Chapter 19: Genetic Technology

Question 1

What is Polymerase Chain reaction (PCR) used for?

Answer 1

To clone and amplify (to produce many copies of a length of) DNA

Question 2

Advantages of PCR

Answer 2

1. Rapid and efficient process
2. Only small sample of DNA needed
3. Produces many copies
4. Automated process in a thermal cycler

Question 3

Components of PCR

Answer 3

1. Template DNA: which is replicated
2. Taq polymerase (enzyme): can withstand high temperatures as they are isolated from bacteria found in hot springs (thermus aquaticus)
3. Buffer: to maintain a stable pH (KCl or MgCl2)
4. Four nucleotides- ATCG in excess
5. Two primers: short 20 base pairs single stranded RNA/DNA: they are complimentary and act as start and end positions for amplification

Question 4

Three stages of PCR

Answer 4

1. Denaturation
2. Annealing
3. Extension

Question 5

Denaturation

Answer 5

Dna strands separate by breakdown of hydrogen bonds.
Bases are exposed, producing template strands for copying. Occurs at high temperatures of 95 degrees.

Question 6

Annealing

Answer 6

60-65 degrees
Primers bind to specific DNA section
Via complementary base pairing
New hydrogen bonds form

Question 6

Role of primers

Answer 6

1. Bind to target region for amplification
2. Starting point for Taq polymerase to bind
3. Reduce reannealing (joining back) of template strands

Question 7

Extension

Answer 7

1. Taq polymerase binds to primer
2. Synthesises new DNA strands
3. Complementary to template DNA strands

Question 8

Advantages of Taq polymerase

Answer 8

1. Very heat stable
2. No need to replace after every cycle

Question 9

What affects annealing temperature?

Answer 9

Sequence of primer

Question 10

How many cycles take place and what happens?

Answer 10

Around 30 cycles, DNA strands double each cycle

Question 11

Number of DNA molecules made from one starting molecule at the end of n cycles

Answer 11

2^n (exponential amplification)

Question 12

Disadvantages of PCR

Answer 12

1. Need to know the precise DNA sequence to design primers
2. Only shorter fragments can be amplified

Question 13

Applications of PCR

Answer 13

1. DNA sequencing (in fossils to amplify tiny amounts of DNA)
2. DNA profiling at crime scene
3. Recombinant gene technology
4. genetic screening (to identify mutations or genetic conditions)

Question 14

Recombinant gene technology steps

Answer 14

1. Obtain mRNA from human insulin (from beta cells from isles of langherham)
2. Use reverse transcriptase to form complementary DNA from mRNA.
3. DNA polymerase used to make double stranded cDNA.
   (gene has no introns so is shorter)
4. DNA may be amplified using PCR
5. Restriction enzyme to cut gene on restriction SITES that are present on both sides of target gene.
6. Obtain plasmids from bacteria
7. Cut plasmid at one restriction site using SAME restriction enzyme
8. Complementary sticky ends created
9. recombine gene to plasmid DNA
10. DNA ligase seals sugar phosphate backbone to form recombinant plasmids.
11. Mix recombinant plasmids with bacteria
12. Treat bacteria with calcium ions
13. Apply heat shock to increase chances of plasmid passing through the membrane
14. Identify modified plasmids
15. Put bacteria in fermented and allow it to multiply
16. insulin extracted and sold on market

Question 15

Why do we have to take mRNA instead of just the gene directly?

Answer 15

Genes are quite complicated and have many introns (non-coding regions), which may interfere with insulin production, so mRNA is taken so avoid the non-coding regions.

Question 16

Restriction endonuclease

Answer 16

From bacteria
Recognise, bind to and cut DNA at specific sequences (restriction sites)
hydrolyses phosphodiester bonds
Different enzymes cut at different restriction sites
e.g ecoR1
Can produce sticky ends (unpaired nucleotides)

Question 17

Restriction sites and characteristics

Answer 17

Specific sequences on which restriction enzyme cuts
Multiple sites present in one plasmid
Restriction sites palindromic

Question 18

Sticky ends

Answer 18

Staggered cuts
Few unpaired nucleotides
Forms H bonds easily to complementary base pairs

Question 19

Blunt ends

Answer 19

NO unpaired nucleotides

Question 20

Why are bacterial plasmids used?

