Biology 6.3

Question 1

What is DNA sequencing and what are the steps?

Answer 1

Reading genome

1. Replicate- PCR
2. Heat- single stranded
3. Primer for DNA polymerase
4. Modified bases- radioactive isotope
5. Terminates at modified base
6. Different lengths
7. Electrophoresis

Question 2

What is pyrosequencing and what are the steps?

Answer 2

Faster way of reading genome

1. Cut- nebuliser
2. Heat- single stranded
3. Immobilised
4. Primer added for DNA polymerase
5. Activated nucleotides (2 extra Ps)
6. PP released when joined
7. PP --> ATP with ATP sulfurylase + APS
8. Luciferin --> oxyluciferin with ATP + luciferase
9. Visible light generated
10. Leftovers degraded- apyrase

Question 3

What are the things needed for pyrosequencing?

Answer 3

Primer
DNA polymerase
Activated nucleotides
ATP sulphurylase
APS
Luciferase
Apyrase

Question 4

What is the polymerase chain reaction (PCR) and what are the steps?

Answer 4

Replication of DNA

1. Heat- single stranded (98)
2. Primers anneal (68)
3. Taq polymerase binds
4. Temp raised (72)
5. Nucleotides added 5'-3'
6. DNA forms
7. Cycle repeats

Question 5

What is an alternative way of replicating DNA?

Answer 5

1. Isolate gene- restriction enzymes
   
   2. Insert into plasmid
   3. Insert into bacteria host
   4. Bacteria replicates

Question 6

How does DNA profiling work?

Answer 6

1. DNA obtained
   
   2. Cut by restriction enzymes at short tandem repeats
   3. Stained
   4. Separated- electrophoresis
   5. Banding pattern formed and compared

Question 7

How does electrophoresis work?

Answer 7

By separating different sized fragments
	1. Agarose gel and buffer
	2. DNA/protein dyed
	3. SDS added if molecule is charged
	4. DNA is negative so moves to positive anode
Different speeds cause different bands

Question 8

What are DNA probes used for?

Answer 8

Locating genes
Identifying genes in different species
Identifying the presence of an allele that causes disease

Question 9

How do DNA probes work?

Answer 9

They are complementary to the section of DNA being investigated
They have a radioactive or fluorescent marker on them so you can see when the sequence of investigated DNA is present

Question 10

What are microarrays and how do they work?

Answer 10

They are a number of different probes on fixed surface

1. DNA amplified- PCR
2. Sample and reference DNA added
3. Both fluorescent colours show sequence is present

Question 11

What is gene therapy?

Answer 11

Inserting functional alleles into a DNA sequence to benefit the organism

Question 12

How does somatic cell gene therapy work?

Answer 12

Eg. for cystic fibrosis

1. Liposomes
2. Sprayed in nose
3. Pass through plasma membrane
4. Pass through nuclear envelope
5. Express CFTR gene
6. Replaced every 10-14 days
7. Not passed to offspring
8. Viruses May be used 
9. Germ line gene therapy alters gametes

Question 13

What is genetic engineering?

Answer 13

Combining DNA

Question 14

How does genetic engineering work?

Answer 14

Combining DNA

1. mRNA taken
2. Reverse transcriptase forms cDNA
3. Primers and DNA polymerase added
4. cDNA becomes double stranded
5. DNA probe locates gene
6. Restriction enzymes cut palindromic sequence on DNA and plasmid
7. Complementary nucleotides added to DNA sticky ends
8. DNA ligase anneals DNA and plasmid
9. Plasmid into attenuated virus (vector)
10. Vector into cell
	e. Gene gun- gold + tungsten shot

Question 15

How do restriction enzymes know where to cut?

Answer 15

They cut at the palindromic sequence - antiparallel base pairs = base pairs that can be read the same backwards and forwards
Cuts out with a hydrolysis reaction

Question 16

How do you get the vector into the cell in genetic engineering?

Answer 16

Heat shock treatment- CaCl2
Electroporation- electrical pulse
Electrofusion- electrical fields
Transfection- bacteriophage
Gene gun- gold + tungsten shot

Question 17

How is insulin made from genetically modified bacteria?

Answer 17

• mRNA from beta cells
  
  • Same method as GM
  • E.coli used
  • If gene present, should be resistant to ampicillin but die in tetracycline
  • Die in ampicillin= no plasmid
  • Survive in tetracycline= no gene

Question 18

What are the two antibiotics to test is the gene for insulin is present?

Answer 18

Ampicillin

Tetracycline