14. Advanced Molec Techniques

Question 1

What is proteomics?

Answer 1

The identification of proteins

Question 2

What is ELISA used to detect?

Answer 2

The concentration of protein in a SOLUTION

Question 3

Enzyme assay of which enzymes can be used to identify liver damage?

Answer 3

Aspartate transaminase

Alanine transaminase

Question 4

What are the potential ethical issues with DNA sequencing?

Answer 4

Who would access your genome information… insurance companies?government?schools?
Who owns the information…someone else might of paid for it, but its your DNA
Can the knowledge be used to prevent illness or not.

Question 5

How can you use PCR with allele-specific primers to identify mutations?

Answer 5

If you know what the mutation is and where it is located, you can create primers complementary to the wild type sequence which will not bind if the mutation is there

Question 6

What can northern hybridisation be used for?

Answer 6

Measuring how much gene is expressed

Question 7

Explain how RT-PCR is used.

Answer 7

mRNA to cDNA using a polyA primer and reverse transcriptase.

cDNA produced can then be analysed with PCR to measure the gene expression.

Question 8

What are the benefits of using microarray analysis?

Answer 8

You can analyse thousands of genes at the same time, so you don’t need to know which gene is affected.
Compare gene expression in 2 conditions - healthy and diseased

Question 9

What regions does DNA profiling/finger printing exploit?

Answer 9

Minisatellites, which are non-coding regions which are repeated in different patterns in every individual.

Question 10

What method could you use to identify downs syndrome?

Answer 10

Karyotyping - there will be an extra chromosome 21

Question 11

What can FISH detect?

Answer 11

If regions if DNA are missing, using probe specific for a particular gene or region.

Question 12

Chromosome painting can help to identify what type of mutation?

Answer 12

Translocations between chromosomes - each chromosome will be a different colour, so if parts have swapped then it will be visible.

Question 13

How does southern blotting work?

Answer 13

It uses DNA probes to identify complementary DNA sequences after gel electrophoresis.

Question 14

Give an example how you could use allele specific probes in southern hybridisation to identify a mutation.

Answer 14

Make 1 probe that is complementary to the sickle cell sequence, or another mutated sequence and only DNA with this mutation will be detected,

Question 15

What steps must have been carried out prior to Southern hybridisation?

Answer 15

Digestion of DNA with restriction enzymes,DNA Gel electrophoresis to separate DNA fragments and transfer to a membrane

Question 16

What might Southern hybridisation be used for?

Answer 16

• Investigate gene structure , large deletions or duplications
• Investigate gene expansions, triplet repeats in huntington’s
• Investigate mutations in genetic tests, e.g using allele specific probes for sickle cell disease.

Question 17

What is a characteristic of the DNA probes in southern blotting?

Answer 17

They do not have to be 100% complementary to the sequence they are detecting.

Question 18

If a DNA probe being used in Southern Hybridisation only has 80% sequence similarity, will it still bind?

Answer 18

Yes, but not as tightly as sequences with 100% similarity.

Question 19

What is another feature of Southern blotting DNA probes?

Answer 19

They do not have to completely align with the target sequence. Probes can still bind and be detected if it has only partial overlap with the target sequence.

Question 20

What method can be used to carry out DNA sequencing?

Answer 20

Sanger chain termination or dideoxy chain termination

Question 21

In a normal dNTP, what chemical group allows DNA polymerase to add another dNTP?

Answer 21

3’ hydroxyl group

Question 22

How does a dideoxynucleotide triphosphate (ddNTP) differ from a dNTP?

Answer 22

It doesn’t have a 3’ hydroxyl group

Question 23

What is the significant of the absence of a 3’ hyroxyl group in ddNTP?

Answer 23

It can still be incorporated into the DNA by DNA polymerase, but it will block further elongation as no phosphodiester bonds will be able to form.

Question 24

How many separate tubes are used in the Sanger method of DNA sequencing?

Answer 24

4. One for each ddNP.

So one type with ddATP, one with ddCTP, ddGTP, ddTTP.