Download Biotechnology and Recombinant DNA I. Tools of Biotechnology

Recombinant DNA Technology
Tortora et al , Chap 9
Biotechnology and Recombinant DNA
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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 improve 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 cell 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)
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Mutation
• Biologists use random mutagenesis to create new strains of a particular
microorganism with enhanced characteristics (e.g., increased penicillin production)
• Site directed mutagenesis can be used to introduce specific changes into a particular
gene
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Repeated rounds of mutagenesis and selection have been used to produce novel
industrial strains with improved characteristics (e.g., production of an antibiotic,
enzyme, amino acid,…)
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.
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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 sites 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 (Sau3AI is from Staphylococcus aureus)
Sau3AI ↓GATC
BamHI
G↓GATCC
PstI
CTGCA↓G
NotI
GC↓GGCCGC
Restriction produces overhanging (sticky ends) or blunt ends
EcoRI
G↓AATTC
SmaI
CCC↓GGG
Modification enzymes
Host must protect its own DNA → modifying enzymes or methylases
Methylases → methylate (-CH3) specific bases within the restriction enzyme recognition
site (usually A or C)
CH3
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BamHI methylase
GGATCC
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Restriction enzymes and methylases are part of restriction-modification system (RMS)
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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). When 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.
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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)
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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 35P and 32S, fluorescent
dye molecules…) and used to detect the presence of complementary sequences.
These small labeled ssDNA molecules are called probes.
Southern blot
Northern blot
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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 of 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
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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 using DNA polymerase.
• A DNA primer is used to initiate synthesis (i.e., the sequencing 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
reagents 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
reaction. 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
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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 tm of primers)
iii) Extension (elongation - usually at 72°C)
Ingredients for PCR reaction
• Buffer containing Mg++
• Enzyme Taq DNA polymerase
• Substrate Template
Primers
Nucleotides
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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 RNA?
Just want mRNA – recall Poly A tail
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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
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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 organism.
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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 EcoRI G|AATTC - easier to clone with but may not always have a
useful site in the appropriate location
o blunt ends SmaI 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’-PO4 reforming the
phosphodiester bonds
Cloning vector – “vehicle” for carrying and replicating introduced DNA fragments.
A variety of vectors 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. Selectable 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.
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iii. Introduction of recombinant molecules into host cells
• Escherichia coli and Saccharomyces cerevisiae are the most common prokaryotic and
eukaryotic cloning hosts
• Transformation 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
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Most bacterial cells will not take up DNA efficiently unless they are exposed to
special chemical or electrical treatments. However naturally transformable 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)
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Most naturally transformable bacteria can take up DNA only late in their growth
cycle - usually stationary phase
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A number of genes are involved; B. subtilis - com genes
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Artificially induced competence
Most bacteria are not naturally transformable, al least not at easily detected levels.
However this does not preclude them from transformation
They can be artificially induced to a state of competence
Methods of Induced Competence
Chemical induction
• CaCl2 - E. coli
• Polyethylene glycol - Bacillus thuringiensis
Electroporation
• formation of transient membrane pores through the use of high voltage fields
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• protocols for many prokaryotic and eukaryotic organisms
Biolistic approach – i.e., gene gun
• Microscopic particles of gold or tungsten are coated with DNA and propelled
by a blast of helium 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 capable 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 transferred 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.
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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 gene 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 expression
of a coding sequence of interest.
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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
Pharmaceuticals – insulin, human growth hormone, vaccines
Medical intervention – gene therapy
Enzymes - fibrolytic enzymes, restriction enzymes...
Substrates - cellulose, dextrans
Food products and additives - Flavr saver tomato, cheese (rennet); Herbicide
tolerant 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?
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