RECOMBINANT DNA TECHNOLOGY----a discipline of BIOTECHNOLOGY
GENE MANIPULATION TECHNOLOGY----a discipline of BIOTECHNOLOGY GENE MANIPULATION TECHNOLOGY----a discipline of BIOTECHNOLOGY Biotechnology - a toolbox to solve problems. Biotechnology is technology harnessing
manipulation of cellular and biomolecular processes to develop technologies and products to help improving the health on earth. Biotechnology leverages the understanding of the natural sciences to create novel solutions for many of the world problems.
HEAL THE WORLD Biotech heals the world by harnessing nature's own toolbox and using human genetic makeup to heal and guide lines of research by: Reducing rates of infectious disease; Saving millions of children's lives; Changing the odds of serious, life-threatening conditions affecting millions around the world;
Tailoring treatments to individuals to minimize health risks and side effects (pharmacogenetics); Creating more precise tools for disease detection and Combating serious illnesses and everyday threats confronting the developing world. FUEL THE WORLD Biotech uses fermentation and harnesses enzymes, yeast, and
other microbes to become microscopic manufacturing plants. Biotech is helping to fuel the world by: Streamlining the steps in chemical manufacturing processes by 80% or more; Lowering the temperature for cleaning cost-effectively ; Improving manufacturing process efficiency to save 50% or more on operating costs; Reducing use of and reliance on petrochemicals;
Using biofuels to cut greenhouse gas emissions by ~ 52% and Tapping into the full potential of traditional biomass waste products. FEED THE WORLD Biotech improves crop using
facilitates of environmentally sustainable farming practices, more effectively. Biotech is helping to feed the world by: Generating higher crop yields with fewer inputs; Lowering volumes of agricultural chemicals required by cropslimiting the run-off of these products into the environment; Using biotech crops that need fewer applications of pesticides and that allow farmers to reduce tilling farmland; Developing crops with enhanced nutrition profiles to overcome
deficiencies; Producing foods free of allergens and toxins such as mycotoxin and Improving food and crop oil content to help improve cardiovascular health. Molecular biotechnology is the use of laboratory techniques to study and modify
nucleic acids and proteins for applications in areas of human and animal health. helps to develop and improve drugs, vaccines, therapies, and diagnostic tests for improving human and animal health. has applications in plant and animal biotech., agriculture, aquaculture, chemical and textile manufacturing, forestry, and food processing, health, agriculture, and the environment.
Background Ancient people adapted some natural processes out of practice and intuition, which are currently depicted as part of biotechnology. Fermentation of glucose to beer and making of bread were carried out about more than 6000 years B.C. Hungarian engineer, Karl Erkey, in 1917 used the term
biotechnologyfor large scale production / fattening of pigs using sugar beets as feed. House-hold preparation of yogurt by adding little previously consumed yogurt (now considered to be seeding lactic acid bacteria from old yogurt), idli and dosa(South Indian food items), soy preparation etc. are few names where biotechnology is used for food item preparation.
Use of immobilized enzymes in Glucometer for determination of blood glucose level from a drop of blood is one of such biotechnology based diagnostic processes. Discovery of restriction endonucleases and DNA ligases etc. in 1960s provided arsenals to Boyer and Cohen to develop recombinant DNA technology for expression F-DNA to synthesize protein in the recipient. Industrial microbiology applies manipulation of
microorganism (like mutations of an organism or gene amplification using vectors e.g. plasmids ) in order to increase yield of a desired product, (e.g. antibiotics, vitamins, enzymes, amino acids, solvents, alcohol etc.) in mass quantities in order to reduce production cost making it affordable to everyone and also to make a profit. RAW MATERIALS (unconventional sources)
Biology and Computer Science MOLECULAR BIOTECHNOLOGY Crops, Livestock, Diagnostics, Drugs Vaccines Starting with Genentech in 1970s, Monsanto, Eli Lilly, Du Pont,
Pfizer , GlaxoSmithKline, Novartis etc. are some of more than 8000 biotechnology companies targeting mostly cardiovascular disorders, tissue engineering, cell replacement, drug delivery, vaccine, gene therapy, antisense drugs, diagnosis, genomics, proteomics etc. Molecular biotechnology can provide opportunities for accurate diagnosis, prevent or cure of a wide range of genetic/infectious diseases. significantly increase nutritionally enriched crop yield through
creating resistant plants, like drought/flood resistant, salinity resistant, insect/fungal/viral diseases resistant plants. develop microbes for large scale production of antibiotics, enzymes, amino acids, chemicals, polymers, food additives etc. develop genetically enriched live-stocks. protect environment from pollution through biotransformation of pollutant and appropriate waste management scheme.
