Recombinant DNA Technology 2023

Recombinant DNA Technology
The Role of Recombinant DNA

Technology in Biotechnology
• Recombinant DNA Technology

– Intentional modification of organisms’ genomes
for practical purposes
– Three goals
• Eliminate undesirable phenotypic traits
• Combine beneficial traits of two or more organisms
• Create organisms that synthesize products humans
need
Bacterial
cell DNA
containing
gene of
interest
Bacterial Plasmi
chromoso d
me
Isolate Gene of
plasmid. interest
Enzymatically
cleave DNA into
fragments.
Overview of
Isolate
fragment with
the gene of
interest.
recombinant DNA Insert gene into

plasmid.
technology Insert plasmid and gene

into bacterium.
Culture
bacteria.
Harvest copies of
Harvest
gene to insert into
proteins
plants or animals
coded by gene
Eliminate Create Produce vaccines,

undesirable beneficial antibiotics,
phenotypic combinatio hormones, or
traits n of traits enzymes
The Tools of Recombinant DNA
Technology
• Mutagens
– Physical and chemical agents that
produce mutations
– Scientists utilize mutagens to
• Create changes in microbes’ genomes to
change
phenotypes
• Select for and culture cells with
beneficial characteristics
– Mutated genes alone can be isolated
Technology
• The Use of Reverse Transcriptase to
Synthesize cDNA
– Isolated from retroviruses
– Uses RNA template to transcribe
molecule of cDNA
– Easier to isolate mRNA molecule for desired
protein first
– mRNA of eukaryotes has introns removed
• Allows cloning in prokaryotic cells
Technology
• Synthetic Nucleic Acids
– Molecules of DNA and RNA produced in
cell-free solutions
– Uses of synthetic nucleic acids
• Elucidating the genetic code
• Creating genes for specific proteins
• Synthesizing DNA and RNA probes to locate
specific sequences of nucleotides
• Synthesizing antisense nucleic acid molecules
Technology
• Restriction Enzymes
– Bacterial enzymes that cut DNA molecules
only at restriction sites
– Categorized into two groups based on type of
cut
• Cuts with sticky ends
• Cuts with blunt ends
Actions of restriction
enzymes-overview
Origin and function
• Bacterial origin = enzymes that cleave foreign
DNA
• Named after the organism from which they were
derived
– EcoRI from Escherichia coli (Gram-)
– BamHI from Bacillus amyloliquefaciens (Gram+)
• Protect bacteria from bacteriophage infection
– Restricts viral replication
• Bacterium protects it’s own DNA by methylating
those specific sequence motifs
Availability
• Over 200 enzymes identified, many

available commercially from
biotechnology companies
Classes
• Type I
– Cuts the DNA on both strands but at a
non- specific location at varying
distances from the particular sequence
that is recognized by the restriction
enzyme
– Therefore random/imprecise cuts
– Not very useful for rDNA applications
Classes
• Type II
– Cuts both strands of DNA within the particular
sequence
recognized by the restriction enzyme
– Used widely for molecular biology procedures
– DNA sequence = symmetrical
Classes
• Reads the same in the 5’ 3’ direction on

both strands = Palindromic Sequence
• Some enzymes generate “blunt ends”
(cut in middle)
• Others generate “sticky ends” (staggered
cuts)
– H-bonding possible with complementary tails
– DNA ligase covalently links the two fragments
together by forming phosphodiester bonds of the
phosphate-sugar backbones
Technology
• Vectors
– Nucleic acid molecules that deliver a gene
into a cell
– Useful properties
• Small enough to manipulate in a lab
• Survive inside cells
• Contain recognizable genetic marker
• Ensure genetic expression of gene
– Include viral genomes, transposons, and
plasmids
mRNA for human
growth hormone
Antibioti Restricti (HGH)
c on site
resistan Reverse
ce gene transcripti
on
cDNA for
Plasmid HGH
(vector)
Restricti Restricti
on on
enzyme enzyme
Sticky
ends
Gene for
human growth
hormone
Producing a
recombinant vector
Ligas
e
Recombinant plasmid
Introduce
recombinant
plasmid into
bacteria.
Bacteri Recombina
chromoso
al nt plasmid
me
Inoculate bacteria
on media
containing
antibiotic.
Bacteria
containing the
plasmid with HGH
gene survive
because they also
have resistance
gene.
Requirements of a vector to serve as
a carrier molecule
• The choice of a vector depends on the design

