Recombinant DNA technology which came into existence in the 1970s, allows for genetic manipulation of organisms by incorporating DNA sequences from different sources into a single recombinant molecule. This revolutionary technology has opened up several applications in plant genomics and clinical research. Learning Objective After interacting with this Learning Object, the learner will be able to, ü List out tools used for gene exploration. ü Utilize the knowledge on creation of a genomic library. ü Recall about transgenic plants & animals.
Tools used for gene exploration Restriction endonucleases cleave double stranded DNA at specific recognition sequences which can be used for isolating genes and cloning new DNA molecules. These enzymes are typically found in prokaryotes where they function as a defence mechanism against foreign DNA. Recognitions sites are typically 4-8 base pairs long and are very often palindromic i.e. they read the same in both directions. Cleavage at a phosphodiester bond can produce either blunt ends or cohesive ends. Two DNA molecules that have been cleaved by the same restriction enzyme can be ligated using DNA ligase to produce a recombinant molecule. The fragments may also be separated using electrophoresis techniques.
Tools used for gene exploration DNA that has been fragmented by restriction enzymes can be separated by agarose gel electrophoresis. The DNA is then denatured and blotted onto a nitrocellulose sheet such that their positions remain intact, a process known as Southern blotting. The DNA strands are then probed with a radiolableled DNA molecule which hybridizes to the complementary sequence and can be detected by autoradiography. A similar procedure for RNA molecules is referred to as Northern blotting.
Tools used for gene exploration The polymerase chain reaction can be used to amplify specific DNA sequences of interest. The reaction consists of multiple cycles of strand separation, primer annealing and strand elongation to generate millions of copies of the target sequence within very short time. The two parent DNA strands are first separated by heating them to 95oC after which large excess of primers are added and the solution cooled abruptly (e.g. 54oC), thereby allowing the primers to anneal to each end of the target strand. Strand elongation then takes place at 72oC with help of heat-stable DNA Polymerase from Thermus aquaticus. Both primers are elongated in 5 to 3 direction beyond the target sequence.
Tools used for gene exploration Multiple rounds of PCR are carried out in this manner with repeated cycles of strand separation, primer hybridization and strand elongation. It is possible to carry out these sequence of reactions continuously in a closed container without further addition of reagents after the first cycle, simply by modifying the temperature of the reaction mixture. Although the first cycle yields strands that are longer than the target sequence, only the target is amplified from the end of the second cycle onwards. The sequence gets amplified 2n-fold at the end of n cycles. This is an extremely useful technique in diagnostics, forensics and molecular evolution.
Tools used for gene exploration A simple and elegant method for DNA sequencing was devised by Frederick Sanger where a collection of DNA fragments are synthesized by means of controlled interruption of enzymatic replication. Four DNA synthesis reactions are carried out simultaneously with the strand whose sequence is to be determined being used as the template. The reaction mixture consists of regular deoxy nucleotides and DNA Polymerase along with a small amount of one labelled dideoxy nucleotide analog being added to each of the four reaction mixtures. A primer is added to begin the DNA synthesis and strand elongation continues until a dideoxy analog gets added instead of the regular dntp. Chain termination occurs at this stage due to the absence of a 3 OH group for formation of the next phosphodiester bond.
Tools used for gene exploration The synthesized strands are separated from each other, after which the differentially labelled strands of various lengths are separated by electrophoresis. The smallest fragments move further in the gel while the larger fragments remain close to the point of application. The different fluorescent labels of each ddntp can then be detected by scanning the gel with a beam of laser. The output sequence obtained is complementary to the template strand, which can be used to deduce the original desired template sequence.
Creation of a genomic library Genomic DNA and DNA of the vector molecule are cleaved with the same restriction enzyme so as to generate complimentary end sequences. These are then ligated by means of the enzyme DNA ligase to generate recombinant DNA molecules.
Creation of a genomic library These recombinant molecules can be packaged in-vitro into suitable phage particles which serve as useful vectors to carry the foreign DNA molecules. Lambda and M13 are two of the most commonly used phage particles for this purpose. DNA inserts upto 10 kilobases can be inserted into these phage particles.
