21.3 - In Vitro Gene Cloning — The Polymerase Chain Reaction

Question 1

What is the polymerase chain reaction (PCR)?

Answer 1

PCR is a method of copying fragments of DNA. The process is automated, making it both rapid and efficient.

Question 2

What are the key components required for PCR?

Answer 2

• DNA fragment to be copied
• DNA polymerase, such as taq polymerase, which is thermostable and does not denature at high temperatures
• Primers, short sequences of nucleotides with bases complementary to those at one end of each DNA fragment
• Nucleotides, containing all four DNA bases
• Thermocycler, a computer-controlled machine that varies temperatures precisely over time

Question 3

What happens in the first step of PCR?

Answer 3

1) The DNA fragments, primers, and DNA polymerase are placed in a thermocycler.
2) The temperature is increased to 95°C, breaking the hydrogen bonds between DNA strands, causing them to separate.

Question 4

What happens in the second step of PCR?

Answer 4

1) The mixture is cooled to 55°C, allowing primers to anneal to their complementary bases at the DNA fragment ends.
2) Primers serve as the starting sequences for DNA polymerase and prevent rejoining of the separated strands

Question 5

What happens in the third step of PCR?

Answer 5

1) The temperature is raised to 72°C, the optimum for DNA polymerase.
2) DNA polymerase adds complementary nucleotides along each separated strand, starting at the primer.
3) The process results in two copies of the original fragment, which then undergo further cycles

Question 6

How does PCR exponentially increase DNA quantity?

Answer 6

Each cycle doubles the number of DNA strands. After 25 cycles, over a million copies are produced, and within a few hours, 100 billion copies can be made.

Question 7

Why is PCR valuable in forensic science?

Answer 7

Even the smallest DNA samples, such as from a single hair or blood speck, can be amplified for forensic analysis and cross-matching.

Question 8

What are the advantages of in vitro gene cloning (PCR)?

Answer 8

• Extremely rapid – Can produce 100 billion copies in hours, valuable for forensic analysis.
• No need for living cells – Requires only a DNA sequence, avoiding complex culturing techniques.

Question 9

What are the advantages of in vivo gene cloning?

Answer 9

1) Can introduce genes into organisms via vectors, used in gene therapy.
2) Minimal contamination risk – Only genes cut by the same restriction enzyme match the plasmid.
3) High accuracy – Fewer errors compared to early PCR methods.
4) Specific gene targeting – Only copies the desired gene, not whole DNA.
5) Mass protein production – Transformed bacteria can produce commercial and medical proteins.

Question 10

What are the key benefits of recombinant DNA technology?

Answer 10

• Microorganisms can produce antibiotics, hormones, and enzymes for medical use.
• Pollution control – Bacteria can break down oil spills and toxic gases.
• Plant pharming – Genetically modified plants produce antibodies and antigens for disease prevention.
• Genetically modified crops can withstand drought, salt, and extreme temperatures.
• Golden rice produces vitamin A, preventing blindness in children.
• Genetically modified animals can produce cheap drugs, hormones, and enzymes.
• Gene therapy may cure disorders like cystic fibrosis and SCID.
• Genetic fingerprinting is useful in forensic science.

Question 11

What are the risks of recombinant DNA technology?

Answer 11

• Unpredictable ecological consequences – Genetically modified organisms could disrupt ecosystems.
• Gene transfer between species – E.g., herbicide resistance could spread to weeds.
• Unknown metabolic side effects – Could create new diseases or cancer risks.
• Antibiotic resistance spread – Marker genes in bacteria could make harmful bacteria resistant.
• Mutation of inserted genes – Could turn an engineered organism into an uncontrollable pathogen.
• Long-term genetic impacts – Reducing genetic diversity could affect evolution.
• Economic consequences – Crops like British-grown bananas could harm Caribbean economies.
• Ethical concerns – Is genetic modification acceptable for intelligence, body traits, or appearance?
• Risk of eugenics – Could lead to racial selection.
• Genetic manipulation in the wrong hands – Could be used for political control or warfare.
• Financial priorities – Should funding go to hunger and poverty instead?
• Forensic misuse – False DNA evidence could lead to wrongful convictions.
• Moral debates – Should we let nature run its course instead of intervening?