BT-510 PCR-vector-plt 2020.pdf

LECTURE II: PCR, RT PCR PCR-Basic principles and uses Hosts & Vectors Edible vaccines DNA Polymerase 5’ 3’ 3’ 5’ Extension 5’ 3’ 3’...

LECTURE II: PCR, RT PCR PCR-Basic principles and uses Hosts & Vectors Edible vaccines DNA Polymerase 5’ 3’ 3’ 5’ Extension 5’ 3’ 3’ 5’ 5’-3’ Extension (error) 5’ 3’ 3’ 5’ 3’-5’ Proofreading 5’ 3’ 3’ 5’ 5’ 3’ Extension 3’ 5’  DNA Polymerase DNA polymerase can add free nucleotides to only the 3’ end of the newly-forming strand. This results in elongation of the new strand in a 5’-3’ direction. No known DNA polymerase is able to begin a new chain (de novo). DNA polymerase can add a nucleotide onto only a pre-existing 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide. Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase. Error correction is a property of some, but not all, DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA. The 3‘- 5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Polymerase Chain Reaction (PCR)  The non-covalent forces that hold the double helix together of DNA may be separated (denatured) simply by heating. The thermal energy provided by heating a DNA sample will break the relatively weak hydrogen bonds connecting the two strand of the helix, but will not affect the covalent linkages that hold each strand together. The heating of double-stranded DNA to produce the single-stranded version is an entirely reversible process. Single-stranded DNA will reform as a duplex when the temperature is reduced. As the temperature falls, the thermal energy that was used to break the hydrogen bonds between the strands is reduced, and random collisions between the complementary strands will result in their re-association. Providing that the temperature is reduced relatively slowly (1–2 ◦C per second), complete duplex formation will result. If the temperature is dropped rapidly, for example by plunging a DNA sample at 94 ˚C directly into ice, correct base pairing will not occur and the DNA will remain in a relatively single-stranded form.  What is the Polymerase Chain Reaction? Discovered by K. Mullis in 1984, for which he was awarded the Nobel prize in Chemistry in 1993 Using this process specific sequences may be amplified from DNA / cDNA. The process consists of a cycle of 3 steps 1. Denaturation (strand separation) at 95 ˚C 2. Primer annealing at the required temperature (50 ˚C- 65 ˚C) 3. And extension (synthesis of new strand) using DNA polymerase It is interesting how some seemingly esoteric or obscure discoveries can, years later, be catapulted to something of immense practical importance. Such is the history of Taq DNA polymerase The original report of this enzyme, purified from the hot springs bacterium Thermus aquaticus, was published in 1976 Roughly 10 years later, PCR was developed and shortly thereafter "Taq" became a household word in molecular biology circles. Currently, the world market for Taq polymerase is in the hundreds of millions of dollars each year.  POLYMERASES FOR PCR Initially DNA Polymerase from E.coli was used in the extension step. Since the enzyme is denatured at 95 ˚C , fresh enzyme had to be added after each cycle. The use of Thermostable polymerase, from Thermus aquaticus, a thermophilic bacterium that was isolated from hot springs and deep vents. The enzyme now commonly referred to as “Taq Pol”, can withstand the denaturing conditions in the PCR. The disadvantage of this enzyme is the poor fidelity of the enzyme. This enzyme also has the property of adding multiple Adenine residues at the end of the newly synthesized chain.  Taq Polymerase Like all polymerases it has a 5’ to 3’ activity. It also has a 5’ to 3’ exonuclease activity Lacks proof reading activity which makes amplification error prone It incorporates roughly 1 mismatch every 10 4- 10 5 bp, but under certain conditions It’s optimal polymerase activity is around 72 ˚C, It remains functional even after the 93 ˚ C denaturation step, but prolonged time points beyond this temperature result in a loss of activity  THERMOSTABLE POLYMERASES Since then, several enzymes have been identified that have improved fidelity due to their 3’ to 5’ exonuclease activity and longer half life at high temperatures. Pfu from Pyrococcus furiosus Pwo from Pyrococcus wossei Tfl from Thermus flavus rTth from Thermus thermophilous Tli from Thermius litoris Tma from Thermus maritima  THE COMPONENTS The DNA template: The material from which the required sequence is to be amplified. Primers: Complementary oligonucleotide sequence that flank the required sequence dNTPS: dATP , dCTP, dTTP, dGTP, required for incorporation into the newly synthesized sequence DNA polymerase: A 5’ to 3’ polymerase that can incorporate