Recombinant DNA Technology Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry A – A good student is liked by teacher G – Greets everyone with smile O – Obedient O – On time for college D – Dresses neatly S – Studies with interest T – Treats everyone with smile U – Understands everything D – Does daily home work E – Eager to know new things N – Never misbehaves T – Talks little in class Biotechnology may be defined as “the method by which a living organism or its parts are used to change or to incorporate a particular character to another living organism” It involves the application of scientific principles to the processing of materials by biological agents. Genetic recombination is the exchange of information between two DNA segments. This is a common occurrence within the same species. But by artificial means, when a gene of one species in transferred to another living organism, it is called recombinant DNA technology. In common, this is known as genetic engineering. Definition of recombinant DNA • Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other • DNA fragments are usually derived from different biological sources Definition of recombinant DNA technology • A series of procedures used to recombine DNA segments. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate. History of recombinant DNA technology • Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973. Development of molecular biology • Early research on prokaryotic genetics and the development of molecular techniques has led to a new discipline called MOLECULAR BIOLOGY • “Tools” have been developed (and still continue to be modified/improved) to enable scientists to examine very specific regions of the genome or genes. • Advances in Molecular Biology – The combination of restriction/modification enzymes and hybridization techniques enable the application of a wide variety of procedures TECHNIQUES and PROCEDURES • • • • • Gene isolation/purification/synthesis Sequencing/Genomics/Proteomics Polymerase chain reaction (PCR) Mutagenesis (reverse genetics) Expression analyses (transcriptional and translational levels) • Restriction fragment length polymorphisms (RFLPs) • Biochemistry/ Molecular modeling • Gene therapy Applications • • • • • • • Quantitative preparation of biomolecules Recombinant Vaccines Antenatal diagnosis of genetic diseases Monoclonal antibodies Cell/tissue culture To identify mutations in genes Xenotransplantation Contd… • To detect activation of oncogenes • Production of next generation antibiotics • Forensics • Biosensors • Genetically modified crops • Bioterrorism detection 1.Quantitative Preparation of Biomolecules • If molecules are isolated from higher organisms, the availability will be greatly limited. • For eg.- To get 1 unit of growth hormone, more than 1000 pituitaries from cadavers are required. • By means of recombinant technology, large scale availability is now assured. 2. Risk of contamination is Eliminated • It is now possible to produce a biological substance without any contamination. • Hepatitis, caused by HBV, is highly contagious. • Preparations of vaccines or clotting factors are free from contaminants such as hepatitis B particles. • RD-Technology provides the answer to produce safe antigens for vaccine production. 3. Specific probes for Diagnosis of Diseases Specific probes are useful for: i. Antenatal diagnosis of genetic diseases. For eg.- many of the single gene defects like cystic fibrosis, phenyl ketonuria etc. Could be identified by taking cell samples from fetus. ii. To identify viral particles or bacterial DNA in suspected blood and tissue samples. Contd… iii. To demonstrate virus integration in transformed cells. iv. To detect activation of oncogenes in cancer. v. To pinpoint the location of a gene in a chromosome. vi. To identify mutations in genes. 4. Gene Therapy • It is an important applications of RD-Technology • Normal genes could be introduced into the patient so that genetic diseases can be cured. • How to find one gene in large genome? • A gene might be 1/1,000,000 of the genome. Three basic approaches: • 1. Cell-based molecular cloning: create and isolate a bacterial strain that replicates a copy of your gene. • 2. Polymerase chain reaction (PCR). Make many copies of a specific region of the DNA. • 3. Hybridization: make DNA single stranded, allow double strands to re-form using a labeled (e.g. radioactive) version of your gene to make it easy to detect. Basic principle of recombinant DNA technology • The DNA is inserted into another DNA molecule called ‘vector’ • The recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced Cell-Based Molecular Cloning • The original recombinant DNA technique: 1974 by Cohen and Boyer. • • Several key players: • 1. restriction enzymes. Cut DNA at specific sequences. e.g. EcoR1 cuts at GAATTC and BamH1 cuts at GGATCC. • – Used by bacteria to destroy invading DNA: their own DNA has been modified (methylated) at the corresponding sequences by a methylase. • 2. Plasmids: independently replicating DNA circles (only circles replicate in bacteria). Foreign DNA can be inserted into a plasmid and replicated. – Plasmids for cloning carry drug resistance genes that are used for selection. – Spread antibiotic resistance genes between bacterial species 3. DNA ligase. Attaches 2 pieces of DNA together. 4. transformation: DNA manipulated in vitro can be put back into the living cells by a simple process . – The transformed DNA replicates and expresses its genes. Best Way to keep COOL in SUMMER ? ! ? II. Restriction Endonucleases A. Origin and function • Bacterial origin = enzymes that cleave foreign DNA • Named after the organism from which they were derived – EcoRI from Escherichia coli – BamHI from Bacillus amyloliquefaciens • Protect bacteria from bacteriophage infection – Restricts viral replication • Bacterium protects it’s own DNA by methylating those specific sequence. B. Availability • Over 200 enzymes identified, many available commercially from biotechnology companies C. Classes • Type I – Cuts the DNA on both strands but at a nonspecific location at varying distances from the particular sequence that is recognized by the restriction enzyme – Therefore random/imprecise cuts – Not very useful for rDNA applications • Type II – Cuts both strands of DNA within the particular sequence recognized by the restriction enzyme – Used widely for molecular biology procedures – DNA sequence = symmetrical • Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence • Some enzymes generate “blunt ends” (cut in middle) • Others generate “sticky ends” (staggered cuts) – H-bonding possible with complementary tails – DNA ligase covalently links the two fragments together by forming phosphodiester bonds of the phosphate-sugar backbones DNA Ligase in Action! III. Vectors for Gene Cloning “To be good & to do good that is the whole of religion”
