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18.1 Recombinant DNA

technology

18.1 Recombinant DNA technology

• Explain recombinant DNA technology / genetic
engineering.
• Differentiate between genomic and cDNA
cloning and genomic and cDNA libraries.
• Describe the vectors used in cloning and their
properties.
• Describe the restriction enzyme (EcoR1 and
SmaI), including its nomenclature, recognition
site (palindromic), importance and the types of
ends generated.

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• Explain reverse transcription, insertion, ligation,

transformation / transduction, amplification and
screening.
• Describe the steps involved in genomic and
cDNA cloning, including the enzymes involved,
and explain human insulin production in E.coli
as an example.

GENE LIBRARY
 ‘A collection of cloned DNA fragments’
 Derived from a single individual
 2 types:
i. genomic library
ii. complementary DNA (cDNA) library

i. Genomic Library

• ‘A collection of cloned DNA fragments that consists of all the DNA sequences of an
organism’
• The DNA fragments represent the entire genome of the organism
• Genomic library saves all the genetic information of an organism
• Researchers can use it as source of any gene of interest or for genome mapping

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i. Genomic Library

• 2 types of genomic library:

i. plasmid library
- complete set of recombinant-plasmid clones (within a set of bacteria),
each type of clones is carrying copies of a particular segment from the initial
genome

ii. phage library

- bacteriophages as the cloning vectors
- collection of phage clones, each type carrying copies of a particular foreign
DNA fragment

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i. Genomic Library

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ii. cDNA Library

• ‘A collection of
complementary DNA
molecules that represents
only part of an organism’s
genome’

• A limited kind of DNA library

~ incomplete, does not
represent the entire genome
of an organism

• cDNA is made in vitro using

a particular mRNA as
template with enzyme
reverse transcriptase

Forming a cDNA
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ii. cDNA Library

• The cDNA of the particular gene will then be cloned and kept in a cDNA library

• Advantages of cDNA:
i. does not contain non-coding introns

ii. size of cDNA is smaller ~ easier to make a cDNA library

iii. fewer types of clones are composed in cDNA library ~ screening process is simpler
and faster

iv. easier for researchers to study a particular gene responsible for a specialized
function of a particular kind of cell

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PRODUCTION OF INSULIN USING cDNA

• The mRNA containing the code for synthesis human insulin were identified and isolated
from β cells of Islet of Langerhans of the pancreas.
• Reverse transcriptase enzyme makes single stranded cDNA using the mRNA as a
template.
• Single stranded cDNA is catalysed by DNA polymerase to form double-stranded cDNA.
• Bacterial plasmid with the AmpR gene and lacZ gene is also isolated.
• AmpR enables the host cell, E.coli to become resistant to the antibiotic amphicillin.
• The restriction site for the restriction enzyme is within the lacZ gene of the plasmid.
• The bacterial plasmid is then cut by a specific restriction enzyme at the restriction site
thus distrupting the lacZ gene, making it incapable of producing β-galactosidase
• The DNA fragment with the insulin gene is also cut with the same restriction enzyme.
• The restriction enzyme creates opened plasmid and DNA fragments with sticky ends.
• Insertion of DNA fragment into plasmid as sticky ends of plasmid join with sticky ends of
DNA fragment.
• DNA ligase joins covalently both DNA molecules forming recombinant DNA or
recombinant plasmid.
• Then rDNA formed is introduced into a host cell , E.Coli through the transformation.
• Amplification occurs when the transformed bacteria undergo binary fission, thus
producing a large amount of bacteria clones.

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• The bacteria or host cell are screened to identify the bacteria carries the recombinant DNA
• The screening process is used is the blue-white screening in which the bacterial colony is
cultivated in nutrient that contains antibiotic ampicillin and X-gal sugar.
• Ampicillin ensures that only bacteria with the recombinant plasmids can survive and form
a colony on the medium.
• X-gal used to identify colonies of bacteria contains plasmids carry DNA fragments.
• Bacteria with functional lacZ gene will produce a blue coloured colony, while bacteria with
rDNA and non-functional lacZ gene will produce white colonies.
• The bacteria with the recombinant DNA are then identified, isolated and cultured.
• Replication of plasmids occur as the bacteria reproduces.
• These bacterial colonies can be used to synthesise insulin in large quantities.

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