the dNTPs into the newly synthesized sequence. Currently thermostable polymerases are routinely used for the purpose. The enzyme may also have a 3’ to 5’ proofreading property. PCR machine: Thermocycler to enable the cyclic denaturation, annealing and extension temperatures required for the process  The Three Basic steps of PCR 1. Denaturation of DNA at 93-95 ˚C At high temperatures the hydrogen bonding between the two strands is unstable, resulting in the “melting” of DNA 2. Primer Annealing which depends on the Tm of the two primers 3. The Extension of the Primers at 72 ˚C  95 ˚C 60 ˚C Pol 72 ˚C Pol THE PCR REACTION  PCR results in an exponential increase in the copy number. Cycles Copies 1 2 2 4 4 16 10 1,024 15 32,768 20 1,048,576 25 33,554,432 30 1,073,741,824 While theoretically a billion copies can be from a single copy of DNA after 35 cycles of amplification, this does not occur due to the limitation of the enzyme and its activity. FACTORS THAT INFLUENCE THE PCR REACTION Denaturation Temperature Annealing Temperature Primer length and composition Enzyme concentration Concentration of MgCl2 Elongation Time & temperature  Tm & Ta Melting temperature (Tm) is the temperature at which 50% of the DNA is denatured The Tm increases with the length of the primer and with the GC content. The Tm of a given primer can be calculated using the formula Tm = 4(G + C) + 2(A + T) oC The annealing temperature (Ta) is approximately chosen about 2-3 oC below the Tm Selection of this temperature is critical for the success of the PCR A low Ta will result is non specific amplification, If the selected temperature is too high the primers will fail to anneal and the yield of PCR product will be low  Tm = 4(G + C) + 2(A + T) oC Every AT base pair in the duplex contributes 2 oC of stabilization the double helix, while every GC pairing contributes 4 oC of stabilization. Therefore, an oligonucleotide of 20 bases of single-stranded DNA 5’-GTCGTAGATTCCGATAGATG-3’ This contains 5 A, 6 T, 3 C and 6 G residues The annealing temperature to its complementary DNA sequence of approximately 2(11) + 4(9) = 58 ˚C. A-T , G-C base pairing  Concentration of MgCl2 Mg2+ is a cofactor for Taq Polymerase The concentration of free Mg2+ is critical to the reaction. Low Mg2+ increases the specificity of the reaction but can affect the amount of amplified product High Mg2+ increases primer annealing and therefore the sensitivity of the reaction  Primer Design Primer length is between 18-22 bases It should be complementary and specific to the gene of interest. It should not bind to other (non specific) regions in the gene or genome The forward and reverse primer for a gene should not show complementarity between each other. Amplification will not occur due to primer-dimer formation. A primer should not have complementarity within the strand The Tm of forward and reverse primers should be similar such that a common Ta can be chosen for amplification. A T G C T A C G Primer dimer Hairpin loop Applications of PCR  Applications of PCR Molecular cloning DNA sequencing Archaeology Forensics Amplification of unknown sequences Clinical pathology Genetic diagnosis Characterizing unknown mutations Fingerprinting/population analysis Genome analysis Quantitative PCR of RNA or DNA.  PCR in detection of Genetic Diseases Detection of Insertion/Deletion mutants Waardenburg syndrome, is an inherited autosomal dominant disease that is characterized by a combination of deafnessand abnormal pigmentation. The disease is responsible for over 2 per cent of the cases of adult deafness, and is often associated with a frontal white blaze of hair and white eyelashes. Certain types of Waardenburg syndrome are caused by mutations in the PAX-3 gene, a transcription factor involved in regulating embryonic development. One of the first mutations found within PAX-3 was an 18 bp deletion in the DNA encoding the DNA binding domain of the transcription factor (Tassabehji et al., 1992). This deletion can be detected in other Waardenburg syndrome patients by PCR. Primers can be designed to flank the site of the deletion. Amplification of the wild-type sequence will yield DNA fragment of156 bp, Amplification of the mutant sequence will yield a smaller DNA fragment (138 bp) The disease is dominant, most sufferers will be heterozygotes, having one copy of the wild-type gene and one copy of the mutant gene. PCR amplification of a heterozygote will yield two DNA fragments of (156 and 138 bp) DIFFERENT TYPES OF PCR Hot Start PCR Long PCR Assymetric PCR In situ PCR Touch down PCR Reverse Transcription-PCR Real Time PCR Nested PCR  Quantitative Real Time PCR Quantitative Real-time reverse-transcriptase (qRT) PCR quantitates the initial