Recombinant DNA technology The first major step is isolation of DNA ligase to"glue together" short strands of DNA by Martin Gellert and his colleagues from the National Institutes of Health. A second major step was the discovery of restriction enzymes, which cleave DNA at specific sequences by Swiss biologist Werner Arber and his colleagues .
Stanford University biochemist Stanley Cohen discovered Plasmid as a DNA carrier or vector. Cohen and Boyer in early 1970s successfully transfer human insulin gene to E coli to produce insulin as biopharmaceuticals commercially produced by Genentech but marketed by Eli Lilly. In standard cloning protocols, the cloning of any DNA fragment essentially involves :
Choice of host organism and cloning vector, Preparation of vector DNA,
Preparation of DNA (F-DNA/desired DNA) to be cloned, Creation of recombinant DNA vector, Introduction of recombinant DNA vector into the host organism to form transformant and Screening for clones with desired DNA inserts and biological properties.
Isolation of DNA of interest (F-DNA) from human chromosome The approach is to physically locate the gene in specific chromosome, separate the chromosome, treat with sp. restriction endonuclease and isolate the specific dsDNA fragment by Southern blotting. If the gene cannot be located, respective mRNA is isolated and using reverse transcriptase, cDNA is synthesized.
If the cell/tissue content is very small, as from cigarrete buts, hair roots collected from victims finger nail or blood/semen sample from victims dress, the isolated DNA can be amplified using PCR (polymerase chain reaction). PCR The steps involve:
Denaturation where upon heating of ds DNA at 950C for 20-30 seconds makes ds DNA into single stranded. Addition of primers and cooling to 500 - 600C for 20-30 seconds allows annealing of primers to complementary flanking regions of the target ss DNA and providing seat for DNA Pol to add on dNTPs as mode of chain elongation. Addition of Taq Pol, excess dNTPs and bringing the
temperature to 740C allows synthesis of DNA strand complementary to targeted template ss DNA. Fragmentation of chromosomal dsDNA: Bacterial restriction endonuclase, type-II with target site within palindromic recognition site ( same meaning when read from either side, like MADAM IN EDEN IM ADAM) fragments dsDNA.
Fragmentation of dsDNA could lead to fragments with cohesive/ sticky ends or with blunt ends: EcoRI digestion produces "sticky" ends, G
+ CTTAA AATTC G Whereas enzymes like Sma I restriction enzyme cleavage produces "blunt" ends:
CCC + GGG GGG CCC
SOUTHERN BLOTTING Isolated dsDNA fragment (F-DNA) with cohesive ends is annealed to vector (plasmid) treated with the same restriction endonuclease generating complementary cohesive ends. Annealing is followed by DNA ligase treatment to seal the entry of F-DNA to plasmid producing transformed plasmid.
This is followed by entry of transformed plasmid to E coli host, the transformed bacteria are screened out and allowed to grow as pure culture. Expression of F-DNA yields specific protein to be expelled from the bacterial cell and can easily be purified in active form for human use. Transformed bacteria act as biological fermenter, growing continuously and producing the desired protein.
Expression vector A major objective of gene cloning is the expression of the cloned gene to produce recombinant proteins. But gene cloning doesnt guarantee successful expression.
Factors that influence gene expression: 1.The nature of the transcriptional regulatable promoter and terminator sequences; 2. The strength of Shine Dalgarno Sequence (ribosome binding site); 3. Efficiency of translation (mRNA stability, mRNA secondary structure..) ; 4. Post-translational processing: glycosylation, proteolytic processing;
5. The intrinsic stability of the protein (misfolding of the proteins? susceptible to proteolysis?) . Bacterial virus -phage with ds DNA genome can also be used as vector for F-DNA and the expressed protein is found in the plaque fluid of phage infected bacterial lawn.
