Recombinant DNA Technology[1]

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Recombinant DNA Technology

XII Biotechnology

Nabaneeta Das


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Recombinant DNA Technology

Genetic engineering Recombinant DNA (rDNA)

Steps involved in RDT

* isolation of DNA fragments that are to be used ormanipulated (insert).

* generation of rDNA by insertion of these DNAfragments into the carrier DNA molecule(vector), that can self replicate in the host.

* transfer of rDNA molecule to an appropriate host.

* selection of the host cells that carry the desired rDNAmolecule and replication so that genetically identicalcells (clones) are created.

The first rDNA molecule was created by Paul Berg, HerbertBoyer, Annie Chang and Stanley Cohen


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Tools of RDT

Enzymes: Restriction Enzymes, other enzymes

Vectors

Host cells


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Restriction Enzymes (Molecularscissors)

Foundations of rDNA technology were laid by thediscovery of Restriction Enzymes.

These enzymes are found in bacteria as a part oftheir defense mechanism called Restriction

Modification system. This system consists of twocomponents:

* restriction enzyme that recognizes a specificsequence.

* modification enzyme that adds methyl groupto one or two bases within the recognitionsequence.

Different bacteria have different RM system.


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Why RE?


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Types of Restriction Enzymes

Type I

Type II

Type III

Only type II restriction enzymes are used for RDTas they recognize and cleave within the specificDNA sequence of 4 to 8 nucleotides.

This specific 4 to 8 nucleotide sequence is the

same when read in a 5-> 3 direction on bothDNA strands. Such a sequence is known aspalindromic sequence.


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Type II restriction enzyme They are named for the bacterium from which

they have been isolated.

The first letter of the enzyme comes from thegenus name of the bacteria.

The next two letters come from the speciesname.

Next part of the name of RE comes form thestrain name.

Last is the roman number signifying the order ofdiscovery.

E.g. EcoRI from E. coli RY13 and it was the firstnucleotide

W. Arber, H. Smith and D. Nathans in 1978received Nobel prize in Medicine or Physiology

for the discovery of Restriction Enzymes


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Sticky / Blunt ends?


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EcoRI
http://upload.wikimedia.org/wikipedia/en/9/95/1QPS.png


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Type II RE, their sources andrecognition site


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Constructing rDNA!


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RFLP


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Other Enzymes: DNA Ligase

Forms phosphodiesterbonds between twonucleotides.

The action of the ligase

requires a phosphategroup at the 5 carbon and

a OH group on the 3

carbon.

T4 DNA ligase which is

encoded by T4 phage.


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Other Enzymes: Alkaline Phosphatase

Phosphatase is used toremove the phosphategroup from the 5 end of

the nucleotide leaving free3 OH group.

This enzyme is isolatedfrom bacteria is BAP orfrom calf intestine (CAP).

It is used to prevent

unwanted self ligation ofvector DNA molecule incloning procedures.


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Vectors

Serves as a vehicle to carry a foreign DNAsequence into a given host cell.

Essential features of vectors:

* origin of replication (ori)* selectable marker. E.g. antibiotic resistance

such as ampicillin resistance or enzymessuch as beta galactosidase.

* polylinker or multiple cloning site (MCS). Thisprovides flexibility in the choice of restrictionenzyme(s) that can be used for cloning.

* relatively small in size


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Plasmids

Extra chromosomal, self replicating, circular,double stranded DNA molecules.

Found naturally in many bacteria and also insome yeast.

Plasmids are not essential for the normal cellgrowth and division, they confer some traits onthe host organism that can be a selectiveadvantage under certain conditions.

They may be present in 1 or 2 copies to multiplecopies.

These have been modified to carry a target geneto the host in RDT. They are the most widelyused vectors.


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Plasmids

One of the earliest plasmid vectors- pBR322. this containstwo antibiotic resistance genes.

One popular series of plasmid vectors is pUC family. Theycontain lacZ gene that codes for the enzyme Betagalactosidase. This region also contains a MCS and thusinsertions in this region causes this gene to lose its

function. This forms the direct basis of selection ofrecombinants.

Both these plasmids replicate in E. coli only.

Shuttle Vectors: vectors which can replicate in botheukaryotic cell and E. coli. These vectors contain two types

of ori site and selectible markers that can function in bothtype of cells. E.g. Yep.

In case of plants, a naturally occurring plasmid of thebacterium Agrobacterium tumefaciens called Ti plasmidhas been suitably modified to act as vector.


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pBR322


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pUC19


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Plasmids

Some times the goal of cloning is to express thecloned gene.

