Manipulating genomes

Question 1

What does the PCR do?

Answer 1

Can be used to select a fragment of DNA and amplify it to produce millions of copies in just a few hours

Question 2

What are the 3 key stages in a PCR?

Answer 2

Denaturation, annealing and extension/ elongation

Question 3

PCR: What happens in denaturation?

Answer 3

The double-stranded DNA is heated to 95°C which breaks the hydrogen bonds that bond the two DNA strands together.

Question 4

PCR: What happens in annealing?

Answer 4

The temperature is decreased to between 50-60°C so that primers can anneal to the ends of the single strands of DNA.

Question 5

PCR: What happens in extension/ elongation?

Answer 5

The temperature is increased to 72°C for at least a minute, as this is optimum temperature for Taq polymerase to build complementary strands of DNA to produce the new double-stranded DNA molecules

Question 6

How many DNA fragment are there after 1 cycle of PCR?

Answer 6

4, because it doubles the amount of DNA (i.e. originally had 2)

Question 7

Why is Taq polymerase used?

Answer 7

Comes from thermophilic bacterium which means it does not denature at high temperature.

Question 8

What does each PCR require?

Answer 8

• Target DNA being amplified.
• Primers
• DNA polymerase
• Free nucleotides
• Buffer solution

Question 9

What is the machine called that a PCR takes place in?

Answer 9

Thermocycler

Question 10

What is electrophoresis?

Answer 10

Is a procedure that uses an electrical current to seperate out DNA fragments, RNA fragments, or proteins depending on their size.

Question 11

Electrophoresis: How is the gel tray in the gel box set up?

Answer 11

1. Electrophoresis is commonly performed using agarose gel that has been poured into a gel a tray and left to solidify. A row of wells is created at one end.
2. Firstly, you need to put the gel tray into a gel box. You need to make sure the end with wells is closest to the negative electrode on the gel box.
3. Then add a buffer solution to the reservoirs at the side of the gel box so the surface gel becomes covered in buffer solution.

Question 12

Electrophoresis: How are the DNA samples loaded into the wells?

Answer 12

1) Take your fragmented DNA samples, and using a micropipette, add the same volume of loading dye to each- loading dye helps the samples sink to the bottom of the wells and makes them easier to see.
2) Next add a set volume of a DNA sample to the first well.
3) Then repeat this process for each of the other DNA samples to other wells in the gel.

Question 13

Electrophoresis is carried out?

Answer 13

1) put the lid on the gel box and connect the leads from the gel box to the power supply.
2) Turn on power supply- this causes an electrical current to be passed through the gel.
3) DNA fragments are negatively charged, so they’ll move through the gel towards the positive electrode at the far end of the gel (called the anode). Small DNA fragments move faster and travel further through the gel, so DNA fragments will seperate according to their size.

Question 14

What are palindromic sequences (recognition sequences?

Answer 14

Sequences that consist of antiparallel base pairs (base pairs that read the same in both directions).

Question 15

What are restriction enzymes ?

Answer 15

Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places.

Question 16

What can different restriction enzymes do?

Answer 16

Cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzymes’s active site.

Question 17

What happens if recognition sequences are present at either side of the DNA fragment you want?

Answer 17

Can use the restriction enzyme to seperate it from the rest of the DNA.

Question 18

How does restriction enzyme used?

Answer 18

The DNA sample is incubated with the specific restriction enzyme, which cuts the the DNA fragment out via a hydrolysis reaction.

Question 19

What can restriction enzymes cutting sometimes leave?

Answer 19

Sticky ends- small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind the DNA fragment to another piece of DNA that ahs sticky ends with complementary sequences

Question 20

How can PCR be used in forensic science?

Answer 20

1) The DNA is isolated from all the collected samples.
2) PCR is used to amplify multiple areas containing sequence reports- primers are used to bind to either side of these reports so the whole repeat is amplified.
3) The PCR products are then run on an electrophoresis gel and the DNA profiles are comapred to see if any match .
4) If the samples match it links a person to the crime scene.

Question 21

How can DNSA profiling be used in medical diagnosis?

Answer 21

In a medical diagnosis, a DNA profile can refer to a unqiue pattern of several alleles.
It can be used to analyse the risk of genetic disorders. It’s useful when the specific mutation isn’t known or where several mutations could have caused one disorder, because it identifies broader, altered genetic pattern.