Transformation of animal Transformation of whole animal i.e. generation of transgenic animal After in vitro fertilization, F-DNA with specific promoter ( say, -lactoglobin promoter) is injected into male pro-nucleus (relatively large in size) of the undivided zygote. This zygote is grown up in the lab to 8-cell stage and then introduced into the uterus of pseudo-pregnant mother or surrogate mother. After stipulated gestation period, the mother would deliver transgenic animal, with
F-DNA in all the cells of the animal. But it would be expressed under appropriate condition depending on the nature of the promoter used. For example, as -lactoglobin promoter is used and introduced in to the zygote of goat/cow, recombinant protein would be found in the milk. GENOMIC LIBRARY A genomic library is a set of cloned fragments that collectively represent the genes of a particular organism, tagging them and
preserving as reference for future use. Particular genes can be isolated from DNA libraries, much as books can be obtained from conventional libraries. Complete genome is fragmented by specific restriction endonuclease, with each fragment being transferred via vector to bacteria and grown as clone. Each clone carrying specific gene can be identified, isolated and characterized, based on the concerned protein synthesis.
This process of dividing the entire genome into functionally clonable entities, as determined through protein synthesis by respective transformants, leads to what is called creation of geneomic library, clone bank or gene bank. The genomic library is used To obtain the sequence of genes for analysis, amplification, cloning and expression.
To synthesize probes, primers etc., once the sequence is known, for further diagnostic works using, say, PCR, hybridization, blot etc. To develop the aspect of gene therapy using the knowledge of the gene sequence. To use specific gene, here as specific clone, to synthesize specific protein. GENOME SEQUENCING IS THE BEGINNING OF THE APPLICATION OF GENE MANIPULATION TECHNOLOGY FOR BETTERMENT OF LIFE.
RDT in diagnosis Precise diagnosis of a disease is pre-requisite for appropriate treatment. Diagnostic protocol demands specificity, sensitivity, accuracy and re-producibilty. Virulence (vir) gene from a pathogen can be transferred to a plasmid, amplified through growth of transformant and
collected in bulk amount. vir gene is denatured and made radioactively labelled, to be used as probe. Pathogen from patients sample is collected, DNA isolated, bound to solid support, denatured and baked. Hybridization with radioactive probe( of known pathogen) followed by autoradiography specifically identify the pathogen
Radioactive probe may not be used in all labs. But for such works non-radioactive probes are used. An oligonucleotide complementary to F-DNA sequence is synthesized using Biotin-tagged deoxyribonucleoside triphosphate, say B-dTTP along with normal nucleotides. Avidin from egg white or streptovidin, a bacterial protein, binds specifically to biotin, here biotin-tagged DNA probe. Hybridization is carried out by transferring the dsDNA to
nitrocellulose sheet, followed by denaturation, baking and hybridization with Biotin-probe. At one stage Enzyme-bound to biotin is added to bind to the hybrid. Hybridization is confirmed by changing of the colourless dye to cloured dye by the enzyme (e.g. if ALP is used, it will convert colourless nitrophenol phosphate to yellow-coloured nitrophenol to be identified).
Diagnosis using non-radioactive probe Immunological technique Genetic disorders Disturbances in unequal distribution of chromosome during gamete
formation and fertilization leads to baby with abnormal chromosome number detected by karyotyping, e.g. Downs syndrome; Turner disease; Klenifelter syndrome. Mutation leading to defective gene expression with abnormal protein synthesis may lead to defective enzyme or defective receptor leading to metabolic abnormalities. Defects could be autosomal/sex-linked, being dominant/recessive. Abnormal symptoms of specific disease can be developed at
different period of life cycle. Diagnosis of genetic disorders Genetic counselling considering screening of the parents. Screening before implantation of embryo in the case of IVF.
Prenatal diagnosis using fetal sample for enzyme assay or detection of abnormal gene: Amniocentesis at the middle of second trimester of conception Chorionic Villus Sampling in the first trimester. Population screening Newborn screening using blood sample:
- Thalassemia among in-risk ethnic group. Huntington disease Autosomal dominant disorder, with 1 : 10000 chance of having defective baby. Heterogygous parent (showing no symptom) can have 50% children defective. Progressive neurodegerative disorder being expressed
around mid 30s of age. Huntington Disease Autosomal dominant disorder, with 1 : 10000 chance of having defective baby. Heterogygous parent (showing no symptom) can have 50% children defective.
Progressive neurodegerative disorder being expressed around mid 30s of age. Results of electrophoresis of PCR in Huntington's disease :6 of the 11 children developed Huntington disease at the age specified, showing CAG triplet level 38 to 100. Autosomal recessive disorder with mutation in CFTR (cystic fibrosis
transmembrane conductance regulator) gene. CFTR with 1480 amino acid residue forms Chloride ion channel across the membrane, regulating flow of salt and water. Mutation/defect in gene causes deletion of a triplet with CFRT synthesized having 508th Phe missing. Defect leads to no regulation, salt accumalation on epithelial cells, mucous built up on cell surface leading to chronic progressive infections like RTI as pathogens are trapped in thick mucous.