Creation of a genomic library There are other vector molecules that can take up DNA fragments from genomic DNA. Plasmids possess sites for various restriction enzymes as well as antibiotic resistance sites which help for screening purposes. Certain plasmids like pbr322 can only take up smaller DNA inserts upto ~ 10kB while larger plasmids known as bacterial artificial chromosomes can take up larger inserts upto 300 kb. The Yeast artificial chromosome is a eukaryotic vector that can take up large DNA inserts and consists of restriction sites, centromer and two telomeric sites.
Creation of a genomic library The phage molecules carrying the recombinant DNA with genomic inserts are used to infect E. coli cells. These molecules then get amplified with each round of replication of the E. coli cell. This collection of bacterial cells harboring the various genomic DNA fragments is known as the genomic library.
Creation of a genomic library Once the library has been created, it is essential to have techniques to retrieve a sequence of interest. The transformed cells are selected and plated on agar, after which the plate is blotted on to a nitrocellulose paper. This is then treated with alkali, bringing about disruption of the cells and denaturation of the dsdna. The denatured DNA is then probed for the sequence of interest by a radiolabelled molecule having a complementary sequence.
Transgenic plants & animals When a plant gets injured, there is release of the phenolic compound acetosyringone, which is detected by Agrobacterium tumefaciens. Upon detection, the virulence genes of tumor-inducing (Ti) plasmid get expressed which encode the enzymes that are essential for transfer of the T DNA into the nucleus of the plant cell. Once the T-DNA gets integrated with the plant chromosome there is release of cytokinins, auxins etc. which brings about tumor formation in the plant.
Transgenic plants & animals The useful property of infection by Agrobacterium has allowed several foreign genes of interest to be introduced into plant cells as per the requirement. One plasmid of the cell is the Ti plasmid without the T-DNA. The other plasmid contains the gene of interest along with antibiotic resistance genes placed in between two repeat units that are essential for gene transfer. The gene of interest such as genes for pesticide resistance, better yield etc. invades the plant at the site of injury. Once this happens, the foreign gene gets inserted into the plant DNA, which is confirmed by plating on to agar containing the suitable antibiotic. Only those which have taken up the gene will grow on such plates.
Transgenic plants & animals Transgenic animals especially mice are commonly used to express foreign DNA molecules. This is often done with the help of retroviruses. Successful expression of these inserts in larger mammals has allowed the development of several recombinant products useful for medicine.
Tools used for gene exploration 1. Restriction analysis: A molecular biology technique that is used to fragment and manipulate DNA into manageable lengths before further analysis. The following components are required for restriction analysis. a) Restriction enzymes: An enzyme that cleaves single or double stranded DNA at specific locations based on the nucleotide sequences recognized. b) Restriction sites: These are regions of the nucleotide sequence that are recognised by the restriction enzymes and subsequently cleaved. c) DNA Ligase: Enzyme that is involved in repairing or joining single stranded breaks or discontinuities in double stranded DNA. d) Cohesive ends: A cohesive or sticky end of DNA refers to those DNA molecules having a 3 or 5 overhang region after they have been cleaved by the restriction enzyme. These overhangs possess nucleotide sequences that have complementary regions and can therefore easily anneal together. e) Blunt ends: Blunt ends of DNA molecules refer to those that do not possess any overhang region but are instead cleaved at the same nucleotide position on both strands thereby producing flat or blunt ends.
Tools used for gene exploration 2. Blotting techniques: These are techniques that have been developed to characterize DNA and RNA. The Southern blot, developed by Edwin Southern, is used to characterize DNA while the Northern blot is used for RNA. a) Electrophoresis: Electrophoresis is a gelbased analytical technique that is used for separation and visualization of biomolecules like DNA, RNA and proteins based on their fragment lengths or charge-to-mass ratios using an electric field. b) Agarose gel: Gels made of agarose are commonly used for separation of nucleotide molecules. These are made up of 0.7-2% agarose dissolved in the electrophoresis buffer but concentrations can vary depending on the length of fragments to be separated. c) Blotting: The process by which nucleotide molecules separated on the electrophoresis gel are transferred on to another surface such as nitrocellulose by placing them in contact with each other. d) Nitrocellulose sheet: A membrane or sheet made of nitrocellulose onto which single stranded DNA molecules separated by electrophoresis are transferred for further probing and analysis. e) Probing & autoradiography: The single stranded DNA molecules bound to the nitrocellulose membrane are probed for specific sequences by means of a radioactive probe molecule. Excess probe molecules are washed off after which the radioactivity is detected by means of autoradiography. f) Autoradiograph: The image pattern produced due to decay emissions from a radioactive material.
