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Semester 1-MCBG > Session 12-Intro To Molecular Techniques > Flashcards

Session 12-Intro To Molecular Techniques Flashcards

1
Q

What are the five ways in which DNA can be analysed at a gene level?

A 
Restriction enzymes 
DNA gel electrophoresis 
PCR variations
Southern blotting 
Microarrays
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2
Q

What are the three ways in which proteins can be analysed?

A

Protein electrophoresis
Immunoassays
Enzyme assays

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3
Q

How is DNA analysed at a nucleotide level?

A

DNA sequencing

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4
Q

What are the two ways in which DNA can be analysed at a chromosome level?

A

Karyotyping

FISH

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5
Q

Why do bacteria produce endonucleases?

A

To recognise and degrade foreign DNA

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6
Q

Which restriction site does the restriction enzyme EcoRI recognise?

A

GAATTC

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7
Q

How does DNA gel electrophoresis separate fragments?

A

Depending on their size

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8
Q

Why does DNA move towards the positive electrode in gel electrophoresis?

A

DNA is negatively charged (PO4 3-)

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9
Q

What are the requirements for gel electrophoresis? (4)

A
  1. Gel
  2. Buffer
  3. Power supply
  4. Stain/detection (fluorescent markers)
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10
Q

Why use restriction analysis? (4)

A
  1. To investigate the size of DNA fragments
  2. To investigate mutations eg. Sickle cell disease
  3. To investigate DNA variation
  4. To clone DNA
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11
Q

What are plasmids?

A

Small circular dsDNA found in bacteria which carry genes to replicate independently

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12
Q

What are the four basic steps of gene cloning?

A
  1. Isolate relevant gene of interest following digestion with restriction enzymes
  2. Insert gene of interest into plasmid vector (recombinant DNA molecule)
  3. Introduce recombinant DNA molecule into suitable host cells eg. E. coli
  4. Identify and isolate clone containing DNA of interest
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13
Q

Why are human genes cloned? (4)

A
  1. To make useful proteins eg. Insulin
  2. To find out what genes do
  3. Genetic screening
  4. Gene therapy
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14
Q

What does reverse transcriptase use as a template for DNA?

A

RNA

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15
Q

What is PCR used for?

A
  1. DNA amplification
  2. To investigate single base mutations eg. Tay Sachs, Sickle cell
  3. To investigate small deletions or insertions eg. Cystic fibrosis
  4. To investigate variation, genetic relationships
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16
Q

What is Taq polymerase?

A

DNA polymerase derived from hot springs bacteria- can withstand the high temperature of PCR

17
Q

Describe the process of PCR (5)

A
  1. Amplification of target DNA
  2. Thermostable DNA polymerase (Taq)
  3. Pair of primers (forward and reverse), uniquely defining the region to be copied
  4. Temperature cycles of denature, anneal and polymerise
  5. Repeated copying results in exponential increase in DNA
18
Q

On what basis are proteins separated in protein gel electrophoresis?

A

Size, shape or charge

19
Q

What is the difference between DNA and protein gel electrophoresis?

A

Protein g.e. Uses a vertical, rather than horizontal, method

20
Q

What is native electrophoresis?

A

Using the intrinsic properties of the protein to separate it

21
Q

What are the methods of separating proteins on the basis of size called?

A

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Two-dimensional electrophoresis (2D-PAGE)

22
Q

What happens in SDS-PAGE?

A

The 3D structure of the protein is broken, the protein is made negative and the disulphide bonds are broken i.e. The secondary and tertiary structure of the protein is gotten rid of so the protein size is known

23
Q

How are proteins separated on the basis of charge?

A

Isoelectric focusing (IEF)

24
Q

What happens in IEF? (3)

A
  1. A stable pH gradient is established in the gel after application of an electric field
  2. Protein solution is added and electric field is reapplied
  3. After staining, proteins are shown to be distributed along pH gradient according to their pI values
25
Q

Describe the process of protein identification (proteomics) (4)

A
  1. Digest protein with trypsin
  2. Perform mass spectrometry
  3. Generate a list of peptide sizes
  4. Use database of predicted peptide sizes for known proteins to identify the protein
26
Q

What is the difference between proteomics and molecular diagnosis?

A

Proteomics = analysis of all proteins expressed from genome

Molecular diagnosis = analysis of a single purified protein

27
Q

Which mnemonic is used for the different types of blots and what they are used for?

A

SNOW DROP:
S - southern blotting corresponds to D (DNA)
N - northern blotting corresponds to R (RNA)
Os cancel out
W - western blotting corresponds to P (protein)

28
Q

Which method is used to measure the concentration of proteins in a solution?

A

Enzyme-linked immunoabsorbent assay (ELISA)

29
Q

Describe the steps of ELISA

A
  1. Antigen-coated well
  2. Specific antibody binds to antigen
  3. Enzyme-linked antibody binds to specific antibody
  4. Substrate added and converted by enzyme into coloured product; rate of colour formation is proportional to amount of specific antibody
30
Q

What is the difference between ELISA and radioimmunoassays?

A

Radioimmunoassay works in the same way as ELISA but uses a radio-labelled primary antibody

31
Q

Name two continuous enzyme assays

A

Spectrophotometry

Chemoluminescence

32
Q

Name two discontinuous enzyme assays

A

Radioactivity

Chromatography

33
Q

What are the serum enzyme markers for liver damage/disease?

A 
Aspartate transaminase (AST)
Alanine transaminase (ALT)
34
Q

What is the serum enzyme marker for pancreatitis?

A

Amylase/lipase

35
Q

What is the serum enzyme marker for bone disorders?

A

Alkaline phosphatase

36
Q

What is the standard for diagnosis of an MI?

A

Measurement of cardiac troponin I (cTnI) by ELISA

37
Q

Give an example of when enzymes are used to measure clinically important metabolites

A

Measurement of glucose concentration with glucose oxidase:
Test strips-H2O2 converted to coloured dye
Glucose monitor-glucose oxidase used as biosensor

38
Q

Why do proteins move in gel electrophoresis?

A

Proteins are charged molecules and will move towards either the anode or cathode if placed in an electric field

Semester 1-MCBG (20 decks)

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