This can be done by inserting the signalsnecessary for the initiation and termination oftranscription and also for translation initiation intothe vector adjacent to the cloning site.

The vectors which contain these signals arecalled expression vectors.


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Vectors based on bacteriophages

Bacteriophages areviruses that infect bacterialcells by injecting their DNAinto these cells.

The bacteriophage DNA isreplicated and expressedin the host bacterial cellresulting in replication ofthe phage virus which

burst out of the cell andinfect the neighbouringcells (lytic cycle).


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Lytic cycle


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Bacteriophage Vector

The ability to transfer DNA to specific bacterialhost is used for specially designing vectors.

Lambda and M13 are the most extensively usedas vectors.


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Lambda Bacteriophage

Double stranded, linear DNA genome of 48, 514bp in which 12 bases at each end are unpairedbut complementary.

These ends are therefore sticky or cohesive andare called cos site or cohesive end site.

These ends are important for packaging DNA intophage heads.

The central region of its genome is not essentialfor the lytic cycle in E. coli and so this region canbe replaced by foreign DNA.

The cloning DNA size can be upto 23kb.


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M13 Bacteriophage

Filamentous phage which infects E. coli having F pili. Its genome is a single stranded, circular DNA of 6407 bp.

Foreign DNA can be inserted into it without disrupting anyof its essential genes.

As the M13 phage DNA enters the bacterial cell, it converts

to a double stranded molecule known as replicative form(RF).

This replicates until there are 100 copies in the cell. At thispoint DNA replication becomes asymmetric and ss copiesof the genome are created and released from the cell asM13 particles.

Advantage: genome size less than 10kb in size and RF canbe purified and manipulated like a plasmid. And the genesare obtained in ss form which is useful for varioustechniques like DNA sequencing and site directedmutagenesis.


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Cosmids

Have been constructed by combining certainfeatures of plasmid and the cos site of lambdaphage.

The simplest cosmid vector contains a plasmidorigin of replication, a selectable marker, suitablerestriction enzyme sites and lambda cos site.

The cosmids can be used to clone DNAfragments upto 45kb in length.


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YAC Vectors

Yeast artificialchromosome.

Used to clone DNAfragments of more than1Mb in size.

Exploited in mapping largegenomes- HGP.

These vectors containtelomeric sequence,centromere and an

autonomously replicatingsequence from yeastchromosome.

Suitable restriction siteand marker gene.


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YAC


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BAC Vectors

Bacterial Artificial Chromosome

Vectors based on the natural, extra-chromosomalplasmid of E.coli- the F factor.

Contains gene for replication and maintenance ofthe F factor, a selectable marker gene andcloning sites.

They can take upto 300-350kb of foreign DNA

They are used in genome sequencing projects. See table 2


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Animal and Plant Viral Vectors

The natural ability of the virus cell to adsorb the cells thatthey infect is exploited to design the viral vectors andintroduce new gene into eukaryotic cell in culture.

Vector based on Simian Virus 40 (SV40) was used in thefirst cloning experiment involving mammalian cells in 1979.

Since then a no. of other viruses have been used asvectors like Adenovirus and Papilloma virus for mammaliancell.

At present retroviral vectors are most commonly usedvector for cloning in mammalian cells.

For plants, viruses like Cauliflower Mosaic Virus, TMV andGemini viruses are used but with limited success.


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Host Cells

The kind of cell depends on the aim of the cloningexperiment.

Gram negative bacteria E. coli is most extensively used inRDT as it is easy to handle and grow, can accept a rangeof vectors. Their doubling time is also short (20 minutes).

For eukaryotic proteins, eukaryotic cells may be preferred.Why cant we use prokaryotic cell to express eukaryoticproteins?

Yeasts have been used extensively for functional forexpression of eukaryotic genes. They offer severaladvantages like they are simplest single celled eukaryoticorganisms, genetically well characterized, easy to grow andmanipulate.

Plant and animal cells can also be used as host in genemanipulation and for protein expression either tissueculture or whole organism is used


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Making Recombinant DNA


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DNA Library


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Introduction of Recombinant DNA intoHost Cells

Transformation: Introduction of rDNA into living cells. Cellsdo not naturally transform. Simple chemical treatmentmakes them competent for transformation. In 1970, Mandeland Higa found that the cells found that E. coli becomescompetent for transformation if briefly suspended in coldcalcium chloride.

Transfection: DNA is mixed with charged substances likecalcium phosphate, cationic liposomes or DEAE dextranand over layered on the recipient host cells.

Electroporation: An electric current is used to createtransient microscopic pores in the cell membrane of thehost through which the foreign DNA enters.


