MGD S7+8 - Molecular Diagnoses

Question 1

Describe the process of DNA sequencingl

Answer 1

1. Fluorescently labelled dideoxyNTP (ddNTPs)
   dNTPs
   DNA polymerase

are added to a DNA template strand to create a complimentary DNA strand

2. Depending on what ddNTP is used the strand will terminate at different places
3. This produces lots of new DNA fragments of different lengths/sizes that can be denatured with heat and separated by gel electrophoresis.

Each of the ddNTPs can then be run in a separate lane and the end result allows us to write the DNA sequence.

Question 2

Why are Dideoxynucleotides useful for DNA sequencing?

Answer 2

Due to removal of the 3’ -OH group the ddNTPs cannot polymerise and cause chain termination during DNA polymerisation.

Question 3

Describe the action of restriction endonuclease enzymes.

Answer 3

Bacterial restriction endonucleases recognise specific DNA sequences called ‘restriction sites’ and cut the DNA at this point.

Cutting of the DNA leaves exposed bases at the ends of fragments called ‘sticky ends’ (can be rejoined by DNA ligase)

Question 4

What is restriction analysis useful for?

Answer 4

In conjunction with electrophoresis can be used to:

Investigate size of DNA fragments
Investigate Mutations
Investigate DNA variation
Gene cloning

Question 5

Describe the process of gene cloning.

Answer 5

Plasmid is cut with restriction enzymes

Gene of interest is added with DNA ligase

This creates what is known as a recombinant DNA molecule

Plasmid is reintroduced into bacterium - Transformation

Bacteria containing Recombinant DNA multiplies

Question 6

How might a researcher select for bacteria that have taken up a plasmid he introduced to the colony?

Answer 6

Plasmid often contains an antibiotic gene so a researcher can select for bacteria which have taken up the plasmid by adding an antibiotic to the culture.

Question 7

What is DNA gel electrophoresis used for?

Hint: keep it general

Answer 7

To separate different sized DNA fragments

Question 8

How is DNA gel electrophoresis performed?

Answer 8

1. Solution of different DNA fragments placed in a well at the negative cathode end of the gel
2. A charge is applied and the negative DNA moves toward the positive anode
3. Larger fragments move slower than smaller fragments and so fragments are separated by size.
4. Fragments of known size then used as a reference

Question 9

What does the process of DNA gel electrophoresis require? (apart from DNA, smartass)

Answer 9

Gel
Buffer
Power supply
Stain

Question 10

What is the Polymerase Chain Reaction (PCR) used for?

Answer 10

To amplify a specific DNA fragment
Investigate single base mutations
Investigate small insertions or deletions

Question 11

How is PCR used to amplify a specific region of DNA?

Answer 11

Requires a specific sequence of cooling and heating DNA as follows

1. Denaturation of DNA at 94-96 degrees
2. Identifies region to be copied with a pair of primers that bind to specific regions on each strand
   Renaturation (Annealing to primers) at 50-65 degrees
3. DNA polymerase is then used to copy the region being amplified.
   DNA synthesis at 75-80 degrees

Question 12

What is DNA hybridisation used for?

Give an example for each use

Answer 12

Investigate Gene structure
Eg. Large deletions/duplications

Investigate Gene expansions, triplet repeats
Eg. Fragile X syndrome

Investigate variation
Eg. DNA fingerprinting

Question 13

How is DNA hybridisation/Southern blotting preformed?

Answer 13

1. Begin with unlabbelled DNA that has undergone gel electrophoresis
2. DNA is transferred from the gel to a sheet of Nylon
3. The DNA fragments are then hybridised with a labelled gene probe to show the DNA sequences being looked for/at

Question 14

What are the three styles of Blotting and what are they used for?

Answer 14

Southern blotting - DNA hybridisation

Northern blotting - Study of RNA

Western blotting - Analysis of proteins

Question 15

How is PCR used in allele specific testing?

Answer 15

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

Question 16

How is restriction analysis used in allele specific testing?

Answer 16

Use restriction endonucleases with restriction sites around the allele of interest

Analysis size of fragments produced to look for deletions/insertions etc

If endonuclease enzymes cut a wild type but not the sample we know the sample’s restriction site is mutated/missing

Question 17

How can DNA hybridisation used in allele specific testing?

Answer 17

Use a DNA probe complementary to wild type or mutated type, see which binds.

Question 18

What methods of gel electrophoresis for proteins are there?

Answer 18

SDS-PAGE
Isoelectric focusing
2D-PAGE

Question 19

Describe the process of SDS-PAGE

Answer 19

The detergent SDS (sodium dodecyl sulphate) denatures proteins and gives the protein a negative charge proportional to molecular weight.

Electrophoresis then takes place, larger proteins that are more negative will travel further toward the cathode.

Question 20

Describe the process of Isoelectric focusing

Answer 20

Proteins applied to a gel that contains a pH gradient

Electric field is applied

Proteins will migrate until it reaches a pH that matches its pI (isoelectric point)

At this point it will have no overall charge and will stop migrating

Question 21

Describe in overview the process of 2D-PAGE

Answer 21

Proteins are applied to gel and are subject to SDS-PAGE and Isoelectric focusing (one at a time)

Between processes the gel is turned 90 degrees to separate bands out in a 2D pattern

Question 22

What is an enzyme assay measuring and why?

Answer 22

Activity of an enzyme

Elevated levels/activity of various enzymes in serum is used as an indicator of tissue damage

Question 23

Why are enzyme assays useful clinically?

Answer 23

They give an indication of whether an enzyme is present at normal levels

Question 24

Under what conditions are all enzyme assays performed?

Answer 24

Optimal pH
Optimal Temp
Optimal ionic strength
Appropriate ions and cofactors present

Question 25

What is measured in an enzyme assay?

Answer 25

Rate of production of product or rate of disappearance of substrate

Question 26

Give some examples of clinically important Serum enzymes and what disease they’re associated with.

Hint: Don’t bother memorising them all, just a couple examples

You know, if you’d like.

Answer 26

Aspartate/Alanine transaminase - Liver disease

Creatine kinase + Lactate dehydrogenase - Myocardial Infarction

Amylase/Lipase - Pancreatitis

gamma-glutamyl transferase - Liver damage, Increased by alcohol

Alkaline phosphatases - Bone disorders

Acid phosphatases - Prostate cancer

Plasma cholinesterase - Decreased in liver disease, Inhibited by organophosphate poisoning

Question 27

What is Western Blotting?

Answer 27

Following SDS-PAGE, proteins are transferred to a nitrocellulose membrane and can be identified by binding with labelled antibodies

Question 28

What is being measured by ELISA?

Describe the process of enzyme linked immunoabsorbent assays (ELISA)

Answer 28

Detects concentration of a protein in the solution being assayed.

Method:

1. Primary antibody (specific to protein) is immobilised on a solid support (Eg. microtitre well)
2. Solution to be assayed applied to surface, assayable protein binds to the immobilised antibodies
3. Secondary antibody added to solution, this antibody is conjugated with an enzyme, this binds with antibody-antigen (protein) complex
4. Surface washed
5. Binding of second antibody and hence concentration of protein is measured by assaying for enzyme activity