PHD Course: Topics in (Nano) Biotechnology

PhD Course
TOPICS IN (NANO)
BIOTECHNOLOGY
Lecture 6
30th October, 2006
Overview
• So we have looked at what is DNA and what is

a gene.
• We also looked at DNA replication and protein
synthesis, and the path from the gene to
protein
• We know what proteins are – and especially
about two specific sets of proteins – enzymes
and antibodies
• This week we will look at Recombinant DNA
technology
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
Discussion of rDNA technology
• Enzymes for Manipulating DNA.

• Vectors for gene cloning.
• Cloning of rDNA.
• Characterization of Cloned Genes
• Construction of Genomic libraries.
• Applications of the rDNA technology
History of Recombinant DNA technology
• In
Antibiotics
just a few
different such
plasmids,
years
as 60-80%
penicillin,
the replication
of the
bacteriaregion
showed
sulfonamides
resistance
encodes traits
notand
not
just streptomycin
essential
to one drug,to the
but
gave
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hope
drugs
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• Antiobiotic
However,
The genesinresistance
responsible
the 50s theye
isfor
onestarting
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of theseto drug
fight
traits.
back, becoming
resistance were increasingly
plasmids, genetic
resistant
elements
to
antibiotics
that could replicate themselves independently.
History of Recombinant DNA technology
In 1971 Cohen, exploited the antibiotic
resistance of the plasmids to
selectively enrich offspring that
contained cell propogating plasmids.
In the late 60s, it was shown that CaCl2 made the cells of
E.coli permeable so that they could take up DNA, but
could not grow E.coli cells with genetic property changes.
In late 1972, Berg reported on methods
for joining fragments of DNA outside of
cells using ligases.
Endonucleases, or restrictions
enzymes, would however, provide the
tool for linking DNA.
Una cerveza y ...
In Nov. 1972, Berg,
Boyer and Cohen met
up at a deli bar in
Honololu, and discussed
the endonuclease that
Boyer was working on,
and that night they
dreamed of the collaborative project that would be the true

start of recombinant DNA technology. In March 1973, the
pair produced DNA fragments using Boyer’s technique and
joined them to plasmids using Berg’s technique, and then
introduced them into bacteria using Cohen’s technique.
The first demonstration of DNA cloning had been
achieved.
But let’s look at it in more
detail....
Recombinant DNA technology
• The two essential elements of recombinant

DNA technology are:
1. Restriction endonucleases
2. Vectors for gene cloning
Restriction endonucleases
What is a restriction enzyme?
• There are two classes of restriction enzymes:
• Type I
• Cuts DNA on both strands but at non-
specific location
• Random imprecise cuts
• Not very useful for rDNA applications
• Type II
• Cuts both strands of DNA within the
particular sequence recognised by the
restriction enzyme
• Restriction enzymes (or endonucleases) are
bacterial enzymes that cut DNA at very
specific sequences
• They generally cut in a ‘staggered’ manner,

leaving sticky ends but some enzymes
generate blunt ends (i.e. Cut DNA in the
middle)
• Their biological function is to destroy invading

foreign DNA
• Each bacteria has different restriction
enzymes
• Enzymes from E.coli cells cut GAATTC/CTTAAG

• Enzymes from B. Amyloloquefaciens cut
GGATCC/CCTAGG
• The restriction enzymes are named after the

organism from which they were derived
• EcoRI from E.coli

• BamHI from B. Amyloloquefaciens
• Restriction enzymes are used to make
recombinant DNA and gene cloning and
genetic engineering were made possible by
these enzymes
• Over 200 different restriction enzymes are

commercially available (some are VERY
expensive)
• DNA ligase ‘sticks’ the ends back together

• Recombinant DNA technology can be used to
isolate a genomic clone from DNA or for the
isolation of human cDNA
• Isolating a genomic clone provides a piece of

DNA identical in base sequence to the
corresponding stretch of DNA in the cell and
is often designed to contain a specific gene
• Isolating human cDNA is used for gene

expression. Human cDNA
(c=complementary) is double stranded DNA
copy of mRNA but WITHOUT introns
Vectors for gene cloning
Vector requirements
• Dependent on design of experimental system
• Most vectors contain a prokaryotic origin of
replication
• Can replicate along with the host cell
• Autonomously
• By integration in the chromosome