Tools used for gene exploration 3. Polymerase Chain reaction (PCR): The polymerase chain reaction is an extremely useful molecular biology tool that allows for amplification of a segment of DNA upto billionfold. This helps in characterization and manipulation of very small quantities of DNA and has found several diverse applications. a) Target sequence: The sequence of interest of DNA that is to be amplified. b) PCR cycle: One round or cycle of PCR refers to strand separation at 95oC, annealing of primers at 54oC and elongation of these primers at 72oC by the polymerase enzyme. Every PCR cycle results in doubling of the number of strands present. c) Primers: A short strand of nucleotides that acts as a starting point for DNA synthesis by providing a free 3 -OH end. These are hybridized to the target DNA and then elongated by means of the polymerase. d) Taq polymerase: This is a thermostable Polymerase enzyme that is obtained from bacterium Thermus aquaticus. It is used for reactions due to its ability to perform elongation even at high temperatures. DNA the PCR DNA
Creation of a genomic library 1. Genomic library: A collection of host bacteria, each carrying a DNA fragment as part of a cloning vector, such that the entire set of DNA fragments when assembled represents the complete genome of an organism. 2. Genomic DNA: The entire DNA sequence of all chromosomes of an organism constitutes its genomic DNA. Genome sequencing projects aimed at deciphering the complete genome sequence of organisms including humans. 3. Splicing & ligation: The process by which the genomic DNA of interest is broken down into fragments using restriction enzymes and then inserted into a suitable vector where it is joined together or ligated using a DNA ligase enzyme. 4. Vector DNA: A vector is a DNA molecule that acts as a vehicle or medium to transfer foreign DNA into another cell. Commonly used vectors include plasmids, phages, bacterial or yeast artificial chromosomes. The DNA fragment of interest is inserted into this vector molecule by means of suitable enzymes. 5. In-vitro packaging: Process by which the vector DNA containing the genomic fragments are taken up by the phage particles and packaged such that they have a protective coating around them. 6. Phage particles: These are viruses that are capable of taking up foreign DNA and infecting bacterial cells, thereby transferring the DNA into them.
Creation of a genomic library 7. Infection: The process by which the phage particles attach themselves to the bacterial cells and insert their genetic material into them. 8. E. coli cells: One of the most commonly used bacterial cells for creation of genomic libraries due to their simple growth requirements and ease of manipulation.
Transgenic plants & animals 1. Ti plasmid: Tumour-inducing or Ti plasmids are plasmid molecules that are carried by the soil bacterium Agrobacterium tumefaciens. This plasmid contains genes that can bring about development of tumour state and therefore synthesis of opines. 5. Crown gall: The lump of tumour tissue that is formed at the site of plant infection upon insertion of genes from the Ti plasmid by the Agrobacterium is known as the Crown gall. 2. T-DNA: The small 20 kb DNA insert of Ti plasmid that gets integrated into the genome of infected plant cells is known as the T-DNA. 6. Release of cytokines, auxins & opines: Formation of the tumour results in release of compounds such as cytokines, auxins and opines, which are metabolized by the infecting bacteria thereby disrupting the plant cell metabolism. 3. Agrobacterium cell: This is a common soil bacterium known as Agrobacterium tumefaciens that is capable of introducing foreign genes into plant cells by means of its plasmid. 7. Transgenic mice: Many transgenic animals have been developed that harbour and express foreign genes of interest. The most commonly used animals are mice due to ease of handling and their convenient growth cycle. 4. vir genes: These are the virulence genes carried by the plasmid that are essential for infection of the plant cells.