amount of the template instead of detecting the final amplified product. It is characterized by the point in time during cycling when amplification of the PCR product of interest is first detected rather than the amount of the PCR product of interest which is accumulated at the end-point after PCR which contained a large number of cycles. Amplification is visualized by monitoring the amount of fluorescence emitted during the PCR reaction. This acts as an indicator of the amount of PCR amplification that occurs during each PCR cycle. Cloning vectors Plant Biotechnology Edible vaccines  Hosts and Vectors Which is Used Where. Bacterial : Cloning and Expression of proteins Yeast : Expression of proteins that are not efficiently expressed in bacteria or for understanding yeast derived components. Mammalian cells: For the expression of mammalian proteins Others: Insect cell lines, Baculovirus expression Retroviral vectors  E.coli a suitable organism for rDNA It is easy to grow in simple, inexpensive growth medium. The organism has a rapid doubling time of about 20–30 minutes during log-phase growth. Its genetics and strains are well defined. It has a fully mapped and sequenced genome. Laboratory strains of E. coli are generally safe and contain mutations that do not allow them to escape the laboratory environment. Extra-chromosomal copies of DNA (plasmids and bacteriophage DNA) can be exploited to carry foreign DNA fragments. The choice of vector depends chiefly on the size of DNA to be inserted Even if the final destination of a cloned DNA fragment is a eukaryotic cell, DNA constructs are invariably produced in E. coli prior to being shuttled into their ultimate host.  Requirements of a Vector Vectors are autonomously replicating DNA molecules that can be used to carry foreign DNA Vectors are based on naturally occuring DNA sequences that have been modified and combined to serve particular functions. Most vectors have been modified to propagate the inserted sequence, express proteins or silence genes Cloning vectors: Vectors used for propagating a given sequence Expression vectors: Vectors that are used for expression of protein from the inserted sequence  Plasmid Vectors All vectors must essentially have the following properties 1.The ability to self-replicate 2.Low molecular weight 3. A selectable marker to determine the presence in the host 4.The presence of a Multiple Cloning Site (MCS) that contains RE sites required for cloning  PLASMIDS AS CLONING VECTORS 1. Plasmids are naturally occurring extra-chromosomal DNA fragments that are stably inherited from one generation to another 2. They are widely distributed throughout prokaryotes and range in size from approximately 1500 bp to over 300 kbp. 3. Most plasmids exist as closed-circular double-stranded DNA molecules that often confer a particular phenotype onto the bacterial cell in which they are replicated. 4. That is, the plasmid will often carry a gene that encodes resistance to either antibiotics or heavy metals, or that produces DNA restriction and modification enzymes, that the bacterium would not normally possess. The components of a vector are represented as a Map which indicates the total size, major features with details of all features such as size of each component, orientation of expression, markers, Tags , promoters and MCS. The replication of the plasmid is often coupled to that of the host cell in which it is maintained, with plasmid replication occurring at the same time as the host genome is replicated. On the basis of copy number plasmids are classified as a) Relaxed or high copy number plasmids (200 per cell) Relaxed plasmids replicate using host derived proteins. b) Stringent or Low copy number (1–10 per cell). Stringent plasmids encode protein factors required for their own replication. On the basis of the host: Most plasmids survive only in a limited number of hosts. Broad host range plasmids can be transferred and maintained bacteria from a large number of species  Nomenclature of Plasmids Commonly, plasmids are named after the discoverer/research group/function/trait pUC plasmids where p stands for plasmid and UC stands for Univ. of California. pBR322 where B stands for Paco Bolivar and R for Ray Rodrigues. 