DNA Technology Part 2 - (Week 10)

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction - It is the method used in the ‘amplification’ step of gene cloning, where the specific DNA fragment is replicated (in vitro)

Question 2

What biological process is PCR based on?

Answer 2

DNA replication

Question 3

Name the 4 steps of PCR

Answer 3

1. Denaturation
2. Primer annealing
3. Extension/amplification
4. Repeat the entire process

Question 4

What happens in the 1st step of PCR?

Answer 4

The DNA is denatured - to separate the two nucleotide strands of the DNA molecule

Question 5

What happens in the 2nd step of PCR?

Answer 5

Annealing - the primers bind (anneal) to complementary base sequences on the single-stranded target DNA

Question 6

What happens in the 3rd step of PCR?

Answer 6

• Extension - free nucleotides are added to the primers (in 5’ to 3’ direction) using DNA polymerase to synthesise complementary strands to original DNA strand (both DNA strands of original double stranded DNA are used as templates)
• A double stranded copy of the target/template DNA is formed

Question 7

What happens the 4th step of PCR?

Answer 7

The entire process of denaturation, annealing & amplification is repeated (about 30-40 cycles)

Question 8

What are restriction enzymes also known as?

Answer 8

Restriction endonucleases

Question 9

What do restriction enzymes do?

Answer 9

• Cleave DNA at specific DNA sequence sites (restriction sites)
• Most cut in a staggered way to produce fragments with ‘sticky ends’

Question 10

What does DNA ligase do?

Answer 10

It is the enzyme that seals the bonds between restriction fragments (between complementary sticky ends of fragments)

Question 11

What is meant by ‘palindromic sequence’ and how is this important in using restriction enzymes to make recombinant DNA plasmid?

Answer 11

Most restriction enzymes recognise palindromic sequences - the complementary strand w/ same sequence in reverse

Question 12

How is a recombinant DNA plasmid made?

Answer 12

• The DNA fragment w/ ‘desired gene’ is amplified through PCR
• Restriction enzymes are used to generate ‘sticky ends’ for amplified DNA
• Same restriction enzymes are to cut plasmid as specific point to form ‘sticky ends’
• DNA ligase is used to seal bonds between the ‘sticky ends’ of the desired DNA fragment and the plasmid (at those points where fragment & plasmid bind, they have complementary base sequences)

Question 13

What are cloning vectors and how are they important in gene cloning?

Answer 13

• Cloning vectors are self replicating DNA

- It is used to carry the gene of interest into a host system where the gene can be expressed

Question 14

What qualities make a cloning vector suitable for gene cloning?

Answer 14

They need to have:

• Selectable marker e.g. antibiotic resistance
• Unique restriction enzyme recognition sites

Question 15

Give at least two reasons why plasmids are often used cloning vectors

Answer 15

• Readily obtained
• Easily manipulated
• Easily introduced into bacterial cells
• Once in the bacteria, they multiply rapidly

Question 16

What is host cell selection for gene amplification dependent on?

Answer 16

It depends on the choice of vector/plasmid and the aim of the cloning (e.g. expression, amplification)

Question 17

What happens in the host cell in gene amplification?

Answer 17

Reproduction of a recombinant DNA plasmid - the host cell copies the cloned DNA using its own replication mechanism to produce multiple copies of a gene

Question 18

Give at least one example of what should be considered when selecting host cells for gene amplification

Answer 18

• Regulatory sequences necessary for vector maintenance (replication) & inheritance
• Regulatory sequences that are necessary for gene expression in host: transcription & translation is dependent on the host enzymes (has to be compatible)