• Antibiotic resistance genes and/or other
selectable markers
• Contain one or more unique sites for insertion
of foreign DNA
Vector types
• Plasmids (upto about 20kb insert)
• Bacteriophage
  vectors
Can insert fragments of DNA up to 25 kb.
Can introduce into cells at a very high efficiency
(10-100kb depending on the type ofbacteriopage)
• Cosmid(35-45kb)
• Combination of bacteriophage and plasmid
• BAC vectors (bacterial artificial chromosomes)

• Contain sequences from the E. coli F plasmid –
present at one copy per cell. Can clone up to 200 kb
per BAC clone
What is a plasmid?
• Plasmids are small, extrachromosomal pieces
of bacterial DNA that are often antibiotic
resistant
• They are ‘shuttle vectors’
to create, produce, and maintain
recombinant DNA
• An example of one of the first
plasmids is pBR322
• Both Amp & Tet resistant, Several unique restriction
sites
• pUC18 now the most commonly used
• Derivative of pBR322
• Smaller, Higher copy number per cell, Multiple
cloning sites
What is a plasmid?
lacZ gene
• Gene encoding for enzyme -galactosidase
• Polylinker resides in the middle
• Enzyme activity can be measured as marker of

gene insertion
• Disrupted gene – nonfunctional – WHITE
• Intact gene – functional – BLUE
• Amp resistance gene still present, Tet

resisitance gene omitted
What is a bacteriophage?
Lambda vector
• Bacteriophage lambda () infects E.coli
• Double stranded linear DNA vector, suitable for

library construction
• Can accomodate large segments of foreign

DNA, central 1/3 is a ‘stuffer’ fragment
• Can be substituted with any DNA fragment of similar
size
• Can accomodate  15kbp of foreign DNA
Lambda vector
Vectors for eukaryotic cells
• Shuttle vectors
- Hybrid molecules designed for use in multiple cell types
- Multiple ORIs allow replication in both prokaryotic and eukaryotic
host cells allowing transfer between different cell types
• YAC vectors (Yeast artificial chromosomes)

- Contains sequences required to replicate and maintain
chromosome in budding
- yeast (like , end up as a linear molecules)
- a yeast origin of replication, a centromere, and a telomere at each
end.
- Can clone >2,000 kb (2 Mb).
- Agrobacterium Tumefaciens
Plants
- Baculovirus
Insect cells
Comparison of different vectors
Getting Recombinant DNA into cells
Procedure for cloning DNA in a plasmid
vector
Insertion into a Plasmid can be Detected by
Disruption of -gal
• Only bacteria which have
taken up plasmid grow on
ampicillin.
• Blue-white selection:
– white colonies have insert
– blue colonies have no
insert
• To see blue color, add IPTG

(an inducer of -galactosidase
expression) and Xgal
substrate.
• Class 6 Video 1
• Class 6 Video 2

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PhD Course

• So we have looked at what is DNA and what is

• DNA fragments are usually derived from

• Enzymes for Manipulating DNA.

dreamed of the collaborative project that would be the true

• The two essential elements of recombinant

• They generally cut in a ‘staggered’ manner,

• Their biological function is to destroy invading

• Enzymes from E.coli cells cut GAATTC/CTTAAG

• The restriction enzymes are named after the

• EcoRI from E.coli

• Over 200 different restriction enzymes are

• DNA ligase ‘sticks’ the ends back together

• Isolating a genomic clone provides a piece of

• Isolating human cDNA is used for gene

• By integration in the chromosome

• BAC vectors (bacterial artificial chromosomes)

• Polylinker resides in the middle

• Enzyme activity can be measured as marker of

• Amp resistance gene still present, Tet

• Double stranded linear DNA vector, suitable for

• Can accomodate large segments of foreign

• YAC vectors (Yeast artificial chromosomes)

• To see blue color, add IPTG

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