322 is the number of plasmid in their collection ColE1 plasmid: Colicin Origin of replication Selection Marker Restriction Enzyme site (MCS) Promoter Tags  Selectable Markers Ampicillin – binds to and inhibits a number of enzymes in the bacterial membrane that are involved in the synthesis of the gram-negative cell wall. The ampicillin resistance gene (AMPR or bla) codes for the enzyme β-lactamase that is secreted into the periplasmic space of the bacterium, where it catalyzes hydrolysis of the β-lactam ring of the ampicillin. Tetracycline – binds to a protein of the 30S subunit of the ribosome and inhibits ribosomal translocation along the mRNA and thereby interferes with protein translation. The tetracycline resistance gene (TETR) encodes a 399 amino acid outer membrane associated protein of gram-negative cells that prevents the antibiotic from entering the cell. Kanamycin. and neomycin – bind to ribosomal components and inhibit protein synthesis. The KANR gene codes for a protein that is secreted into the periplasmic space and interferes with the transport of these antibiotics into the cell. Like tetracycline resistance, the KANR gene does not destroy the antibiotic.  BACTERIAL TRANSFORMATION & COMPETENCE Transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of naked foreign DNA. Competence refers to the ability of bacterial cells to take up foreign DNA.  Transformation By the end of the 1960s, DNA could be cut and pasted. But scientists needed a mechanism to copy it in order to maintain a large enough sample to work with. That breakthrough came in 1971, when scientists found an efficient way to introduce plasmids into E. coli.  NATURAL COMPETENCE Is the ability of some bacteria to take up naked DNA from their environment. This process was first identified by experiments by Fredrick Griffith (1928) in Streptococcus pneumoniae  Oswald Avery, Colin Mc Leod & MacLyn McCarty (1944). Experiments at the Rockefeller Institute, New York confirmed this transformation was heat sensitive, but not sensitive to freeze/thaw. Samples were then treated individually to RNAse, proteases or DNase and then tested for their ability to transform cells. Transformation was also attempted using purified components. DNA WAS IDENTIFIED AS THE TRANSFORMING FACTOR Since then natural transformation has been reported in other Gram +ve bacteria (Bacillus subtilis) and Gram -ve (Neisseria gonorrhoeae and Haemophilus influenzae)  Regulation of Bacterial Competence S. pneumoniae is known to be regulated by atleast one extracellular peptide (17 aa) factor. Competence decreases at the stationary phase of growth. B.subtilis, several genes have been shown to regulate competence and DNA uptake. Competence here may be regulated by nutritional signals and growth phase. At least two extracellular factors have been identified in this process N.gonohorrea is constitutively competent  Evolutionary Significance of Transformation In acquisition of new traits from genetically distinct organisms Transformation can assist to evade the host defense mechanism In the repair of damaged DNA As a nutrient source  E.coli A rod shaped Gram –ve bacteria, commonly found in gut flora Strains of E.coli have been used for recombinant DNA technology and cloning Plasmids may be easily propogated in E.coli It has been exploited to express numerous proteins such as insulin Though more complex proteins cannot be expressed in this organism  ARTIFICIAL COMPETENCE This process is not encoded by the cells genes, instead it is induced in the laboratory using different procedures that do not normally occur in nature. 1. CHEMICAL TRANSFORMATION OR 2. PHYSICAL TRANSFORMATION (ELECTROPORATION) Chemical Transformation by Heat Shock, Transformation by Electroporation  Factors that affect Electroporation 1. Cell Growth: E.coli has optimal transformation at mid-log phase. 2. The quality of DNA: Purity of the DNA is a vital factor in transformation 3. Electroporation media : Glycerol as a cryoprotectant : Concentration of ionic compounds : pH of the media : volume of the media 4. For microorganisms, a short pulse duration at High voltage is optimal Genetic Modification of plants can be used to incorporate Disease resistance, Nutritive value, drought resistance and salt tolerance. In addition plants may be used for the large scale synthesis of a particular protein. The major advantage of using plants is cellular totipotency, the ability of individual cells to regenerate into a completely new plant. Plant tissue culture systems are well established  Applications of Plant rDNA Floriculture Resistance to herbicides, insects, fungi and viruses Improving the Nutritive value of food Salt and Drought tolerant crops Cold tolerance Phytoremediation  Agrobacterium tumefaciens A bacterial plasmid, the tumour inducing (Ti) plasmid of the soil microorganism Agrobacterium tumefaciens, has been used extensively to introduce genes into plant cells. A.tumefaciens is responsible for crown gall disease in a variety of dicotyledonous plants – such as tomato, tobacco, potato, peas, beans etc. A wound on the stem of the plant allows the bacteria to invade and cause a cancerous proliferation of the stem tissue. A plasmid carried within the bacterium is responsible for its ability to cause crown gall disease (Zaenen et al., 1974; Van Larebeke et al., 1974). This tumour inducing (Ti) plasmid is large (∼200 kbp) and carries a number of genes that are required for the infection process (Suzuki et al., 2000).  