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Recombinant DNA Technology
Recombinant DNA Technology
• The Recombinant DNA technique was engineered by Stanley
Norman Cohen and Herbert Boyer in 1973
• They published their findings in a 1974 paper entitled
"Construction of Biologically Functional Bacterial Plasmids in
vitro”
– Which described a technique to isolate and amplify genes or DNA
segments and insert them into another cell with precision, creating a
transgenic bacterium
• Recombinant DNA technology was made possible by the
discovery of restriction endonucleases by Werner Arber,
Daniel Nathans, and Hamilton Smith
– For which they received the 1978 Nobel Prize in Medicine
Recombinant DNA technology
– a set of techniques for combining genes from different
sources
• recombinant DNA,, is made by connecting or
recombining, fragments of DNA from different sources (
often different species )
• These methods form part of genetic engineering, the
direct manipulation of genes for practical purposes
(Process of altering the genetic material of cells or
organisms to make new substances)
• DNA technology has launched a revolution in
biotechnology, the manipulation of organisms or
their components to make useful products
Uses for Recombinant DNA
Recombinant DNA technology is not only an important tool in scientific
research, but it has also impacted the diagnosis and treatment of diseases
and genetic disorders in many areas of medicine.
It has enabled many advances, including:
• Isolation of large quantities of pure protein
• Diagnosis of affected and carrier states for
hereditary diseases
• Transferring of genes from one organism to another
• Mapping of human genes on chromosomes
• Identification of mutations
– People may be tested for the presence of mutated proteins that may be
associated with breast cancer, retino-blastoma, and neurofibromatosis
Tools & Techniques of genetic
engineering
• enzymes for dicing, splicing, & reversing
nucleic acids
• analysis of DNA
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Enzymes for dicing, splicing, & reversing
nucleic acids
1. restriction endonucleases – recognize
specific sequences of DNA & break
phosphodiester bonds
2. ligase – rejoins phosphate-sugar bonds cut
by endonucleases (phosphodiester bonds)
3. reverse transcriptase – makes a DNA copy of
RNA - cDNA
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Some enzymes used in recombinant
DNA technology
Analysis of DNA
• gel electrophoresis- separates DNA fragments based
on size
• nucleic acid hybridization & probes – probes base
pair with complementary sequences; used to detect
specific sequences
• DNA Sequencing – reading the sequence of
nucleotides in a stretch of DNA
• Polymerase Chain Reaction – way to amplify DNA
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DNA Amplification
•
DNA amplification is based on two different
techniques :
a- Cell-based DNA cloning(DNA cloning)
involving a vector/replicon and a suitable
host cell .
b- in vitro DNA cloning (PCR)
DNA Cloning
(cell-based cloning)
The goal of molecular cloning is large amounts of pure DNA
that can be further manipulated and studied.
A clone is an identical copy. This term originally applied
to cells of a single type, isolated and allowed to reproduce
to create a population of identical cells.
•
Principles of cell-based cloning:
Four steps in cell-based cloning
- Construction of recombinant DNA molecules. Involves the use of
endonuclease restriction enzymes, ligation, and a replicon (vector).
- Transformation in appropriate host cells and cloning .
- Selective propagation of cell clones. This step takes advantage of
selectable markers.
- Isolation of recombinant DNA from cell clones followed by molecular
characterization .
Major Steps in the Cloning of DNA
• Fragmentation of DNA :
– Appropriate restriction endonucleases is used to ensure
that the gene fragment in question is excised completely
from the source of DNA .
• Construction of recombinant DNA ( rDNA)molecule :
• · The target gene fragment is ligated to a DNA vector
(i.e. a plasmid), making a recombinant DNA molecule
• · The DNA recombinant molecule replicates itself
autonomously
• Transformation of recombinant DNA into the host
cell .
• Cell cloning .
Major Steps in the Cloning of DNA
• The selection of the successfully transformed cells
-Isolation of successfully transformed bacterial cells with the
recombinant DNA using a marker, such as antibiotic resistance
genes
-If colonies grow, despite the existence of such antibiotics,
then the recombinant DNA vector was successfully
transformed
• The surviving colonies are isolated and are grown in culture to
produce multiple copies of the incorporated recombinant
DNA
• Isolation of recombinant DNA from cell clones followed by
molecular characterization
Fragmentation of DNA
Restriction Enzymes
• Restriction enzymes are Bacterial origin = enzymes that cleave foreign
DNA
• classified as endonucleases. Their biochemical activity is the hydrolysis
("digestion") of the phosphodiester backbone at specific sites in a DNA
sequence. By "specific" it means that an enzyme will only digest a DNA
molecule after locating a particular sequence.
