Molecular Diagnosis

Question 1

How is our own DNA protected from restriction enzymes?

Answer 1

Methylation.

Question 2

How can we isolate particular pieces of DNA from a sequence?

Answer 2

Use multiple restriction enzymes.

Question 3

What is DNA electrophoresis?

Answer 3

It is a method which can be used for visualising colourless DNA. Fragments are separated using a negative charge. Fragments have to move on the plate so larger molecules move less far, gradient of molecule sizes created.

Question 4

What are the four requirements for gel electrophoresis?

Answer 4

Gel - allows separation, Buffer - maintains charge, Power supply - creates charge difference, stain - visualise DNA.

Question 5

What is the function of DNA ligase in molecular diagnosis?

Answer 5

When complementary base sequences align, this can lead to the formation of new bonds.

Question 6

What is used in gene cloning?

Answer 6

Plasmids from Bacteria are used in gene cloning.

Question 7

What is recombinant DNA?

Answer 7

This is the plasmid ring with the DNA in it as well.

Question 8

What is the use of cloning genes?

Answer 8

We can use cloned genes in order to make useful proteins such as insulin.

Question 9

What is genetic screening?

Answer 9

This is where we are looking for a specific gene. To look at this properly we clone genes.

Question 10

What is PCR?

Answer 10

Polymerise chain reaction. It is a method for obtaining large amounts of a particular piece of DNA for analysis.

Question 11

What is a primer?

Answer 11

This is a short molecule which is complementary to part of the DNA sequence. It is a site where DNA replication begins.

Question 12

Explain the cycle process of PCR.

Answer 12

First DNA is heated to 95 degree so that it is denatured. It is then cooled to 60 so that primers anneal and then heated to 75 when replication takes place. The DNA is then reheated.

Question 13

What is used so that DNA polymerase denaturing is not a problem in PCR?

Answer 13

Taq, thermostable DNA polymerase so that it does not have to be replaced with each cycle.

Question 14

What is the use of PCR?

Answer 14

PCR allows amplification of a specific piece of DNA which can then be investigated for base mutations or deletions/insertions.

Question 15

State the difference between protein and DNA gel electrophoresis.

Answer 15

Protein gel electrophoresis is done vertically rather than horizontally so proteins move down the plate.

Question 16

What is Isoelectric focusing in protein gel electrophoresis?

Answer 16

This is where proteins are separated on the basis of their charge. When they reach the pH = pI they cease to have a charge and so they do not move as they are not attracted to either dipole.

Question 17

How are proteins separated in SDS-Page?

Answer 17

In this process all proteins are denatured and given a uniform negative charge. They are then separated by size as larger proteins will move more slowly across the gel.

Question 18

What is 2D PAGE?

Answer 18

This is where proteins are initially separated by Isoelectric focusing and then they are separated by size.

Question 19

What methods can we use to identify proteins after they have been separated?

Answer 19

Digest with trypsin and perform mass spectrometry, enzymatic cleavage, chemical cleavage or antibodies which will recognise a few amino acids on protein.

Question 20

What is the difference between polyclonal and monoclonal antibodies?

Answer 20

Polyclonal antibodies are antibodies which can detect one antigen but there are multiple different antibodies which recognise different epitopes. Monoclonal antibodies are specific to one antigen but there is one antibody and one epitope.

Question 21

What is a restriction enzyme?

Answer 21

This is a specific endonuclease which cuts a specific DNA sequence, usually a palindrome.

Question 22

What is western blotting?

Answer 22

This is where there is a nitrocellulose replica of the glee electrophoregram which is incubated with primary antibodies and then and enzyme linked to a second antibody is used to detect this binding.

Question 23

What is an Enzyme-linked immunoabsorbent assay?

Answer 23

This is where there is an antigen coated well to which a specific enzyme linked antibody can bind. When substrate binds it is converted to a coloured product by the enzyme, therefore indicating how much antibody was bound.

Question 24

What is a radioimmunoassay?

Answer 24

This is the same as an ELISA however it uses a radio labelled primary antibody.

Question 25

Why is measurement of enzymes an important diagnostic technique?

Answer 25

We can detect: metabolic disorders of tissues, and also we can diagnose disease. There are many enzymes which when they become present in the blood indicate disease.

Question 26

How can enzymes be used to measure clinically important metabolites?

Answer 26

On test strips, enzymes can convert these metabolites to product. This product can then be detected and measured.

Question 27

What is DNA sequencing?

Answer 27

This is a process by which we can find out the genomic code and look to see if there are mutations and insertions or deletions.

Question 28

Describe briefly the process of DNA sequencing.

Answer 28

Dideoxynucleotides are added to the mixture rather than just deoxy ones. This means that in replication, there is a chance that each time it is a specific base the dideoxy form will take the space. This means that the sequence stops. By running it the fragments are separated and we can see the order of the bases.

Question 29

What is DNA hybridisation?

Answer 29

This is where we can denature DNA and then insert a radioactively labelled DNA probe into the mixture. This allows us to identify fragments of DNA.

Question 30

What is the difference between southern blotting and northern blotting.

Answer 30

Both are processes which involve using DNA probes to find complementary base sequences after gel electrophoresis. Southern uses DNA to find DNA, Northern uses DNA to find RNA.

Question 31

Why after gel electrophoresis is there transfer to a nylon membrane in southern blotting?

Answer 31

DNA needs to be transferred to a solid support. this can be done by either capillary action or it can also be done by using a voltage to move the negatively charged DNA from the gel to filter.

Question 32

Once DNA is on the filter, what happens in southern blotting?

Answer 32

The filter is placed in a solution with labelled DNA probes. These will bind to complementary base sequence. The filter is then washed before the probes are visualised.

Question 33

Explain the properties of probes in southern blotting.

Answer 33

Probes do not have to be 100% complementary because they will still bind just not as tightly. They also do not have to completely align as long as overlap is sufficient. Probe position will not affect the position of the band identified.

Question 34

What is an allele specific primer in PCR?

Answer 34

This will only produce a PCR product when the correct sequence is present - allele specific.

Question 35

How can we analyse RNA?

Answer 35

RNA is an unstable molecule so first it is converted to DNA by RNA reverse transcriptase. In order for this we simply need a T primer which can bind to the polyA tail.

Question 36

What is a microarray?

Answer 36

This is where dots of either red, green or brown can be seen. mRNA from two cells is used and then fluorescent probes are used. This allows us to see where there is missing RNA and in which cell this occurs.

Question 37

What is an important process in DNA fingerprinting?

Answer 37

PCR. The amount of DNA we have is very limited, so it must be amplified by PCR.

Question 38

What is Karyotyping?

Answer 38

This is looking at banding patterns on Chromosomes.

Question 39

What is FISH?

Answer 39

Fluorescent in situ hybridisation. This uses fluorescent probes to look inside a cell at the chromosomes. Particular probes can highlight specific portions of genes and this is chromosome painting.