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Recombinant DNA Technology



Tortora et al
, Chap
9


1

Biotechnology and Recombinant DNA




Development of this technology was initiated in the early 1970s following the
discovery of restriction enzymes.



In 1972 Jackson, Symons and Berg generated recombinant DNA molecules



Plasmid vectors that carry foreign
DNA fragments were developed in 1973



Southern developed the Southern blot procedure in 1975



Stanley Cohen and Herbert Boyer patent filed in 1978 entitled “Biologically
Functional Molecular Chimeras”


This technology has lead to the development of a number
of important tools for the study
of life (e.g., Gene cloning (recombination), Hybridization analysis, Nucleotide sequence
analysis, mutagenesis, gene expression, DNA fingerprinting).


This technology relies on several basic principles



DNA as the hereditary

material of all living cells
-

Conserved mechanisms for DNA
replication



Universality of the Genetic Code


What does this technology allow us to do and how does it do it?




Biotechnology



"The industrial use of living organisms or their components to impro
ve human health
and food production" (Campbell et al., 1999)



"Those processes in which living organisms are manipulated, particularly at the
molecular genetic level, to form useful products" (Prescott et al., 2008)



“the use of microorganisms, cells or cel
l components to make a product.” (Tortora et
al., 2010)



I.
Tools
of Biotechnology


1.

Selection



Humans use artificial selection to select desire breeds of animals or strains of plants
to cultivate.



Similarly, microbiologists can select specific strains
of bacteria or fungi that produce
a desired product (e.g., antibiotic, enzyme, fermentation end
-
product)


2.

Mutation



Biologists use random mutagenesis to create new strains of a particular
microorganism with enhanced
characteristics

(e.g., increased penic
illin production)



Site directed mutagenesis can be used to introduce specific changes into a particular
gene



Recombinant DNA Technology



Tortora et al
, Chap
9


2



Repeated rounds of mutagenesis and selection ha
ve

been used to
produce novel
industrial strains with improved characteristics (e.g., production of
an antibiotic,
enzyme, amino acid,…)


3.

Restriction Enzymes


How do prokaryotic cells deal with foreign DNA?


One mechanism common to prokaryotic cells is the production of restriction
endonucleases (restriction enzymes). Discovered by Arber and Smith in
the late 1960s.




Most common kind of restriction enzymes
-

type II enzymes that recognize very
specific DNA sequences and cause double stranded breaks in the DNA.



>3000 restriction enzymes have been identified from >200 species.



Most of these recognition s
ites show a two
-
fold symmetry around a given point
(palindrome
-

reads the same from the left or right).



Recognition sequence length varies
-

4, 6, 8… cutters.



Named after the producing organisms (
Sau
3AI is from
S
taphylococcus
au
reus
)

Sau
3AI


GATC


Bam
HI


G

GATCC


Pst
I


CTGCA

G


Not
I


GC

GGCCGC




Restriction produces overhanging (sticky ends) or blunt ends


Eco
RI


G

AATTC



Sma
I


CCC

GGG




Modification enzymes

Host must protect its own DNA


modifying enzymes or methylases


Methylases


methylate (
-
CH
3
)
specific bases within the restriction enzyme recognition
site (usually A or C)






CH
3






|

Bam
HI methylase


GGATCC





Recombinant DNA Technology



Tortora et al
, Chap
9


3


Restriction enzymes and methylases are part of
restriction
-
modification system

(RMS)




One cell type may contain more than one
RMS.



Different strains of the same species may contain different RMS



Restriction enzymes have been extensively characterized and are an integral part of
recombinant DNA technology.



Used to characterize DNA
-

restriction enzyme analysis (physical map). W
hen cut by
a restriction enzyme, any particular fragment will yield a limited number of pieces of
DNA. These pieces of DNA can be resolved by gel electrophoresis.




Used in cloning of DNA fragments



Web tools can be used to determine restriction sites in
known nucleotide sequences (e.g.
NEBcutter at
www.neb
.com
)



4
.

Hybridization

(Fig 9.16)



Recall that dsDNA can be denatured into two complementary strands and the ssDNA
molecules can form
hybrids

with other complementary DNA
or RNA molecules



Small ssDNA molecules can be labeled (i.e., with isotopes
35
P and
32
S, fluorescent
dye molecules…) and used to detect the presence of complementary sequences.
These small labeled ssDNA molecules are called probes.


Southern blot



Northern blot





5
.

Sequencing and synthesis of DNA


i) Nucleotide sequence analysis



Two methods were developed that, using a specific fragment of DNA, generate 4
pools of labeled random DNA fragments (i.e., radioisotopes); each pool contains
fragments o
f DNA ending at only one of the four bases. The pools of DNA
fragments are resolved by gel electrophoresis and the sequence is read directly from
the gels.


a) Maxim and Gilbert technique creates the pools of fragments chemically and is not
used anymore



Recombinant DNA Technology



Tortora et al
, Chap
9


4


b) Sanger dideoxy method creates the pools enzymatically (i.e., uses DNA
polymerase) and has been adapted to automated sequencing methods. A copy of the
DNA fragment is made us
ing DNA polymerase
.