• All restriction enzymes cut DNA between the 3’ carbon and the phosphate
moiety of the phosphodiester bond.
• The term restriction comes from the fact that these enzymes were
discovered in E. coli strains that appeared to be restricting the infection by
certain bacteriophages
• Over 400 enzymes identified, many available commercially from
biotechnology companies
• Names typically begin with 3 italicized letters ex. EcoRI source E. coli RY13 ,
BamHI source Bacillus amyloliquefaciens H
Restriction Enzymes
Origin and function
• Bacterial origin = enzymes
that cleave foreign DNA
• Protect bacteria from
bacteriophage infection
– Restricts viral replication
– However, certain bacteriophages
have evolved to use methylation
as a way to avoid digestion
by restriction enzymes
• Bacterium protects it’s
own DNA by methylating
those specific sequence motifs
Restriction Enzymes
• Restriction enzymes bind to, recognize, and cut (digest) DNA
within specific sequences of bases called a recognition
sequence or restriction site
• These recognition sequences are palindromes
( arrangement
a
of nucleotides reads the same forwards and
backwards on opposite strands of the DNA molecule )
• They typically recognize restriction sites with a sequence of
four or six nucleotides Eight-base pair cutters have also been
identified
• They produce either Blunt Ends or Staggered ends:
Staggered ends
Blunt Ends
Staggered ends
Blunt Ends
Restriction Enzymes
• Enzymes that produce cohesive ends are often favored over
blunt-end cutters for many cloning experiments
• But blunt ends can be converted to sticky ends by linkers .
– DNA fragments with cohesive ends can easily be joined
together .
• In the simplest sense, the discovery of restriction enzymes
provided molecular biologists with the "scissors" needed to
carry out gene cloning
linkers
• Blunt ends can be converted to sticky ends.
• A short double-stranded molecules called
linkers or adaptors are attached to the blunt
ends. Linkers and adaptors work in slightly
different ways but both contain a recognition
sequence for a restriction endonuclease and
so produce a sticky end after treatment with
the appropriate enzyme
LINKER
Another way to create a sticky end
• Homopolymer tailing
- Nucleotides are added one after the other to
the 3′ terminus at a blunt end .
• The enzyme involved is called terminal
deoxynucleotidyl transferase .
End-modification enzymes
• Terminal deoxynucleotidyl transferase from calf
thymus tissue, is one example of an end-modification
enzyme.
• It is a template-independent DNA polymerase
because it is able to synthesize a new DNA
polynucleotide without base-pairing of the incoming
nucleotides to an existing strand of DNA or RNA. Its
main role in recombinant DNA technology is in
homopolymer tailing
• Two other end-modification enzymes are also
frequently used , alkaline phosphatase and T4
polynucleotide .
Restriction Enzymes
Classes of Restriction Enzymes
Type I
Type II
Type III
cleavage occurs 400-7000 bp
from recognition site
cleavage occurs adjacent or
within recognition site
cleavage occurs 25-27 bp
from recognition site
• type II enzymes are powerful tools
in molecular biology
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
Type III (similar to type I , few examples )
- Recognition sequence: 5-7 bp
- Cleavage site: 25-27 bp downstream of recognition site
• 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
• Reads the same in the 5’ 3’ direction on both strands =
Palindromic Sequence
Some Examples of Restriction
Endonucleases
Vectors
• A vector is a DNA molecule into which foreign fragments of DNA is
inserted
• A vector functions like a “molecular carrier”
– Which will carry fragments of DNA into a host cell
– Vector DNA functions to insert and amplify the DNA of intersite.
• Vectors should contain an origin of replication
– Enables the vector, together with the foreign DNA fragment
inserted into it, to replicate
• they contain one or more single (unique) restriction endonuclease
sites that provide a choice of possible insertion (cloning) sites
• vectors have one or more genes (selectable markers) that enable
host cells with DNA constructs to be distinguished from cells that
either do not carry a DNA construct or carry a cloning vector
without an insert.
• Small size in comparison with host’s chromosomes
Cloning Vectors
•
•
•
•
•
Plasmids
Phage
Cosmids
BACs
YACs
Main types of vectors
Choice of vector
• Depends on nature of protocol or experiment
• Length of the DNA molecule .
• Type of host cell to accommodate rDNA
– Eukaryotic
- Prokaryotic
• Some vectors contain inducible or tissue-specific
promoters permitting controlled expression of
introduced genes in transfected cells or transgenic
animals.
