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MOLECULES GENES AND DISEASE > Molecular techniques > Flashcards

Molecular techniques Flashcards

0
Q

What are the steps in genetic cloning?

A
  • Digest gene and a vector with restriction enzymes to create complementary sticky ends
  • Add DNA ligase so the gene becomes incorporated into the vector
  • Introduce the recombinant vector to bacteria
  • Allow Replication
  • Screen for the vector
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1
Q

What is gene cloning?

A

-The production of exact copies of a length of DNA for a gene

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2
Q

What are often used as vectors?

A

-Plasmids-> circular dsDNA in bacteria

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3
Q

Why are plasmids often used to make recombinant DNA?

A
  • Easily accessible and cheap
  • Can transfer to other bacteria
  • Often carry antibiotic resistance which allows for selective screenin
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4
Q

Why must a plasmid be fully sequenced before use?

A

-Need to know origin of replication and restriction sites

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5
Q

What is gene cloning used for?

A
  • Protein production eg insulin
  • Investigation of gene function eg Huntington’s
  • Genetic screening
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6
Q

What is restriction analysis?

A

-Using restriction enzymes coupled with another technique to carry out DNA analysis

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7
Q

What are restriction enzymes?

A

-Enzymes which recognise and cut specific DNA sequences

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8
Q

When is restriction analysis used?

A
  • Gene cloning
  • Gel electrophoresis to examine mutations
  • DNA fingerprinting
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9
Q

What is DNA sequencing?

A

-Determining the order of the bases in a length of DNA

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10
Q

When is DNA sequencing used?

A
  • Base mutations

- Polymorphisms

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11
Q

Why is DNA sequencing avoided?

A

-Expensive!

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12
Q

Describe DNA electrophoresis

A
  • Digest DNA with restriction enzymes
  • Inject into wells
  • Pass a current across the gel
  • The -vely charged DNA will migrate towards the anode
  • Migration distance and speed will be based on the size of the DNA fragments
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13
Q

Why is a buffer needed in electrophoresis?

A

-To account for different charges

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14
Q

When would you use DNA electrophoresis?

A

-To investigate mutations

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15
Q

Describe the basis of PCR

A
  • Take DNA of interest
  • Heat to 95c to denature
  • Add primers, Taqpol and dNTPs
  • Cool to 60c to allow annealing
  • Repeat
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16
Q

What is PCR used for?

A

-Quick amplification of DNA

17
Q

What does vertical protein electrophoresis separate proteins based on?

A
  • Size
  • Shape
  • Charge
18
Q

What does serum protein electrophoresis separate proteins based on?

A
  • Size
  • Shape
  • Charge
19
Q

How is serum protein electrophoresis useful?

A
  • Compare proteins in patients serum to a standard reference
  • Provides information on albumin and globins
  • Can detect monoclonal gammopathies such as multiple myeloma due to change in globins
20
Q

Why does SDS-PAGE only seoarate proteins based on size?

A

-SDS is a detergent which denatures the protein and thus removes any charges

21
Q

What is isoelectric focusing?

A
  • Separation of proteins based on charge
  • The gel has a decreasing pH gradient and proteins migrate to the pH which is equal to the pI and then stop migrating as they have ni overall net charge
22
Q

What is 2D-PAGE?

A

-Electrophoresis which separates proteins based on two intrinsic properties in different dimensions, i.e. one direction based on decreasing pH and the other based on size

23
Q

When is 2D-PAGE used?

A
  • To analyse complex mixtures of proteins

- To compare diseased tissues with normal eg, suspected tumour vs normal

24
Q

What is the purpose of mass spectrometry

A

-Generates a list of peptide sizes and composition in order to identify a protein by using a database

25
Q

What is the difference between polyclonal and monoclonal antibodies?

A
  • Polyclonal recognises multiple epitopes on antigens

- Monoclonal recognises one epitope on antigens

26
Q

Describe western blotting

A
  • Preform protein electrophoresis
  • Make a nitrocellulose replica of the plate
  • Use a primary antibody agains the protein of interest
  • Use a secondary antibody which is enzyme linked against the first antibody
  • Immunoblot and identify protein
27
Q

When are ELISAs used?

A

-For proteins in solution, eg hormones

28
Q

What are enzyme assays?

A

-Tests to measure enzyme/product concentration, often used to calculate rate of reaction

29
Q

Name a continuous enzyme assay

A
  • Spectrophotometry

- Chemiluminescence

30
Q

Name a discontinuous enzyme assay

A
  • Chromatography

- Radioactivity

31
Q

When are enzyme assays used?

A
  • In metabolic disorders
  • Diagnosis eg AST and ALT in liver disease, amylase and lipase in pancreatitis, alkaline phosphatase in bone, enzymes associated with MI
32
Q

What is southern hybridisation?

A

Label a specific probe and hybridise to separated DNA
If probe sequence is specific to normal DNA it will only bind to normal DNA
If probe specific for mutated DNA it will only bind to mutated DNA

33
Q

How can PCR be used allele-specifically?

A

The primer to the DNS to be amplified is specific
Using a variety of primers whose sequence is specific to different alleles will only produce a PCR product if the specific allele is present

34
Q

What is norther hybridisation?

A

-Same as southern but analysis of RNA expression

35
Q

What is the difference with real time PCR?

A

-Uses fluorescent probe to see rt-PCR product

36
Q

What is the benefit of microarray?

A
  • Can analyse thousands of genes at once, ie it is genome-wide
  • can analyse healthy cells compared to diseased cells
37
Q

What is karyotyping?

A

-systematic sorting of chromosomes via lining metaphase chromosomes up and grouping them together according to size

38
Q

What is karyotyping used for?

A

To show aneuploidy, polyploidy, abnormal sex, translocation and deletions

39
Q

When can fish be used?

A

To probe for specific chromosomal loci to show duplications or deletions

MOLECULES GENES AND DISEASE (12 decks)

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