Ti plasmid The Ti plasmid carries T DNA, a 23 kb fragment that contains the genes responsible for the cancerous properties of the transformed cells (e.g. those controlling the production of the plant hormones auxin and cytokinin that stimulate cell division and growth). It also carries genes needed for opine utilization. The opines produced by the infected plant, e.g. nopaline, which is formed through the reductive condensation of arginine and α-keto-glutarate, can be used by Agrobacterium cells as their sole source of carbon and energy. ̰ genes are required for virulence (vir) involved in infection and A series of 35 integration of T DNA An Agrobacterium origin of replication (ori).  BioPharm from Plants Product Definition Examples Antibodies Proteins for immune Specific antibodies developed defense responses to fight cancer, treat inflammation, and fight viral and bacterial diseases. Antigens Stimulate production of Vaccines for protection (vaccines) antibodies that protect against cholera, diarrhea against disease (Norwalk virus), and hepatitis B Enzymes Proteins that catalyze Enzymes used to treat and to biochemical reactions diagnose disease. Hormones Chemical messengers Insulin for diabetics Structural Proteins for structural Collagen is a structural proteins support to cells or tissues protein found in animal connective tissues and used in cosmetics Anti-disease Variety of proteins The anti-infection agents agents interferon and lactoferrin, and aprotinin have been engineered in plants Edible vaccines  Vaccines Vaccines are designed to elicit an immune response without causing a disease. Typical vaccines are composed of killed or live pathogens or their components. Successful vaccination are useful in curbing the spread of disease. Most available vaccines are injectables.  Disadvantages of existing vaccines Extensive requirement for proper storage to retain stability Since these are injectables, sterility is an essential factor The cost involved is an important factor in developing countries where vaccination is needed the most  What is an Edible Vaccine ? A vaccine that can be eaten rather than injected. It involves the development of transgenic plants that express a subunit or part of a pathogen. The availability of this pathogen protein is capable of eliciting an immune response in the host Advantages of Edible Vaccines Edible means of administration. Reduced need for medical personnel, sterile conditions. Economical in mass production and transport. Free of pathogens and toxins. Subunit vaccines, therefore improved safety. Storage near site of use. Heat stable, No refrigeration required. Generation of systemic and mucosal immunity. Enhanced compliance (especially in children). Delivery of multiple antigens in one dose. General strategy for Edible Vaccines  Plants & Vectors Plants Vectors Tobacco Agrobacterium tumefaciens Lettuce Plant based viral vectors Potato Animal models Maize Mice Banana Tomato  Examples of Edible Vaccines S.no Vaccine Vector used Disease condition it is used on 1. Hepatitis B virus Tobacco Hepatitis B Potato Lettuce 2. Norwalk virus Tobacco Diahorrea, Nausea Potato Stomach cramps 3. Rabies virus Tobacco Rabies 4. Corona virus Tobacco Gastroenteritis Maize 5. Rabbit hemorrhagic Potato Hemorrhage disease virus 6. HIV Virus Tomato AIDS 7. Vibrio cholera Potato Cholera  Hepatitis B vaccine in Banana Expression of hepatitis B surface antigen in transgenic banana plants G. B. Sunil. Kumar, T. R. Ganapathi, C. J. Revathi, L. Srinivas and V. A. Bapat  Limitations of Plant vaccines Development of Immune tolerance to vaccine peptide or protein. Variation in dosage from fruit-to fruit or plant to plant and over generations Stability of vaccine in fruit is not known. Evaluating dosage is difficult Certain plant such as potato cannot be eaten raw, cooking may dilute the antigenic ability of the expressed gene.  Internal assessment (BT-510) Date: 24th DECEMBER, 2020 (THURSDAY) Time: 6:30 pm (30 min duration) Mode: Google Form Link will be sent at 6:25 pm to (please check the correctness of email addresses): 1. [email protected] [NP] 2. [email protected] [PA] 3. [email protected] [PC] 4. [email protected] [PE] 5. [email protected] [PTF] The class representatives will forward the link to their classmates by email, with a copy to [email protected] Submission of responses will be closed after 30 minutes. Course: All lectures completed till 21st December, 2020