Question 22

What is a transformed organisms?

Answer 22

Organisms that have had their DNA altered by genetic enginnering.

Question 23

What is a transgenic organism?

Answer 23

An organism that has been genetically engineered to include a gene from a different species.

Question 24

Genetic enginnering: How is the desired DNA fragment inserted into a vector?

Answer 24

1) The DNA fragment is inserted into vector DNA. They can be plasmids (small circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria).
2) The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing the desired gene. So the ends are complementary.
3) The vector and the DNA are mixed together with DNA ligase. DNA ligase joins up the sugar-phosphate backbones of the two bits. This process is called ligation.
4) The new combination of bases in the DNA (vector DNA + DNA fragment ) is called recombination DNA.

Question 25

Genetic enginnering: How does the vector transfer the gene into the bacteria?

Answer 25

• A suspension of bacterial cells is mixed with the plasmid vector and placed in a machine called an electroporator.
• The machine is switched on and an electrical field is created in the mixture, which increases the permeability of the bacterial cell membranes and allows them to take in the plasmids.

Question 26

What are the two types of gene therapy?

Answer 26

Somatic therpay and germ line therpay

Question 27

Gene therapy: What happens in germ line therapy?

Answer 27

Involves altering the alleles in the sex cells. This means that every cell of any offspring produces from these cells will be affected by the gene therpay and they won’t inherit the disease. Germ line therpay in humans is currently illegal though.

Question 28

Gene therapy: What happens in somatic therapy?

Answer 28

This involves altering the alleles in body cells, particualry the cells that are most affected by the disorder. Somatic therapy doesn’t affect the individual’s sex cells (sperm or eggs) through, so any offspring could inherit the disease.

Question 29

Gene therapy: Example of somatic therpay for cystic fibrous?

Answer 29

Cystic fibrous (CF) is a genetic disorder that’s very damaging to the respiratory system, so somatic therpay for CF targets the epithelial cells lining the lungs.

Question 30

Gene therapy: Positive ethical issues?

Answer 30

• Could prolong the lives of people with genetic disorders
• Could give people with genetic disorders a better quality of life
• Carriers of genetic disorders may be able to conceive a baby without that disorder or risk of cancer (only in germ line therapy).
• It could decrease the number of people who suffer from genetic disorders (only in germ line therapy).

Question 31

Gene therapy: Negative ethical issues?

Answer 31

• The technology could potentially be used in other way other than for medical treatment, such as for treating cosmetic effects of aging.
• There is the potential to do more harm than good by using the technology (e.g. risk of overexpression of genes)
• There is the concern that gene therpay is expensive- some people believe that health service resources could be better spent on other treatments that have passed clinical trails.

Question 32

Gene therpay: how would you alter the alleles inside the cell if a genetic disorder was caused by a dominant allele?

Answer 32

If it’s caused by a dominant allele you can ‘silence’ the dominant allele (e.g. by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore).

Question 33

Gene therpay: how would you alter the alleles inside the cell if a genetic disorder was caused by tow recessive alleles?

Answer 33

If it’s casued by two recessive alleles you can add a working dominant allele to make up for them.

Question 34

Gene therpay: Disadvantages?

Answer 34

• The effects of treatment may be short-lived (only in somatic therpay).
• The patients might have to undergo multiple treatments (only in somatic therapy).
• It might be difficult to get the allele into specific body cells
• ## The body could identify vectors as foreign antibodies and start an immune repsonse against them

Question 35

What is DNA sequencing?

Answer 35

Identify the base sequence of DNA fragment

Question 36

How have DNA sequencing methods changed over time?

Answer 36

• Used to be a manual process, however it has now become very automated
• Entire genomes can now be read

Question 37

Benefits of genome wide comparsions?

Answer 37

• Comparing between species allows us to determine evolutionary relationships
• Comparing between individuals of the same species allows us to tailor medical treatment to the individual

Question 38

How can DNA sequencing be used in synthetic biology?

Answer 38

• Knowing the sequence of the gene allows us to predict the sequence of amino acids that make up the polypeptide it produces.
• This in turn allows for the development of synthetic biology