11.

Gene technology
CAPE Biology Unit 1
Module 2
Tutor: Nehru Narine
Terms

 Gene technology – the use of DNA or genes to produce a useful product.

 Genetic engineering – the use of technology to transfer a gene from one species to another.
 Genome – the complete set of genetic material of a species.
 Recombinant DNA (rDNA) – DNA that was created by combining the DNA of two different species.

DNA from goldfish + Glow in the dark DNA from jellyfish Glow in the dark recombinant DNA

 Genetically Modified Organism (GMO) – an organism that has its DNA changed or altered by gene technology. A
GMO may or may not have DNA from a different species.
 Transgenic organism/Recombinant organism – an organism that has a piece of DNA that came from a different
species.
Diagram of a bacterium
Requirements for Genetic engineering
(Recombinant DNA technology)

• (i) Reverse transcriptase – uses mRNA to make a single stranded

complementary copy of DNA (cDNA).
• (ii) DNA polymerase – add free nucleotides to cDNA to make double
stranded cDNA.
• (iii) cDNA (Complementary DNA) – DNA that has been made from
mRNA using reverse transcriptase.
• (iv) Restriction enzymes – enzymes that cut DNA at specific base
sequences. Restriction enzymes would usually make staggered cuts on
DNA called ‘sticky ends. Sticky ends are the ends of a gene that can
easily form hydrogen bonds with other DNA molecules.
Requirements for Genetic engineering
(Recombinant DNA technology)

• (iv) Restriction enzymes – enzymes that cut DNA at specific base

sequences. Restriction enzymes would usually make staggered cuts
on DNA called ‘sticky ends. Sticky ends are the ends of a gene that
can easily form hydrogen bonds with other DNA molecules.
Requirements for Genetic engineering
(Recombinant DNA technology)

• (v) Plasmid – a circular piece of DNA that contains antibiotic

resistant genes. Plasmids can be cut by restriction enzymes so that
the desired gene (insulin gene) can be inserted.

• (vi) Ligase – enzyme that joins two pieces of DNA.

• (vii) Bacteria and fermenter
Steps in Recombinant DNA technology (Gene
cloning)

Isolation of gene Insertion of gene into plasmid Insertion of

plasmid into bacterium Synthesis
 Steps in Recombinant DNA technology (Gene
cloning)
(i) Isolation of insulin gene (cDNA)
mRNA molecules are obtained from
pancreatic cells and then reverse
transcriptase is added to convert the mRNA
into singled stranded cDNA. DNA polymerase
then add free nucleotides to the single
stranded cDNA form double stranded cDNA.
Transcription vs. Reverse transcription

Features Transcription Reverse transcription

Enzymes involved    
 
Precursor molecule    

Product    
Steps in Recombinant DNA technology (Gene
cloning)

(ii) Insertion of insulin gene into plasmid vector

The plasmid is cut using restriction enzymes and then ligase is used
to join the plasmid and insulin gene.
Steps in Recombinant DNA technology (Gene
cloning)

(iii) Insertion of plasmid into bacteria

• The recombinant plasmid is then
added to a culture of bacteria and
Ca2+ are added to increase the
permeability of the cell membrane of
the bacteria. Some of the bacteria
then absorb the recombinant plasmid.
Antibiotics are then added to identify
the transformed bacteria.
Steps in Recombinant DNA technology (Gene
cloning)

(iv) Synthesis
• The culture of transformed bacteria is then added to a nutrient
rich fermenter and as the bacteria reproduce insulin is made as a
product. Insulin is then extracted and purified (Humulin).
Questions
Questions

1. Contrast the role of restriction enzymes in bacterial cells and

genetic engineering.
Questions

2. Explain the steps involved in the use of recombinant DNA

technology for the production of human insulin under the following
headings:
(i) Gene isolation
(ii) Gene insertion into vector