Recombinant DNA technology involves intentionally modifying organisms' genomes for practical purposes such as eliminating undesirable traits, combining beneficial traits from different organisms, or creating organisms that synthesize useful products. The key steps involve isolating a gene of interest, inserting it into a plasmid, introducing the plasmid into bacteria, and harvesting copies of the gene or its protein products. Common tools used include mutagens, reverse transcriptase to synthesize cDNA, synthetic nucleic acids, restriction enzymes to cut DNA at specific sites, vectors to deliver genes into cells, and gene libraries containing collections of cloned genes.

1. Recombinant DNA
Technology
Anas Bahnassi PhD RPh CDE CDM

2. 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

3. Bacterial cell
DNA containing
gene of interest
Bacterial Plasmid
chromosome
Isolate plasmid. Gene of interest
Enzymatically cleave
DNA into fragments.
Isolate fragment
with the gene of
interest.
Overview of Insert gene into plasmid.
recombinant
Insert plasmid and gene into
DNA bacterium.
technology
Culture bacteria.
Harvest copies of
gene to insert into Harvest proteins
coded by gene
plants or animals
Eliminate Create Produce vaccines,
undesirable beneficial antibiotics,
phenotypic combination hormones, or
traits of traits enzymes

4. 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

5. The Tools of Recombinant DNA
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

6. The Tools of Recombinant DNA
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

7. The Tools of Recombinant DNA
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

8. Actions of restriction
enzymes-overview

9. Origin and function
• Bacterial origin = enzymes that cleave foreign DNA
• Named after the organism from which they were
derived
– EcoRI from Escherichia coli
– BamHI from Bacillus amyloliquefaciens
• Protect bacteria from bacteriophage infection
– Restricts viral replication
• Bacterium protects it’s own DNA by methylating
those specific sequence motifs

10. Availability
• Over 200 enzymes identified, many available
commercially from biotechnology companies

11. 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

12. 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

13. 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

14. The Tools of Recombinant DNA
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

15. mRNA for human
growth hormone (HGH)
Antibiotic Restriction
resistance site
gene
Reverse
transcription
cDNA for HGH
Plasmid (vector)
Restriction Restriction
enzyme enzyme
Sticky ends
Gene for human
growth hormone
Producing a
recombinant
Ligase
vector
Recombinant plasmid
Introduce recombinant
plasmid into bacteria.
Bacterial Recombinant
chromosome plasmid
Inoculate bacteria
on media containing
antibiotic.
Bacteria containing
the plasmid with
HGH gene survive
because they also
have resistance gene.

16. 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.

17. 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.

18. 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.

19. 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.

20. Main types of vectors
• Plasmid, bacteriophage, cosmid, bacterial
artificial chromosome (BAC), yeast artificial
chromosome (YAC), yeast 2 micron plasmid,
retrovirus, baculovirus vector……

21. Choice of vector
• Depends on nature of protocol or experiment
• Type of host cell to accommodate rDNA
– Prokaryotic
– Eukaryotic

22. 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)

23. The Tools of Recombinant DNA
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

24. Genome
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.

25. 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

26. 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

27. Steps involved in isolating a particular
DNA fragment from a complex
mixture of DNA fragments or
molecules
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 #)

28. Steps involved in isolating a particular
DNA fragment from a complex
mixture of DNA fragments or
molecules
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

29. Steps involved in isolating a particular
DNA fragment from a complex
mixture of DNA fragments or
molecules
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

30. Pharmaceutical
Biotechnology
Anas Bahnassi PhD RPh
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