needed)

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19
Q

What enzyme is commonly used in PCR?

A

Taq polymerase because it is thermostable.

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20
Q

What does PCR do?

A

Amplifies DNA fragments

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21
Q

What apparatus is used to produce the required temperatures in PCR?

A

Thermocycler.

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22
Q

What are the primers used in PCR called?

A

Forward primer and reverse primer

23
Q

Why use PCR?

A
  • To amplify a specific DNA fragment
  • To investigate single base mutations
    e. g. Tay Sachs, Sickle Cell disease
  • To investigate small deletions or insertions
    e. g. Cystic Fibrosis
  • To investigate variation, genetic relationships
    e. g. DNA profiling, DNA typing
24
Q

What is protein gel electrophoresis?

A
  • Proteins are charged molecules and will move towards the anode or the cathode if placed in an electric field
  • Proteins can be separated on the basis of size, shape or charge
25
Q

What are the requirements for protein gel electrophoresis?

A
  1. Gel: A matrix that allows separation of the protein sample
  2. Buffer: Maintains charge on the protein samples
  3. Power supply: Generates charge difference across the gel
  4. Stain/detection: To identify the presence of the separated proteins
26
Q

What is an SDS-PAGE?

A

Sodium dodecyl sulphate polyacrylamide gel

electrophoresis.

27
Q

What is used in an SDS-PAGE?

A
  • Beta Mercaptoethanol
  • Sodium dodecyl sulphate (SDS)
  • Polyacrylamide gel (PAGE)
28
Q

What does an SDS-PAGE allow separation of proteins by?

A

Just by SIZE:

  • SDS is a detergent which breaks down secondary structure and adds negative charge (removes intrinsic charge)
  • all proteins have negative charge - so just separarting by size
  • b-ME breaks disulphide bonds
  • stained
  • compare to standard sizes of molecules to determine what a molecule is
29
Q

what is Serum protein electrophoresis

A

Assessing serum proteins - separated by size and charge

30
Q

How is proteomics done?

A 
A. Digest protein with trypsin
B. Perform mass spectrometry
C. Generate list of peptide sizes
D. Use database of predicted peptide sizes for known proteins to:
E. Identify protein
31
Q

What is isoelectric focusing?

A

A specialised form of electrophoresis where proteins are separated on the basis of isoelectric pH.

32
Q

How does isoelectric focusing work?

A
  • A gel with a pH gradient is loaded into a tube.
  • A charge is run through the gel
  • proteins move through the gel until they reach the pH that is equal to their isoelectric point.
33
Q

What do antibodies bind to?

A

Antigens.

34
Q

What is proteomics?

A

study of all the proteins - Protein identification

35
Q

Define epitope

A

the part of an antigen molecule (few amino acids) to which an antibody attaches itself.

36
Q

What are the two different types of antibody?

A

Polyclonal and monoclonal

37
Q

What are polyclonal antibodies?

A
  • Produced by many B lymphocytes
  • Multiple different antibodies
  • Specific to 1 antigen
  • Multiple epitopes
38
Q

What are monoclonal antibodies?

A
  • Produced from 1 B lymphocyte
  • 1 identical antibody
  • Specific to 1 antigen
  • 1 epitope
39
Q

What is a western blot?

A

Separation of proteins via electrophoresis, followed by identification of specific protein by binding of primary antibody and an enzyme linked secondary antibody

40
Q

What is the ELISA test?

A

Enzyme-Linked Immunosorbent Assay:
- antigen coated well
- specific antibody binds to antigen
- enzyme linked secondary antibody binds to specific antibody
- substrate is added and is converted by enzyme into coloured product
(wash in between each stage)

Rate of colour formation is proportional to the amount of specific primary antibody

41
Q

What is a continuous enzyme assay? give examples.

A

one assay giving the rate of reaction

e.g.
• Spectrophotometry
• Chemoluminescence

42
Q

What is a discontinuous enzyme assay? give examples.

A

samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples

e.g.
• Radioactivity
• Chromatography

43
Q

What are enzyme assays and what are they used for?

A
  • measure the rate of enzyme activity in tissues or serum
  • can get standard rates and compare in metabolic disorders to diagnose diseases linked with low activity of enzymes
  • can see if enzymes are in the wrong place —> diagnosis of disease
  • can detect enzyme elevations after acute MI
44
Q

Give an example of when serum protein electrophoresis is used?

A

In monoclonal gammopathies e.g. multiple myeloma —> see changes in amount of globulins/ albumin —> big increase in gamma globulin

45
Q

What are ELISA tests used for?

A

Measure the concentration of proteins in solution e.g. hormones - insulin, cortisol, TSH

The more Ab that binds the more protein that is present

46
Q

What important enzymes are markers for liver damage?

A

AST ALT

Gamma-glutamate transferase (increased by alcohol)

47
Q

What enzymes are markers for pancreatitis?

A

Amylase lipase

48
Q

What enzyme is a marker for bone disorders?

A

Alkaline phosphatase

49
Q

What can follow PCR?

A
  • restriction analysis
  • gel electrophoresis
  • DNA sequencing
  • southern blotting (probe)
  • another PCR - nested PCR
50
Q

What is the open reading frame?

A

includes the start codon, the part that codes for amino acids and the stop codon - introns removed

51
Q

What mutation in sickle cell anaemia destroys a restriction site?

A

Single base substitution

A —> T converting Glu to Val

Destroys MstII restriction site so restriction enzyme wont cut it

52
Q

What chromosome and gene is mutated in sickle cell aneamia?

A

HBB gene chromosome 11

53
Q

Can PCR be used on RNA? What is the alternative, if any?

A

No, taq polymerase does not recognise RNA template

Alternative: take out mRNA - convert to cDNA - then to DS DNA to use as a template for PCR

54
Q

What are primers in PCR and what is their function?

A
  • small section of DNA complimentary to a certain sequence of DNA - anneal to sequence to show Taq DNA polymerase where to bind for amplification

Semester 1 - MCBG (18 decks)

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