MGD S7+8 - Molecular Diagnoses Flashcards Preview

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Flashcards in MGD S7+8 - Molecular Diagnoses Deck (28):
1

Describe the process of DNA sequencingl

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.

2

Why are Dideoxynucleotides useful for DNA sequencing?

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

3

Describe the action of restriction endonuclease enzymes.

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)

4

What is restriction analysis useful for?

In conjunction with electrophoresis can be used to:

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

5

Describe the process of gene cloning.

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

6

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

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.

7

What is DNA gel electrophoresis used for?

Hint: keep it general

To separate different sized DNA fragments

8

How is DNA gel electrophoresis performed?

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

9

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

Gel
Buffer
Power supply
Stain

10

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

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

11

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

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

12

What is DNA hybridisation used for?

Give an example for each use

Investigate Gene structure
Eg. Large deletions/duplications

Investigate Gene expansions, triplet repeats
Eg. Fragile X syndrome

Investigate variation
Eg. DNA fingerprinting

13

How is DNA hybridisation/Southern blotting preformed?

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

14

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

Southern blotting - DNA hybridisation

Northern blotting - Study of RNA

Western blotting - Analysis of proteins

15

How is PCR used in allele specific testing?

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

16

How is restriction analysis used in allele specific testing?

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

17

How can DNA hybridisation used in allele specific testing?

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

18

What methods of gel electrophoresis for proteins are there?

SDS-PAGE
Isoelectric focusing
2D-PAGE

19

Describe the process of SDS-PAGE

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.

20

Describe the process of Isoelectric focusing

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

21

Describe in overview the process of 2D-PAGE

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

22

What is an enzyme assay measuring and why?

Activity of an enzyme

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

23

Why are enzyme assays useful clinically?

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

24

Under what conditions are all enzyme assays performed?

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

25

What is measured in an enzyme assay?

Rate of production of product or rate of disappearance of substrate

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.

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

27

What is Western Blotting?

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

28

What is being measured by ELISA?

Describe the process of enzyme linked immunoabsorbent assays (ELISA)

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