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Recombinant DNA Technology

•Also known as genetic engineering

•Isolate and manipulate genes in vitro

•Cloning of specific genes using vectors

•Endow engineered cells with new synthetic capabilities

•Vector
•A DNA molecule into
which a DNA fragment
can be introduced and
which can replicate in a
suitable host organism

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•Vector

• The most generally used vectors have three properties

1. The vector DNA can be introduced into a host cell relatively

easily

2. The vector and the DNA it contains can be replicated inside

the host cell

3. Cells containing the vector can be identified in a

straightforward manner, most conveniently through a novel
phenotype conferred on the host by DNA sequences present
in the vector (i.e. antibiotic resistance)

•Bacterial cloning

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•Bacterial cloning

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•Bacterial cloning (contd.)

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•Bacterial cloning (contd.)

Types of Vectors
1. Plasmid vector – carries
up to 10 kb

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2. λ phage vector (~20 kb)

• Substitutes the
central gene
cluster, which is
not essential for
infection and
phage propagation

3. Cosmid vector (~50 kb)

• Act as a plasmid but also contains phage single-stranded
sticky ends that tend to bind to each other

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4. Bacterial Artificial
Chromosomes (BACs)
(~300 kb)

• oriS – plasmid regulation and

regulation of copy number
• parA, parB– for partitioning
plasmid to daughter cells
during division and ensures
stable maintenance of the BAC
• CMR– selection marker, i.e.
chloramphenicol resistance
• T7, Sp6– phage promoters

5. Yeast Artificial
Chromosomes (YACs) (100 -
1,000 kb)

• built using an initial

circular plasmid, which is
typically broken into two linear
molecules using restriction
enzymes

• DNA ligase is then used to ligate

a sequence or gene of interest
between the two linear
molecules, forming a single large
linear piece of DNA

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•Examples of recombinant DNA technology

1. Genetically-engineered glyphosate-resistant plants

•Examples of recombinant DNA technology

1. Genetically-engineered glyphosate-resistant plants

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•Examples of recombinant DNA technology

2. Synthesizing human recombinant insulin

•Examples of recombinant DNA technology

2. Genetically-engineered glyphosate-resistant plants

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•Examples of recombinant DNA technology

3. Gene therapy to treat SCID
•SCID – severe combined immunodeficiency disease

– antibodies and T-cells are either missing or

disabled

– with a number of different causes

– one cause due to lack of adenosine deaminase (ADA)

– substrates of ADA accumulates and kills

immature lymphoid cells

3. Gene therapy to treat SCID

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•Some examples of genetically modified foods (GMF)

Golden rice – produces beta-carotene Long lasting tomatoes

Bt corn and Bt eggplant – produces own

pesticide derived from Bacillus thuringiensis

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•Some ethical considerations

•Humans have been manipulating the genetic character of

diverse species for their own ends
•Corn •Cattle
•Wheat •Poultry
•Rice •Dogs
•Potato •Horses

•Medical practices are also forms of genetic manipulations

Genomics

•Development and application of new mapping, sequencing

and computational procedures for the analysis of the entire
genome of organisms

•E.g. Human genome project

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•Fields of Genomics
•Structural genomics
•Genetic mapping, physical mapping and sequencing of
entire genomes

•Functional genomics
•Comprehensive analysis of the functions of gene and
non-gene sequences in entire genomes

•Comparative genomics
•Comparison of entire genomes of different species to
understand the functions of each genome, including its
evolutionary relationships

•Timeline of genome sequencing

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•Two approaches to genome sequencing

•Mapping approach
•Generate high-resolution genetic and physical maps of
the genome, then sequence the segments in an orderly
manner

•Whole genome shotgun approach

•Break up the genome into random, overlapping
fragments and then sequence and assemble the
fragments using computer algorithms

Mapping approach

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Whole genome shotgun approach

•What we know of the human genome

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•Ethical issues raised by the Human Genome Project

•Should a patient be told if a test for an incurable genetic
disease is positive?
•Should pregnant mothers abort their unborn children
with known genetic disorders?
•Should parents be eventually allowed to design their own
babies?

•Should employers be able to ask for results of a genetic

test as a basis for employment?

•Should governments be allowed to collect genetic data

on their populace?

Some say that the integrity of the DNA of

each species is inviolate and should not be
tampered with. But as seen, nature itself
alters DNA. Consider whether we as a
species have the wisdom to bring about
beneficial changes without causing harm
to ourselves and to the environment.

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