A DNA primer is used to initiate synthesis (i.e., the sequ
encing reaction).



The polymerase reaction generates random fragments of DNA with known
nucleotides at the ends, by incorporating dideoxy analogs of the dNTPs used as
substrates.



The dideoxy analogs (ddATP, ddCTP, ddGTP, dTTP) are chain termination
reagen
ts and separate reactions are set up for each ddNTP. Thus each reaction
generates pools of fragments ending at a particular base.



Fragments of varying lengths are obtained



The pools of fragments are separated by electrophoresis. One lane for each
react
ion. The fragments have been labeled (e.g., isotope or fluorescent dyes for
automated sequencing) for detection and reading of sequence.





ii) DNA synthesis



Automated DNA synthesizers can make ssDNA oligonucleotides several to 100 nt in
length









6
.

Polymerase Chain reaction



in vitro

replication

of define sequences of
DNA



Developed by Kary Mullis (1984; Nobel prize in 1992)


Repeated cycling of the following steps in an automated thermal cycler

i) Denaturation (94


98

C)

ii) Annealing (temperature

depends upon t
m

of primers)

iii) Extension (elongation
-

usually at 72

C)


Ingredients for PCR reaction



Buffer containing Mg
++



Enzyme

Taq

DNA polymerase



Substrate

Template




Primers



Nucleotides




Recombinant DNA Technology



Tortora et al
, Chap
9


5







Real
-
time PCR



Newly synthesized DNA is tagged with fluorescent dye and the levels
of fluorescence are measured by the real
-
time instrument


quantitative PCR



7
.

Gene Cloning


What is gene cloning?


Why clone a gene?


A basic concept in recombinant DNA technology is

that of gene cloning. This involves
in vitro

recombination followed by replication of recombinant DNA. We need some way
of reproducing these hybrid molecules in such a way as we can produce enough of them
to study.



Steps involved in cloning a gene


i.
Extraction of DNA or nucleic acids of interest



Both DNA and RNA may be used


RNA must first be converted to DNA through the
use of reverse transcriptase to form complementary DNA (cDNA or copy DNA).


Recall

that eukaryotic organisms may have introns

in their genes. This complicates
the cloning of complete gene sequences. Post
-
transcriptional modification removes
introns and produces the mature mRNA that is translated to produce the polypeptide


How do we distinguish between the different types of R
NA?

Just want mRNA


recall Poly A tail




DNA may come from a variety of sources, including genomic DNA, cDNA or PCR
products

or it may be synthesized. Synthetic genes may be made nowadays for as
little as $0.75/bp




Cloning may involve introducing a single

gene fragment into a suitable cloning
vector. It may be more complicated and involve the creation of a genomic library. A
genomic library

is a collection of clones that is large enough to ensure that at least
one clone exists for every gene in the organi
sm.






Recombinant DNA Technology



Tortora et al
, Chap
9


6

ii. Insertion of DNA fragments into a suitable
cloning vector
. This is often accomplished
in the following two steps.


a)
Restriction digestion using restriction endonucleases


o

sticky ends
Eco
RI G|AATTC

-

easier to clone with but may not always ha
ve a
useful site in the appropriate location

o

blunt ends
Sma
I CCC|GGG

-

do not have to have compatible ends but more
difficult to clone with


b) Ligation


T4 DNA ligase joins the 3’
-
OH to the 5’
-
PO
4

reforming the
phosphodiester bonds




Cloning vector



“vehicle” for carrying and replicating introduced DNA fragments.


A variety of
v
ectors

may be used including



plasmid


most common cloning vectors



phage/viruses



Cosmid



BACs


bacterial artificial chromosomes/YACs


yeast artificial chromosomes


linear

plasmids



Vector features

1.

Origin of replication (
ori
) and other replication functions for stable maintenance in
host cells
.
Shuttle vector
: can replicate in several different species (e.g.,
E. coli

and
Saccharomyces cerevisiae



2.

S
electable markers


to identify cells carrying the vector



Ampicillin resistance (
bla
) is the most common selectable marker on plasmid
vectors



Other antibiotic resistance genes have been used as well as auxotrophic
markers


3.

cloning

sites


unique restriction sites


multiple cloning sites (MCS). Vectors
often have features that allow one to identify recombinant vectors by a process
known as insertional inactivation





4.

Most cloning vectors are also small in size. For plasmids,

phage vectors and
cosmids, there are limits on how large a fragment can be cloned.