Plasmid vector
• Closed, circular, double stranded DNA molecules that occur
naturally and replicate extrachromosomally in bacteria
• a multiple cloning site or MCS
• Many confer drug resistance to bacterial strains
• Origin of replication present (ORI)
• pBR322 is the basis
of most engineered plasmids
Plasmid vector
Another example of a typical E. coli cloning vector is pUC19 (2,686-bp). The
pUC19 plasmid features:
a. High copy number in E. coli, with nearly a hundred copies per cell,
provides a good yield of cloned DNA.
b. Its selectable marker is ampR.
c. It has a cluster of unique restriction sites, called the polylinker
(multiple cloning site).
d. The polylinker is part of the lacZ (β-galactosidase) gene. The pUC19
plasmid will complement a lacZ- E. coli, allowing it to become lacZ+.
When DNA is cloned into the polylinker, lacZ is disrupted, preventing
complementation from occurring.
e. X-gal, a chromogenic analog of lactose, turns blue whenβgalactosidase is present, and remains white in its absence, so bluewhite screening can indicate which colonies contain recombinant
plasmids
The plasmid cloning vector pUC19
Chapter 7 slide 33
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Plasmid vector
• Artificial plasmids have been created that possess
a unique region that can be cut by many
restriction enzymes
• The recognition sites of many restriction enzymes
have been positioned very close together in this
one area and are not found anywhere else on the
plasmid’s DNA sequence – the site is called the
multiple-cloning site
• The recognition site exists in only one area of the
plasmid which means that the DNA can only be
cut at one location
Bacteriophage Vectors
Lambda vector
• Bacteriophage lambda (λ) infects E. coli
• Double-stranded, linear DNA vector – suitable for library
construction
• At each end of the λ chromosome are 12 nucleotide sequences
called cohesive sites (COS) that can base pair with each other
- When λ infects E. coli as a host, the λ chromosome uses these COS sites to
circularize and then replicate
• Can accommodate large segments of foreign DNA
• Central 1/3 = “stuffer” fragment
– Can be substituted with any DNA fragment of similar size without affecting
ability of lambda to package itself and infect E. coli
– Accommodates ~15kbp of foreign DNA
– Foreign DNA is ligated to Left and Right Arms of lambda Then either:
• 1) Transfected into E. coli as naked DNA, or
• 2) Packaged in vitro by combining with phage protein components
(heads and tails) (more efficient, but labor intensive and
expensive)
using phage λ DNA as a cloning vector
Chapter 7 slide 37
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Cosmid cloning vectors
1. Cosmids are constructed vector with features from
both plasmids and phages. These features include:
a.
b.
c.
d.
An E. coli ori sequence.
A selectable marker such as ampR
Convenient restriction sites.
A phage cos site, allowing the DNA to be pakaged into a phage
head for introduction into E. coli.
2. Cosmid as small as 5kb are available, and 32-47kb of
DNA can be inserted into them. Recombinant cosmids
< 37kb or > 52 kb cannot be packaged, don’t enter E.
coli, and therefore are not replicated.
Cosmid cloning vector
Yeast Artificial Chromosomes (YACs)
• Bacterial plasmids are not good vectors for yeast
hosts because prokaryotic and eukaryotic DNA
sequences use different origins of replication.
• A yeast artificial chromosome (YAC), has been
made that has a yeast origin of replication, a
centromere sequence, and telomeres, making it a
true eukaryotic chromosome.
• YACs have been engineered to include specialized
single restriction sites and selectable markers.
• YACs can accommodate up to 1.5 million base
pairs of inserted DNA.
YACs
• Yeast artificial
chromosomes
• Have centromere,
telomeres and an
origin of replication,
plus selectable
markers
Yeast Artificial Chromosome
Example of a yeast artificial chromosome (YAC) cloning vector. A YAC vector contains a yeast
telomere (TEL) at each end, a yeast centromere sequence (CEN), a yeast selectable marker
for each arm (here, TRP1 and URA3), a sequence that allows autonomous replication in
yeast (ARS), and restriction sites for cloning.
Bacterial Artificial Chromosomes (BACs)
1. BACs are used for cloning fragments up to about 200 kb
in E .coli. BAC vectors contain:
a. the ori of an E. coli plasmid called the F factor.
b. A multiple cloning sites.
c. A selectable marker.
d. Other features
2. BAC can be handles like regular bacterial plasmids, but
the F factor ori keeps copy number at one BAC molecule
per cell.
