6.1.3 Manipulating Genomes

Question 1

Name the 3 techniques for studying genes (DNA profiling, genetic engineering, gene therapy and DNA sequencing)

Answer 1

1. Polymerase Chain Reaction
2. Cutting out DNA fragments using restriction enzymes
3. Gel Electrophoresis

Question 2

What is the function of the polymerase chain reaction?

Answer 2

Can be used to select a fragment of DNA (containing the gene or bit of DNA desired) + amplifies it to create millions of copies in only a few hours

Question 3

What is the method for polymerase chain reactions (PCR)?

Answer 3

1. Reaction mixture is set up including DNA sample, primers, free nucleotides and DNA polymerase
2. DNA mixture is heated to 95 degrees to break H bonds between 2 DNA strands, DNA polymerase does not denature at this temp. The mixture is then cooled to about 50-65 degrees so that primers can bind (anneal) to complementary DNA strands
3. The mixture is then heated to 72 degrees so that DNA polymerase can work - binds to primer, where it lines up complementary free nucleotides to the DNA strands, creating phosphodiester bonds between the nucleotides so that new complementary strands are formed (extension)
4. 2 new copies of the fragment of DNA are formed with one cycled of PCR complete, then cycle repeats as heated to 95 degrees again, whereby all 4 strands will now act as templates

Question 4

What is a primer?

Answer 4

Relatively short pieces of DNA that are complementary to the bases at the start of the DNA fragment you want

Question 5

Why does DNA polymerase not denature at high temperatures and why is this good?

Answer 5

It is a thermostable enzyme so does not denature at high temp which is good so that the same enzyme can be reused in the PCR so many cycles of PCR can occur without having to use a new enzyme each time

Question 6

How does the PCR make many copies?

Answer 6

In each new cycle PCR cycle, the amount of DNA doubles 
1st cycle - 2
2nd cycle - 4
3rd cycle - 8
4th cycle - 16
nth term - 2 to power of n

Question 7

Why are we using TAQ polymerase (from particular bacteria) instead of normal/human DNA polymerase?

Answer 7

TAQ polymerase is thermophilic (does not denature at high temp) - more efficient as one can use the same enzyme consistently instead o having to use another one after each cycle

Question 8

Advantages of PCR

Answer 8

Very rapid + does not require living cells (only needs base sequence, no culturing required)

Question 9

List the 7 Applications of PCR

Answer 9

1. Tissue typing - donor + recipient tissues can be matched to reduce the risk of rejection
2. Detection of Oncogenes - detect the type of mutation leading to cancer - can inform medication
3. Detecting mutations - detecting genetic diseases, parents may do this before conceiving
4. Identifying viral infections - Verify the type of viral infections present
5. Monitoring the spread of infectious disease - also monitoring the emergence of new strains
6. Forensic science - small quantities of DNA can be amplified to identify criminals or ascertain parentage
7. Research - amplifying DNA from extinct organisms e.g. mammoths