Answers

1. Vector transfers genes (DNA) from one organism to

another
 In vitro gene cloning 19 June 2024
The polymerase chain reaction
Learning objectives
Describe and explain the polymerase chain reaction
Summarise the advantages and disadvantages of in vitro and in vivo gene
cloning

Starter
Review the in vivo cloning process with
your partner and recap the use of
genetic markers
DNA Technology

1. Isolation – of the DNA containing the required

gene
2. Insertion – of the DNA into a vector
3. Transformation – Transfer of DNA into a
suitable host
4. Identification – finding those host organisms
containing the vector and DNA (by use of gene
markers)
5. Growth/Cloning – of the successful host cells
 Method by which we can copy fragments of
Polymerase chain reaction DNA
It is an automated process, meaning it is rapid
and efficient

Requirements
- DNA fragment (with required gene to be copied)
- DNA polymerase (taq polymerase in specific, which is thermostable -
tolerant to high temperatures)
- Primers – short sequences of nucelotides with a set of bases
complementary to those at each end of the two DNA strands
- Nucleotides
- Thermocycler – computer-controlled machine that varies the temperatures
over a period of time
3 main stages of PCR:
1. Separation of DNA
strand
2. Addition (annealing) of
primers
3. Synthesis of DNA
TASK
Annotate the diagram using the
information, giving a summary of
the PCR

https://www.youtube.com/watch?v=MyLrs_h1OlE
Separation
 Place the DNA fragments, DNA
polymerase and primers into the
thermocycler
 Increase temperature to 95°C –
this causes the two strands of
the DNA fragments to separate
due to the breaking of the
hydrogen bonds
Addition (annealing) of the primers
 Mixture is cooled to 55°C – this
causes the primers to join to
their complementary bases at
the end of the DNA fragment
 Primers:
- Provide the starting sequence
for DNA polymerase to begin
copying
- prevent two separate strands
re-joining
Synthesis of DNA
 Temperature increased to 72°C –
optimum temperature for DNA
polymerase to add complementary
nucleotides along each DNA of
the separated strands
 Begins at the primer and adds
nucleotides in sequence until it
reaches the end of the chain
Result
 Leads to 2 identical copies of the original
fragment
 Process is then repeated by subjecting them
to the temperature cycle from the beginning
 This leads to 4 strands
 This then continues exponentially

 One temperature cycle takes approximately

2 minutes
 Over a million copies of DNA can be made in
just 25 cycles (less than an hour!)
Advantages of in vitro and in vivo
What do you think are some of the advantages
of:
• in vivo
• in vitro
gene cloning?

TASK
Create a table to summarise the advantages of both
methods using the information provided

https://www.youtube.com/watch?v=MyLrs_h1OlE
Evaluating the ethical, social and financial issues