Recombinant DNA Technology

XII Biotechnology Nabaneeta Das

Recombinant DNA Technology

Genetic engineering Recombinant DNA (rDNA) Steps involved in RDT * isolation of DNA fragments that are to be used or manipulated (insert). * generation of rDNA by insertion of these DNA fragments 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 rDNA molecule and replication so that genetically identical cells (clones) are created. The first rDNA molecule was created by Paul Berg, Herbert Boyer, Annie Chang and Stanley Cohen

Tools of RDT
Enzymes: Restriction Enzymes, other enzymes Vectors Host cells

Restriction Enzymes (Molecular scissors)

Foundations of rDNA technology were laid by the discovery of Restriction Enzymes. These enzymes are found in bacteria as a part of their defense mechanism called Restriction Modification system. This system consists of two components: * restriction enzyme that recognizes a specific sequence. * modification enzyme that adds methyl group to one or two bases within the recognition sequence. Different bacteria have different RM system.

Why RE?

Types of Restriction Enzymes

Type I Type II Type III Only type II restriction enzymes are used for RDT as they recognize and cleave within the specific DNA sequence of 4 to 8 nucleotides. This specific 4 to 8 nucleotide sequence is the same when read in a 5-> 3 direction on both DNA strands. Such a sequence is known as palindromic sequence.

Type II restriction enzyme

They are named for the bacterium from which they have been isolated. The first letter of the enzyme comes from the genus name of the bacteria. The next two letters come from the species name. Next part of the name of RE comes form the strain name. Last is the roman number signifying the order of discovery. E.g. EcoRI from E. coli RY13 and it was the first nucleotide W. Arber, H. Smith and D. Nathans in 1978 received Nobel prize in Medicine or Physiology for the discovery of Restriction Enzymes

Sticky / Blunt ends?

EcoRI

Type II RE, their sources and recognition site

Constructing rDNA!

RFLP

Other Enzymes: DNA Ligase

Forms phosphodiester bonds between two nucleotides. The action of the ligase requires a phosphate group at the 5 carbon and a OH group on the 3 carbon. T4 DNA ligase which is encoded by T4 phage.

Other Enzymes: Alkaline Phosphatase

Phosphatase is used to remove the phosphate group from the 5 end of the nucleotide leaving free 3 OH group. This enzyme is isolated from bacteria is BAP or from calf intestine (CAP). It is used to prevent unwanted self ligation of vector DNA molecule in cloning procedures.

Vectors
Serves as a vehicle to carry a foreign DNA sequence into a given host cell. Essential features of vectors: * origin of replication (ori) * selectable marker. E.g. antibiotic resistance such as ampicillin resistance or enzymes such as beta galactosidase. * polylinker or multiple cloning site (MCS). This provides flexibility in the choice of restriction enzyme(s) that can be used for cloning. * relatively small in size

Plasmids
Extra chromosomal, self replicating, circular, double stranded DNA molecules. Found naturally in many bacteria and also in some yeast. Plasmids are not essential for the normal cell growth and division, they confer some traits on the host organism that can be a selective advantage under certain conditions. They may be present in 1 or 2 copies to multiple copies. These have been modified to carry a target gene to the host in RDT. They are the most widely used vectors.

Plasmids
One of the earliest plasmid vectors- pBR322. this contains two antibiotic resistance genes. One popular series of plasmid vectors is pUC family. They contain lacZ gene that codes for the enzyme Beta galactosidase. This region also contains a MCS and thus insertions in this region causes this gene to lose its function. This forms the direct basis of selection of recombinants. Both these plasmids replicate in E. coli only. Shuttle Vectors: vectors which can replicate in both eukaryotic cell and E. coli. These vectors contain two types of ori site and selectible markers that can function in both type of cells. E.g. Yep. In case of plants, a naturally occurring plasmid of the bacterium Agrobacterium tumefaciens called Ti plasmid has been suitably modified to act as vector.

pBR322

pUC19

Plasmids
Some times the goal of cloning is to express the cloned gene. This can be done by inserting the signals necessary for the initiation and termination of transcription and also for translation initiation into the vector adjacent to the cloning site. The vectors which contain these signals are called expression vectors.

Vectors based on bacteriophages

Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. The bacteriophage DNA is replicated and expressed in the host bacterial cell resulting in replication of the phage virus which burst out of the cell and infect the neighbouring cells (lytic cycle).

Lytic cycle

Bacteriophage Vector
The ability to transfer DNA to specific bacterial host is used for specially designing vectors. Lambda and M13 are the most extensively used as vectors.

Lambda Bacteriophage
Double stranded, linear DNA genome of 48, 514 bp in which 12 bases at each end are unpaired but complementary. These ends are therefore sticky or cohesive and are called cos site or cohesive end site. These ends are important for packaging DNA into phage heads. The central region of its genome is not essential for the lytic cycle in E. coli and so this region can be replaced by foreign DNA. The cloning DNA size can be upto 23kb.

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 any of 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 this point DNA replication becomes asymmetric and ss copies of the genome are created and released from the cell as M13 particles. Advantage: genome size less than 10kb in size and RF can be purified and manipulated like a plasmid. And the genes are obtained in ss form which is useful for various techniques like DNA sequencing and site directed mutagenesis.

Cosmids
Have been constructed by combining certain features of plasmid and the cos site of lambda phage. The simplest cosmid vector contains a plasmid origin of replication, a selectable marker, suitable restriction enzyme sites and lambda cos site. The cosmids can be used to clone DNA fragments upto 45kb in length.

YAC Vectors
Yeast artificial chromosome. Used to clone DNA fragments of more than 1Mb in size. Exploited in mapping large genomes- HGP. These vectors contain telomeric sequence, centromere and an autonomously replicating sequence from yeast chromosome. Suitable restriction site and marker gene.