Answer 20

1. They are small so can be easily inserted
2. Circular so not easily damaged but host cells, more stable
3. Easy to extract from bacteria
4. Great vector to delivered desired genes into bacteria
5. Easy to be taken up by bacteria
6. High copy number: many copies form inside bacteria
7. Able to clone any gene inserted int them
8. Essential DNA sequence present

Question 21

Origin of replication

Answer 21

Allows bacterial DNA polymerase to bind and replication of plasmids within the bacteria to be initiated

Question 22

Promoter

Answer 22

A specific DNA sequence that tells the cell where to start transcription

Question 23

How does a promoter work?

Answer 23

1. RNA polymerase binds to promoter
2. Transcription starts after the RNA polymerase starts to make complementary mRNA strand
3. Different promoters determine different levels of transcription

Question 24

Marker gene

Answer 24

Used to identify presence of recombinant plasmids in bacteria or transgenic organisms

Question 25

Why is bacteria treated with calcium ions?

Answer 25

Bacteria have a negatively charged cell membrane. DNA (including plasmids) is also negatively charged. It repels the membrane. Calcium ions neutralize these negative charges, making the membrane more permeable and allowing plasmids to enter.

Question 26

Why conduct a heat shock?

Answer 26

Bacteria are placed in an ice-cold solution with Ca²⁺ and plasmids. A quick heat shock (42°C for ~30–60 sec) is applied. This creates a temporary pressure difference, making pores open in the membrane. The plasmid rushes into the bacterial cell.

Question 27

What % of plasmids are recombinant?

Answer 27

Around 1%

Question 28

How do marker genes work?

Answer 28

A marker gene is inserted alongside the desired gene (using same method) so that: ✔ If the marker gene is expressed, it means the desired gene is also present. ✔ If the marker gene is missing, the cell didn’t take up the desired gene.

Question 29

Ways to identify transformed bacteria: antibiotic selection

Answer 29

1. uses two marker genes -ampicillin resistant gene, tetracycline resistant gene Bacteria are spread onto an agar plate containing an antibiotic (e.g., ampicillin). Only bacteria that successfully took up the plasmid survive because they have antibiotic resistance. Bacteria without the plasmid die. Make a replica plate using a sponge/velvet pad. Recombinant plasmids do not have tetracycline resistance gene, SO: Bacteria with this recombinant plasmid will NOT survive on tetracycline plates.

Question 30

Problems

Answer 30

1. Spread of antibiotic resistance -Plasmids easily transferred via conjugation

Question 31

Ways to identify transformed bacteria: Green Fluorescent protein (GFP)

Answer 31

Helps identify bacteria that have successfully taken up a plasmid. GFP gene is inserted into the plasmid alongside the desired gene. Bacteria that take up the plasmid will express GFP and glow under UV light. Bacteria without the plasmid will not fluoresce.

Question 32

Ways to identify transformed bacteria: easily stained substance

Answer 32

Some marker genes produce substances that can be easily stained for quick identification of transformed bacteria. Example: β-Galactosidase (LacZ) Marker The LacZ gene (from the lac operon) codes for β-galactosidase enzyme. If the plasmid has been successfully taken up, the enzyme will be expressed. When bacteria are grown on a medium with a special non-blue substrate, the enzyme breaks it down, producing a blue color. If the desired gene was inserted into the LacZ gene, it disrupts its function, and the bacteria remain white instead of blue.

Question 33

Advantages of GFP

Answer 33

1. Avoids antibiotic use 2. More economical and time saving 3. Enables identification of transgenic organisms as well

Question 34

Advantages of producing human insulin by gene technology:

Answer 34

1. Chemically identical to human insulin → exact fit to insulin receptors on target organs → does not stimulate the immune system → faster response → fewer side affects → less / no risk of allergic reaction 2. Effective in people who have developed tolerance to animal-derived insulin 3. Avoid ethical issues related to religion & use of animal products → no killing of animals 4. Lower cost of purification and processing 5. Mass production = large and constant reliable supply all year round 6. Less risk of contamination/infection → no risk of transfer of disease 7. Potential to engineer / improve recombinant proteins

Question 35

Applications of Recombinant DNA Technology

Answer 35

Production of pharmaceuticals → No modifying of protein in bacteria (bcs no membrane-bound organelles) → Can genetically modify eukaryotic cells to produce human proteins E.g. yeast cells, insect larvae cells, mammalian cells