Gene manipulation tech. in vaccine development Through deletion of virulent gene retaining immunogenicity. Creation of a live non-pathogenic carrier system having antigenic determinant of a pathogen. Production of sub-unit vaccine by using transformed nonpathogenic host to synthesize pathens antigenic determinant, e.g. HBsAg-vaccine. In vitro packaging virus to generate pseudo capsid to be used
as vaccine, e.g Gardasil, a HPV vaccine generated through quadrivalent In vitro assembly of capsid proteins of four different strains/serotypes. Biopharmaceutical production Insulin, growth hormone etc. Dnase I and alginate lyase as probable option to clrear thick mucous developed on lungs of Cystic Fibrosis patient.
-antitrypsin to inhibit bacterial/viral polyprotein processing, would be an option to treat CMV infection. Anti-sense RNA / miRNA synthesized against some important mRNA of pathogen (produced in infected cells) can be used to control pathogen growth and hence infectious diseases. DNA fingerprinting
Every individual has specific minisatellites of different sizes (e.g. tandem repeat of 9-70 bp), also called STR (short tandem repeat), of different number in specific chromosome in the non-coding region. Specific promoter complementary to flanking regions of specific minisatellite can be used for PCR dependent amplification. Even flanking region could be the target of specific restriction
endonuclease. Using different minisatellites of different chromosomes and using Southern blotting and minisatellite specific radioactive probe, banding pattern of individual can be developed for comparison. The Combined DNA Index System (CODIS) blends computer and DNA
technologies, using 13 primers, into an effective tool for comparing DNA profiles, indicating that it is unlikely to have two individual having similar banding pattern among the total population on earth. CODIS utilizes computer software to automatically search its two indices for matching DNA profiles. CODIS software enables State, local, and national law enforcement crime laboratories to compare DNA profiles electronically, thereby
linking serial crimes to each other and identifying suspects by matching DNA profiles from crime scenes with profiles from convicted offenders. The success of CODIS is demonstrated by the thousands of matches that have linked serial cases to each other and cases that have been solved by matching crime scene evidence to known convicted offenders. To solve the forensic problems, data base of the population as well as criminals should be preserved.
DNA fingerprinting Crime solving Some application of gene manipulation technology It helps in developing genomic library as reference for future use.
It helps in diagnosis of diseases, specially infectious and genetic disorders. The technology contributes in vaccine development. The technology helps design, development and production of biopharmaceuticals. Concept of personalized/tailored drugs developed as its application. DNA fingerprinting, an application helps
Solving forensic investigation of crimes Solving paternity suits Identification of mutilated bodies of victims. The technology is applied in agriculture: Development of stress resistant cultivars. Utilization of bio-fertilizers for improving soil qualitiese.g. use of Rhizobium spp., Azolla spp. Etc. Application of biopesticide, like BT-toxin through introduction
of cry-gene into seed to kill pests at larval stage. Development of herbicide resistant herbs e.g. Through introduction of glyphosate resistant 5-enolpyruvylshikimate-3phosphate (EPSP) synthase gene from E coli to ,say rice seed. Developing system to synthesize antisense RNA for acetyl cyclopropane synthetase gene preventing /slowing ethylene synthesis for fruit ripening. Similar method is used to develop Flvr-Savr Tomato now consumed in USA.
Development of Golden rice through introduction into rice seed of psy (phytoene synthetase) gene from daffodil and crot(carotene desaturase) gene from Erwinia spp. To synthesize -carotene in rice to overcome vitamin A deficiency, specially in children. THIS GENE MANIPULATION TECHNOLOGY WOULD DEFINITELY IMPROVE FURTHER THE QUALITY OF HUMAN LIFE IN THE VERY NEAR FUTURE. THE FUONDATION FOR THE
DEVELOPMENT OF THIS TECHNOLOGY IS BASED ON THE SKILL IN THE IMPLEMENTATION OF BIOCHEMICAL KNOWLEDGE OF LIVING ORGANISMS. FELLOW BIOCHEMISTS, IT IS YOUR TURN TO RAISE THIS TECHNOLOGY AND DERIVE ITS FRUITS. THANK YOU
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