YAC

BAC Vectors
Bacterial Artificial Chromosome Vectors based on the natural, extra-chromosomal plasmid of E.coli- the F factor. Contains gene for replication and maintenance of the F factor, a selectable marker gene and cloning sites. They can take upto 300-350kb of foreign DNA They are used in genome sequencing projects. See table 2

Animal and Plant Viral Vectors

The natural ability of the virus cell to adsorb the cells that they infect is exploited to design the viral vectors and introduce new gene into eukaryotic cell in culture. Vector based on Simian Virus 40 (SV40) was used in the first cloning experiment involving mammalian cells in 1979. Since then a no. of other viruses have been used as vectors like Adenovirus and Papilloma virus for mammalian cell. At present retroviral vectors are most commonly used vector for cloning in mammalian cells. For plants, viruses like Cauliflower Mosaic Virus, TMV and Gemini viruses are used but with limited success.

Host Cells
The kind of cell depends on the aim of the cloning experiment. Gram negative bacteria E. coli is most extensively used in RDT as it is easy to handle and grow, can accept a range of 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 eukaryotic proteins? Yeasts have been used extensively for functional for expression of eukaryotic genes. They offer several advantages like they are simplest single celled eukaryotic organisms, genetically well characterized, easy to grow and manipulate. Plant and animal cells can also be used as host in gene manipulation and for protein expression either tissue culture or whole organism is used

Making Recombinant DNA

DNA Library

Introduction of Recombinant DNA into Host Cells

Transformation: Introduction of rDNA into living cells. Cells do not naturally transform. Simple chemical treatment makes them competent for transformation. In 1970, Mandel and Higa found that the cells found that E. coli becomes competent for transformation if briefly suspended in cold calcium chloride. Transfection: DNA is mixed with charged substances like calcium phosphate, cationic liposomes or DEAE dextran and over layered on the recipient host cells. Electroporation: An electric current is used to create transient microscopic pores in the cell membrane of the host through which the foreign DNA enters.

Introduction of Recombinant DNA into Host Cells

Microinjection: Exogenous DNA can be introduced directly into animal or plant cell nucleus without the use of special eukaryotic vectors using a glass micropipette. It was first used in animal cells and subsequently in plant cells. Biolistics: Foreign DNA can be introduced inside host cell by using a gene gun or particle gun. Microscopic particles of gold or tungsten are coated with DNA of interest and bombarded on the cells. Bacteriophage vectors are naturally infective and hence no special treatment of the host cell.

Identification of Recombinants
Antibiotic Resistance Insertional Inactivation Blue- white selection: insertional inactivation of lac Z gene which codes for beta galactosidase. This enzyme cleaves a colourless, synthetic substrate 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 the positive clones with the same restriction enzyme, by PCR and hybridization or sequencing.

Blue-white selection

Replica Plating Technique

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 strands separate giving rise to ssDNA molecules. These single stranded molecules can be copied by DNA polymerase and thus we can generate multiple copies of the original DNA.

Requirements of PCR
DNA template to be amplified. Primers: oligo-nucleotides usually 10-18 nucleotides long. Bind to the each template strand. Two primers are required: forward primer and reverse primer. They are oriented with their ends facing each. The template region in between is amplified. DNA polymerase: it should be stable at high temperatures ~92oC. The polymerase that is generally used is Taq polymerase. This enzyme is derived from Thermus aquaticus. Deoxynucleotides, dATP, dGTP, dCTP, dTTP

Three Basic Steps in PCR

Denaturation: The target DNA is heated upto 94oC resulting in separation of the two strands. Each of these strands act as the templates. Annealing: As the temperature is reduced the primers anneals 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 optimum temperature 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 are 2 to the n no. of molecules where n denotes the no. of cycles.

PCR

Applications of PCR
Detection of pathogens. Detection of genetic basis of disease like mutations. Used in generating abundant amount DNA for DNA fingerprinting. Detecting specific microorganisms from soil samples, sediments and water. These assays offer a great sensitivity.

DNA Probes
The probe is a relatively short sequence of DNA that recognizes and binds to its complementary sequence. 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. The probe is labeled to make its detection easier. We can stain a gel with ethidium bromide, but this cannot detect small amounts of DNA. The sensitivity is higher in case of probes. The probes are labeled by radioactive isotopes of P, S or a fluorescent molecule.

Hybridization Techniques
Probes are designed to detect the presence and also determine the amounts of complementary sequences in complex mixtures like cellular DNA or RNA. Southern Hybridization: originally described by Edward Southern in 1975 and has been named Southern Blotting.

Southern Hybridization

DNA Sequencing
The most fundamental method of analyzing structure of DNA is determining its sequence of bases. The first complete nucleotide sequence to be carried was for alanine-tRNA residues of yeast. It was 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

DNA Sequencing

Sanger DNA Sequencing

Dideooxy nucleotide

Dye Termination Sequencing

In early 1990s, automated sequencing machines were developed. Fluorescent dyes are conjugated with the ddNTPs such that chain termination is marked by the presence of a dye which is a unique chemical group. The products are electrophoresed on a single lane acrylamide gel. The migrating DNA fragments are illuminated with laser and they on excitation emit a spectral emission of a specific wavelength which is recorded and the computer generates a tracing of electrophorogram. As different dyes correspond to different bases, the emission data can be converted to a nucleotide sequence of the DNA molecule The main advantage of this technique is it is fast and accurate.

Site Directed Mutagenesis

Mutation is an alteration of a base in DNA sequence which may lead to a defective protein or 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 cause change in it effectively. This technique is extensively used by Biotechnologists to design novel proteins.

Site-Directed Mutagenesis

Thank you!