Expression Vectors
• Protein expression vectors allow for the high -level synthesis
(expression) of eukaryotic proteins within bacterial cells
– They contain a prokaryotic promoter sequence adjacent to the site
where DNA is inserted into the plasmid
• Bacterial RNA polymerase can bind to the promoter and
synthesize large amounts of RNA (for the insert)
– Which is then translated into protein
• Protein may then be isolated using biochemical techniques
Ti Vectors
• Ti vectors are naturally occurring plasmids (around
200 kb in size)
– Isolated from the bacterium Agrobaderium tumefaciens
• Which is a soilborne plant pathogen that causes a condition in
plants called crown gall disease
• When A. tumefaciens enters host plants, a piece of
DNA (T-DNA) from the Ti plasmid (Ti stands for
tumor-inducing) inserts into the host chromosome
Ti Vectors
• T-DNA encodes for the synthesis of a hormone called
auxin, which weakens the host cell wall
– Infected plant cells divide and enlarge to form a tumor
(gall)
• Plant geneticists recognized that if they could
remove auxin and other detrimental genes from the
Ti plasmid, the resulting vector could be used to
deliver genes into plant cells
• Ti vectors are widely used to transfer genes into
plants
Shuttle Vectors
1.A cloning vector capable of replicating in two
or more types of organism (e.g., E. coli and
yeast) is called a shuttle vector. Shuttle vectors
may replicate autonomously in both hosts, or
integrate into the host genome
Properties of Good Vector
1. It should be able to
replicate autonomously.
2. It should be easy to
isolate and purify.
3. It should be easily
introduced into the host
cells.
4. The vector should have
suitable marker genes that
allow easy detection and/or
selection of the
transformed host cells.
5. For gene transfer, it should
integrate itself or the DNA
insert in it into the genome
of the host cell.
7. A vector should contain
unique target sites for as
may restriction enzymes
8. For expression of the
DNA insert, the vector
should contain suitable
control elements, e.g.,
promoter, operator and
ribosome binding sites.
Construction of recombinant DNA
( rDNA)molecule
• As the DNA of interest and the vector are digested by the
same restriction enzyme ,they have a complementary ends .
• The target DNA fragment is ligated to a DNA vector (i.e. a
plasmid) by ligase enzyme, making a recombinant DNA
molecule
Transformation
• Recombinant DNA molecules are transferred
into appropriate host cells (e.g. bacteria) for
propagation. Normally a single recombinant
DNA exists per cell but sometimes cotransformation may result in two or more
recombinant DNA molecules per host cell.
Characteristics of cloning hosts
1. Rapid overturn, fast growth rate
2. Can be grown in large quantities using ordinary
culture methods
3. Nonpathogenic
4. Capable of accepting plasmid or bacteriophage
vectors
5. Maintains foreign genes through multiple generations
6. Will secrete a high yield of proteins from expressed
foreign genes
Transformation
• Transformation
– Process for inserting foreign DNA into bacteria
– the bacteria that has accepted a foreign plasmid is referred to as
being transformed .
• I Cells (generally E. coli) and plasmid DNA are incubated
together at 0 C in a calcium chloride solution then the
mixture is subjected to a shock by rapidly shifting the
temperature to 43 C.
Result : Plasmid DNA entered bacterial cells
• Once inside bacteria, plasmids replicate and express their
genes
Transformation
• II
Electroporation :
– Involves applying a brief (millisecond) pulse of high-voltage electricity
to create tiny holes in the bacterial cell wall that allow DNA to enter
– Can be used to introduce DNA into mammalian cells and to transform
plant cells
• Ligation of DNA fragments and transformation by any method are some
what inefficient
• During ligation, some of the digested plasmid will ligate back to itself to
create a recircularized plasmid that lacks foreign DNA
• During transformation, a majority of cells will not take up DNA
Selection
• How can recombinant bacteria (those transformed with a
recombinant plasmid) be distinguished from a large number
of non transformed bacteria and bacterial cells that contain
plasmid DNA without foreign DNA?
– Screening process is called selection
• Designed to facilitate the identification of (selecting for)
recombinant bacteria
• Example "blue
blue-white" screening by using the plasmid
cloning vector pUC19 . ( Fig )
Practical Features of DNA Cloning Vectors
•
Transformed bacteria are plated on agar
plates that contain a chromogenic (colorproducing) substrate for β-gal called X-gal
– (5-bromo-4-chloro-3-indolylβ-[5]Dgalactopyranoside)
•
X-gal is similar to lactose in structure and
turns blue when cleaved by β-gal
•
As a result, non-recombinant bacteria, which
contain a func-tionallac z gene, produce β-gal
and turn blue
•
Conversely, recombinant bacteria are
identified as white colonies
•
Because these cells contain plasmid with
foreign DNA inserted into the lac z gene, β-gal
is not produced, and these cells cannot
metabolize X-gal.
Recombinant selection with pUC8
. •
Selection by Antibiotic Marker
Selection by Antibiotic Marker
Recombinant selection with pBR322