of the experimental system and how the
cloned gene will be screened or utilized
subsequently
• Most vectors contain a prokaryotic origin of
replication allowing maintenance in bacterial
cells.
Requirements of a vector to serve as
a carrier molecule
• Some vectors contain an additional
eukaryotic origin of replication allowing
autonomous, episomal replication in
eukaryotic cells.
• Multiple unique cloning sites are
often included for versatility and
easier library construction.
Requirements of a vector to serve
as a carrier molecule
• Antibiotic resistance genes and/or other

selectable markers enable identification of
cells that have acquired the vector construct.
• Some vectors contain inducible or tissue-
specific promoters permitting controlled
expression of introduced genes in transfected
cells or transgenic animals.
Requirements of a vector to serve
as a carrier molecule
• Modern vectors contain multi-functional

elements designed to permit a
combination of cloning, DNA sequencing,
in vitro mutagenesis and transcription and
episomal replication.
Main types of vectors
• Plasmid, bacteriophage, cosmid,

bacterial artificial chromosome (BAC),
yeast artificial chromosome (YAC),
yeast 2 micron plasmid, retrovirus,
baculovirus vector……
Choice of vector
• Depends on nature of protocol or

experiment
• Type of host cell to accommodate rDNA
– Prokaryotic
– Eukaryotic
Plasmid vector
• Covalently closed, circular, double stranded DNA

molecules that occur naturally and replicate
extrachromosomally in bacteria
• Many confer drug resistance to bacterial strains
• Origin of replication present (ORI)
Technology
• Gene Libraries
– A collection of bacterial or phage clones
• Each clone in library often contains one gene
of an organism’s genome
– Library may contain all genes of a
single chromosome
– Library may contain set of cDNA
complementary to mRNA
Genom
e
Isolate
genome or
organism.
Generate fragments
using restriction
enzymes.
Production of a Insert each

fragment into a
vector.
gene library-
overview Introduce
vectors into
cells.
Culture recombinant
cells; descendants are
clones.
Definition of recombinant DNA
• Production of a unique DNA molecule by

joining together two or more DNA
fragments not normally associated with
each other
• DNA fragments are usually derived
from different biological sources
Steps involved in isolating a particular
DNA fragment from a complex mixture of
DNA fragments or molecules
1. DNA molecules are digested with

enzymes called restriction
endonucleases which reduces the size of
the fragments Renders them more
manageable for cloning purposes
2. These products of digestion are inserted

into a DNA molecule called a vector
Enables desired fragment to be
replicated in cell culture to very high
levels in a given cell (copy #)
3. Introduction of recombinant DNA molecule into

an appropriate host cell
– Transformation or transfection
– Each cell receiving rDNA = CLONE
– May have thousands of copies of rDNA
molecules/cell
after DNA replication
– As host cell divides, rDNA partitioned into daughter
cells
4. Population of cells of a given clone is expanded, and
therefore so is the rDNA.
– Amplification
– DNA can be extracted, purified and used for
molecular analyses
• Investigate organization of genes
• Structure/function
• Activation
• Processing
– Gene product encoded by that rDNA can be
characterized or modified through mutational
experiments

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Recombinant DNA Technology

The Role of Recombinant DNA

• Recombinant DNA Technology

recombinant DNA Insert gene into

technology Insert plasmid and gene

Eliminate Create Produce vaccines,

• Over 200 enzymes identified, many

• Reads the same in the 5’ 3’ direction on

• The choice of a vector depends on the design

• Antibiotic resistance genes and/or other

• Modern vectors contain multi-functional

• Plasmid, bacteriophage, cosmid,

• Depends on nature of protocol or

• Covalently closed, circular, double stranded DNA

Production of a Insert each

• Production of a unique DNA molecule by

1. DNA molecules are digested with

2. These products of digestion are inserted

3. Introduction of recombinant DNA molecule into

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