Recombinant DNA Technology



Tortora et al
, Chap
9


7

iii. Introduction of recombinant molecules into host cells



Escherichia coli

and
Saccharomyces cerevisiae

are the most common prokaryotic and
eukaryotic cloning hosts



Tra
nsformation is the most common method for introducing foreign DNA into host
cells
. One can also use conjugal and viral systems.


Transformation




uptake of naked DNA directly from the environment



DNase sensitive



DNA is originally derived from donor cell and

taken up by recipient which is then
called the transformant



Cornerstone technique of molecular biology




Most bacterial cells will not take up DNA efficiently unless they are exposed to
special chemical or electrical treatments. However naturally transfor
mable bacteria
can take up DNA from their environment without special treatment


a)
Natural competence



The naturally transformable bacteria take up DNA when they are in the state of
natural competence



A number of Gram positive and Gram negative bacteria
are capable of natural
competence


Examples

Bacillus subtilis

Haemophilus influenzae

Streptococcus pneumoniae

(Griffith
-

1928 and Avery, McCarty, MacLeod, 1944)




Most naturally transformable bacteria can take up DNA only late in their growth
cycle
-

usual
ly stationary phase




A number of genes are involved;
B. subtilis

-

com

genes


b)
Artificially induced competence



Most bacteria are not naturally transformable, al least not at easily detected levels.



However this does not preclude them from transformatio
n



They can be artificially induced to a state of competence


Methods of Induced Competence


Chemical induction



CaCl
2

-

E. coli



Polyethylene glycol
-

Bacillus thuringiensis


Electroporation



Recombinant DNA Technology



Tortora et al
, Chap
9


8



formation

of transient membrane pores through the use of high voltage fields



protocols for many prokaryotic and eukaryotic organisms

Biolistic ap
proach


i.e., gene gun




Microscopic particles of gold or tungsten are coated with DNA and propelled
by a blast of heliu
m into cells



protocols for many eukaryotic organisms


Microinjection



Small drawn glass micropipette are used to inject DNA solutions through the
plasma membrane of animals cells


Agrobacterium tumefaciens

mediated plant transformation



This bacterium naturally infects plants and introduces foreign DNA into the
plant cell nucleus resulting in neoplastic growth
(produces a plant gall


i.e.,
tumour)
and the abnormal production of amino acid derivatives known as
opines. The bacterium

is cap
able of metabolizing opines as a source of carbon
and nitrogen.



The genetic information necessary for the transfer of the bacterial DNA into
the plant cell is encoded on the Ti (tumour inducing) plasmid. Only a small
portion of the Ti plasmid (T
-
DNA) is t
ransferred into the plant cell.



This system has been well characterized and is now used to introduce foreign
DNA into plants as well as some animal cells.



iv. Screening or Detection of Recombinant Molecules



May be creating a scenario not much different
than the proverbial “needle in the
haystack”. This technology is only useful if you can recover the desired molecules.
If you have made a gene library (collection of all genes in an organism) you want
some way to identify the genes that you are after.




Gene Probes
-

use one gene sequence to probe a library for similar sequences



Expression of the gene product
-

phenotype
-

e.g., enzymes



PCR amplification



Reporter genes
-

encode easily detectable traits such as an enzyme activity.
Promoters can be cloned

using this technology


v. Gene Expression



It is possible to use the above techniques to introduce foreign genes into almost any
organism. It is also possible to introduce foreign gene constructs that are expressed
by the host, thereby making a desired g
ene product.



Expression vectors are specialized vectors that have been designed to promote gene
expression in a particular host. These vectors contain the appropriate regulatory
sequences (e.g., promoter, operator, terminators) that can be used to drive e
xpression
of a coding sequence of interest.





Recombinant DNA Technology



Tortora et al
, Chap
9


9


II.
Application of Recombinant DNA Technology


Genetic analysis


Gene Mapping


Gene Cloning


Systematics


Genome
Sequencing
Projects


Gene Cloning


Fingerprinting


Mutagensis

random




directed




site
-
directed


Gene silencing


RNA interference (RNAi)


small interfering RNAs (siRNA) are
introduced into a cell where the siRNA bind to mRNA causing the enzymatic
destruction of the mRNA thereby silencing the expression of the gene


Products


Pharmaceutica
ls


insulin, human growth hormone
, vaccines


Medical intervention


gene therapy


Enzymes
-

fibrolytic enzymes, restriction enzymes...


Substrates
-

cellulose, dextrans


Food products and additives
-

Flav
r saver tomato, cheese (rennet);

Herbicide

toleran
t
Canola (glyphosate resistance);

Insect resistant corn, Soy bean,

cotton and potatoes


Industrial products
-

solvents, ethanol...


Cloned livestock


Nanotechnology


Forensic Medicine



Ethics and safety of genetically modified organisms?