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Introduction of Recombinant DNA intoHost Cells

Microinjection: Exogenous DNA can beintroduced directly into animal or plant cellnucleus without the use of special eukaryoticvectors using a glass micropipette. It was first

used in animal cells and subsequently in plantcells.

Biolistics: Foreign DNA can be introduced insidehost cell by using a gene gun or particle gun.Microscopic particles of gold or tungsten are

coated with DNA of interest and bombarded onthe cells.

Bacteriophage vectors are naturally infective andhence no special treatment of the host cell.


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Identification of Recombinants

Antibiotic Resistance

Insertional Inactivation

Blue- white selection: insertional inactivation oflac Z gene which codes for beta galactosidase.

This enzyme cleaves a colourless, syntheticsubstrate X-Gal into a blue coloured product.Thus cells carrying rDNA will form white colonies.

The above mentioned techniques are used

especially in E.coli. There are several methods to detect the rDNA

like digesting the plasmids obtained from thepositive clones with the same restriction enzyme,by PCR and hybridization or sequencing.


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Blue-whiteselection


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Replica Plating Technique


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PCR

Polymerase Chain Reaction.

Invented by Kery Mullis in 1985.

Selective amplification of a specific region of

DNA. Basic principle is that when dsDNA molecule is

heated to a high temperature, the two strandsseparate giving rise to ssDNA molecules. These

single stranded molecules can be copied by DNApolymerase and thus we can generate multiplecopies of the original DNA.


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Three Basic Steps in PCR

Denaturation: The target DNA is heated upto 94oCresulting in separation of the two strands. Each of thesestrands act as the templates.

Annealing: As the temperature is reduced the primersanneals to the template strand. The temperature of

annealing depends on the sequence of the primer. Extension: The Taq polymerase synthesizes DNA between

the primers using the dNTPs and Mg2+ ions. The optimumtemperature of extension is 72oC.

The second cycle the DNA is heated again and all the steps

take place subsequently. At the end of each cycle there are2 to the n no. of molecules where n denotes the no. ofcycles.


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PCR


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Applications of PCR

Detection of pathogens.

Detection of genetic basis of disease likemutations.

Used in generating abundant amount DNA forDNA fingerprinting.

Detecting specific microorganisms from soilsamples, sediments and water.

These assays offer a great sensitivity.


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DNA Probes

The probe is a relatively short sequence of DNAthat recognizes and binds to its complementarysequence.

The binding of the DNA probes is highly specific

with its complementary nucleotide sequence. Thus it is used in hybridization experiments to

detect the specific nucleotide sequence. Theprobe is labeled to make its detection easier.

We can stain a gel with ethidium bromide, but thiscannot detect small amounts of DNA.

The sensitivity is higher in case of probes. Theprobes are labeled by radioactive isotopes of P, Sor a fluorescent molecule.


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Hybridization Techniques

Probes are designed to detect the presence andalso determine the amounts of complementarysequences in complex mixtures like cellular DNAor RNA.

Southern Hybridization: originally described byEdward Southern in 1975 and has been namedSouthern Blotting.


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Southern Hybridization


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DNA Sequencing

The most fundamental method of analyzingstructure of DNA is determining its sequence ofbases.

The first complete nucleotide sequence to be

carried was for alanine-tRNA residues of yeast. Itwas done by Robert Holleys group at Cornell

University in 1965.

Two methods used for DNA sequencing

developed in 1977 are:

Sanger Method

Maxam Gilbert Method


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DNA Sequencing


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Sanger DNA Sequencing


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Dideooxy nucleotide


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Dye Termination Sequencing

In early 1990s, automated sequencing machines weredeveloped.

Fluorescent dyes are conjugated with the ddNTPs suchthat chain termination is marked by the presence of a dyewhich is a unique chemical group.

The products are electrophoresed on a single laneacrylamide gel.

The migrating DNA fragments are illuminated with laserand they on excitation emit a spectral emission of a specificwavelength which is recorded and the computer generates

a tracing of electrophorogram. As different dyes correspondto different bases, the emission data can be converted to anucleotide sequence of the DNA molecule

The main advantage of this technique is it is fast andaccurate.


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Site Directed Mutagenesis

Mutation is an alteration of a base in DNAsequence which may lead to a defective proteinor prematurely terminated non-functional protein.Mutations are rare and may or may not be

spontaneous. If we desire some particular property in a protein

we can change its coding sequence and causechange in it effectively. This technique is

extensively used by Biotechnologists to designnovel proteins.


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Thank you!

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Recombinant DNA Technology[1]