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Flashcards in Molecular Techniques & Diagnosis Deck (33):

Describe DNA sequencing (Sanger chain termination method)

1. Fluorescent/radioactive ddNTPs are added with dNTPs to a DNA template strand with DNA polymerase to create a complementary DNA strand.

2. Depending which ddNTP is used, the new strand will stop at different places.

3. This produces DNA fragments with different lengths. They are denatured with heat and separated using gel electrophoresis.


What is different about a ddNTP to a normal nucleotide?

ddNTPs lack the 3' OH group so polymerisation cannot occur.


What is a restriction endonuclease?

An enzyme that recognises and cuts DNA at a specific sequence (the restriction site).


What does DNA ligase do?

Repairs the double stranded break made by restriction endonucleases. Can also insert a new gene into the section.


Describe DNA gel electrophoresis.

1. DNA fragments of different sizes are added to the gel at the negative anode.

2. Negatively charged DNA moves towards the positively charged anode when electric field is switched on.

3. Large fragments move more slowly.

4. Fragments of known size are used as a reference.

5. DNA is visualised under UV light when ethidium bromide is added to the gel.


Describe process of PCR

1. DNA heated to 95*C to separate strands of the template.

2. Sample cooled to 55*C and sequence specific primers are annealed to DNA.

3. Sample is heated 72*C to allow DNA synthesis by DNA polymerase.

4. Cycle is repeated many times and there is an exponential increase in fan with each cycle.


What can PCR be used for?

-Amplify a specific DNA fragment
-Investigates single base mutations eg sickle cell or Tay Sachs disease
-Investigate small deletions/insertions eg CF
-Detect early stages of infection by pathogenic micro organisms or viruses
-Detect presence of tumour cells


What is a plasmid?

A small circular loop of DNA
Can transfer to other bacteria
Can contain antibiotic genes


How is gene cloning done?

1. Plasmid is cut using restriction enzymes and the gene of interest is added to produce a recombinant DNA molecule - requires DNA ligase

2. This is reintroduced to a bacterium - transformation

3. Bacteria are placed in an environment so that they can multiply

4. Bacteria often contain an antibiotic resistant gene, to positively select for those that have taken up the plasmid.


What do Western, Southern and Northern blotting each look at?

Southern - DNA
Northern - RNA
Western - proteins


What can Southern blotting be used for?

Gene structure eg large deletions or duplications

Gene expansions - triple repeats eg Huntington's, Fragile X syndrome

Mutations in genetic tests using allele specific tests eg sickle cell

Investigate variation and genetic relationships eg DNA fingerprinting


How can PCR be used for an allele specific test?

Use primers specific for either side of the allele of interest to amplify that allele


How can restriction analysis be used in an allele specific test?

Use restriction enzymes with restriction sites around/within the allele. Analyse the size of the fragments produced. If the restriction enzyme cuts the wild type but not the sample, the restriction site is mutated/missing


How can DNA hybridisation be used in an allele specific test?

Use a DNA probe that is complementary to the wild type or mutated allele. See which binds.


In gel electrophoresis for proteins, what does protein movement depend on?



What is SDS-PAGE?

A method of electrophoresis which allows proteins to be separated based only on their molecular mass.


Describe the process of SDS-PAGE

The detergent SDS is added to proteins which denatures them and carries a large negative charge, masking whatever charge is on the protein

Electrophoresis then takes place with migration from the negative to positive electrode.

Proteins are visualised by adding a dye which binds to the proteins and not the gel.


In SDS-PAGE, what travels furthest? Lightest or heaviest proteins?

Heaviest because the have a greater negative charge from the SDS because there is more SDS bound to the protein.


How does isoelectric focusing work?

A form of electrophoresis.
Protein is placed on a gel with a pH gradient.
Protein will stop moving at its isoelectric point because this is the point at which it has no overall charge.


What does 2D-PAGE allow?

Separation of proteins which have an identical pI but different molecular weights or vice versa


Describe Southern blotting.

After electrophoresis, nylon is used to transfer DNA fragments. This is then hybridised with a labelled gene probe to show the specific DNA fragments.


What is ELISA used for?

Find the concentrations of a protein in a complex mixture.


How is ELISA carried out?

1. An antibody to the protein is immobilised on a solid support.
2. Solution to be assayed is applied to the antibody coated surface.
3. Antibody binds the protein and all others are washed away.
4. A second antibody specific to the protein binds to the complex.
5. Binding of the second antibody is measured by assaying the activity of the enzyme linked to it.


What can enzyme assays show?

If an enzyme is present at a normal level.


What conditions are needed for enzyme assays?

Optimum pH, temperature and ionic strength for the enzyme.
Any cofactors and appropriate ions needed for the enzyme.
High concentration of substrate.


What is hybridisation?

A process whereby two complementary strands of DNA reform hydrogen bonds between the bases to form a double strand of DNA


At what stage is cell division stopped at when analysing chromosomes and why?

During late prophase/early metaphase
Chromosomes are highly condensed and replicated
Not yet organised at metaphase plate


What is a karyotype?

A picture of the full set of stained metaphase chromosomes of an individual, organised by chromosome number.


What does FISH allow?

The investigation of specific DNA sequences on a chromosome


What is used in FISH?

Fluorescent probes for a specific gene or probes for specific DNA stretches can be used.


What is chromosome painting?

A different colour fluorescent probe is used to visualise each chromosome.


What is Array CGH used for?

Screens for sub-microscopic deletions for which the locus cannot be deduced from the patient's phenotype.


How is Array-CGH carried out?

1. An array of DNA probes covering the entire genome are applied to the surface of a solid matrix.

2. Patient DNA and normal control DNA are labelled with different colours of fluorescent probe.

3. Equal amounts of the DNA are then hybridised to the probe array and the hybridisation signals are detected and compared.

4. For probes where the signal of normal DNA is greater than for the patient's DNA, the patient has a deletion in the chromosomal region from which that probe was derived.