Molecular techniques and diagnosis

Question 1

How does the Sanger Dideoxy Chain Termination Method of DNA sequencing work?

Answer 1

Fluorescent ddNTPs are added to a DNA template strand (with DNA polymerase) to create a complimentary DNA strand
The new chain terminates at different places depending on which ddNTP is used.
Creates lots of DNA fragments which can be denatured and separated using gel electrophoresis.

Question 2

Why does ddNTP cause the DNA chain to terminate during synthesis?

Answer 2

Because ddNTP is missing an OH group polymerisation cannot occur therefore the chain terminates.

Question 3

What are restriction endonucleases?

Answer 3

Restriction enzymes that are bacterial and recognise specific DNA sequences (restriction sites) and cut the DNA here

Question 4

What can restriction analysis (combined with electrophoresis) be used for?

Answer 4

See size of DNA fragments (Useful in seeing deletions)
Investigate mutations
Investigate DNA variation
Genetic cloning

Question 5

What are plasmids?

Answer 5

Small sections of circular DNA that can transfer to other bacteria and sometimes contain antibiotic genes.

Question 6

How can plasmids be used in Genetic cloning?

Answer 6

The plasmid is cut using restriction enzymes.
Gene of interest is added to create recombinant DNA molecule
Placed into the bacterium (Transformation)
Placed into an environment to multiply

Question 7

Why does the plasmid often contain an antibiotic gene?

Answer 7

So that the bacterium which takes up the plasmid can easily be selected.

Question 8

What is DNA gel electrophoresis used for?

Answer 8

Separation of different sized fragments

Question 9

What direction do the DNA fragments move in DNA gel electeophoresis?

Answer 9

From the negative well to the positive anode. Heavier fragments travel slower.
Fragments of known size are used as reference.

Question 10

What does DNA gel electrophoresis require?

Answer 10

A gel
A stain
A buffer
A power supply
Fragments of known size for reference

Question 11

How does PCR amplify small DNA segments?

Answer 11

Repeated copying of target DNA using thermostable DNA polymerase and a pair of primers.

Question 12

What are the 3 main steps to PCR amplification?

Answer 12

Denaturation at high temperatures
Renaturation at lower temperatures
DNA synthesis at a medium temperature

Question 13

What can PCR be used for?

Answer 13

Amplification of a specific DNA fragment
Investigate single base mutations
Investigate small deletions or insertions

Question 14

What is Southern blotting used for?

Answer 14

Marking unlabelled DNA from gel electrophoresis

Question 15

How does Southern blotting work?

Answer 15

Nylon transfers the fragments from gel electrophoresis
Then this is hybridised with a labelled gene probe
Shows specific DNA fragments
Radioactive probes can be used to mark specific complimentary DNA fragments

Question 16

What is Northern blotting?

Answer 16

The same process as Southern blotting, but it uses RNA instead of DNA

Question 17

What is Western blotting?

Answer 17

The same principple as Southern blotting, but it analyses Proteins.

Question 18

What is blotting used for?

Answer 18

Investigate gene structure
Investigate gene expansions
Investigate variation

Question 19

How can PCR be used in Allele specific testing?

Answer 19

Use primers specific to the sequences either side of the allele then amplify that allele

Question 20

How can Restriction analysis be used in Allele specific testing?

Answer 20

Use restriction enzymes that have restriction sites around and within the allele.
Analyse the size of the fragments produced
The restriction site is missing/mutated if the restriction enzyme cuts the wild type.

Question 21

How can DNA hybridisation be used in allele specific testing?

Answer 21

Use DNA probe that is specific to either the wild type allele or mutated allele then see which binds.

Question 22

What is SDS page?

Answer 22

A technique that allows proteins to be separated by their molecular weight only.

Question 23

How does SDS page work?

Answer 23

SDS denatured the protein molecules
Protein has a negative charge which is proportional to molecular weight
Electrophoresis - heavier molecular weight means more negatively charged so moves further

Question 24

What is isoelectric focusing?

Answer 24

Where proteins can be separated using their isoelectric point

Question 25

How does isoelectric focusing work?

Answer 25

Proteins are applied to a gel with a pH gradient

The protein migrates until it reaches a pH which matches its pI (At this point there is no overall charge)

Question 26

What is 2D page?

Answer 26

Allows separation of proteins with the same pI, but different molecular weights (or visa versa)

Question 27

Why is the measurement of activity of an enzyme clinically important?

Answer 27

Indicates if the particular enzyme is present at normal levels

Question 28

What pH, temperature and ionic strength are enzyme assays performed at?

Answer 28

Optimal pH
Optimal temperature
Optimal ionic strength
Appropriate co factors or ions are included

Question 29

What does an enzyme assay measure?

Answer 29

Production of product or disappearance of substrate

Rate of action can then be calculated

Question 30

What is often measured with enzyme assays?

Answer 30

Activity of various enzymes in serum because they are elevated in tissue damage so can indicate if there is any tissue damage

Question 31

What serum enzyme is increased after a MI?

Answer 31

Creatinine Kinase

Question 32

What serum enzyme is increased in bone disorders?

Answer 32

Alkaline phosphatase

Question 33

How can antibodies be used to identify proteins?

Answer 33

They are attached to a fluorescent label and the proteins undergo Western blotting after SDS page.

Question 34

How do Enzyme Linked Immunoabsorbent Assays work?

Answer 34

They can determine the concentration of a protein by analysing binding of its corresponding antibody.

Question 35

What is the method of Enzyme Linked Immunoabsorbent Assays?

Answer 35

The Primary antibody (specific to a protein) is immobilised on a solid
Solution to be assayed is added to solid
The secondary antibody (conjugated with an enzyme) binds to the antibody - antigen complex
This is then measured by assaying for the enzymes activity.