Genetic engineering- definition, objectives and basicPresentation methodologyout line Recombinant DNA technology- definition, objectives, history and enzyme tools. Restriction enzymes - features, types of cuts. Restriction mapping- applications Vector DNA – types, characteristics and practical features Host cell types Recombinant DNA technology- procedure Gene transfer technology Screening technology Applications
Genetic engineeringDefinition -1• A set of techniques capable to allow the identification, manipulation and multiplication of genes of living organisms.Definition -2• Changing of genes by using in vitro processes.Other names• Gene manipulation, gene cloning• Recombinant DNA technology, genetic modification
Objectives of Genetic Engineering Basic research on gene structure and function Production of useful products and services Generation of transgenic plants and animals Investigation of human genome for gene therapy
Recombinant DNA technology r DNA technology • The technology of preparing r DNA in vitro by cutting up DNA molecules and splicing them together fragments from more than one organism.
Meaning r DNA technology Modifying the genetic make up an organism by: • Adding new genes • Changing the existing genes
Objectives of Artificially synthesize newr DNA genes.Technology Altering the genome of an organism. Bring about new gene combinations not found in nature. Understanding the hereditary diseases and their cure. Improving human genome.
Discovery of DNA structure- Watson and Crick in 1953 Isolation of DNA ligase in 1967History ofrecombinantDNA Isolation of REase in 1970technology Jackson, Symons, and Berg (1972) generated first recombinant DNA molecules. Cohen and Boyer (1973) produced first plasmid vector capable of being replicated within a bacterial host
Biological tools of r DNA technology Foreign DNA/ Vehicle/vector Culture media,Enzymes Host cells Synthetic DNA DNA Buffers, reagents
DNA or RNA polymerase-Chemical replicating or annealing a DNAknives in chain.molecular Reverse transcriptase – synthesizecarpentry c DNA from RNA template.Enzymes are DNA ligase – joining DNA strandschemical knives together.in r DNA Nuclease-breaks phospho-diestertechnology bonds within free ends (exonucleases) or in an interior position( Endonucleases ). Restriction endonuclease – recognize a specific base sequence and cuts the DNA.
A special class of sequence –Restriction specific enzymesendonucleases Found in bacteria which protect its(RE ases) genetic material from the invasive attacks of viruses. Site-specific - cleave DNA molecules only at specific nucleotide sequences. REases recognize DNA base sequences that are palindromes. REases make two single stranded breaks, one in each strand. RE ases make staggered cuts with complementary base sequences for easy circularization.
Recognition sequences of RE ases are palindromesRestriction A palindrome is a word, phrase,Enzymes- number or other sequence of unitsTypes of cuts that can be read the same way in either direction Cohesive (sticky) ends – overhanging single-stranded ends Blunt ends – double-stranded, non-overhanging ends
Salient features Bacterialof Restriction Endonucleases enzymesendonucleases(RE ases) Different REases Names of REases Isolated from are derived different from source bacteria bacteria. Cut DNA into Basic tool of defined and Genetic reproducible fragments Engineer
A map showing the unique sitesRestriction of cutting of the DNA of amapping particular organism by a single REase enzyme. A particular REase generates a unique set of DNA fragments with specific base sequence. Another enzyme will generate a different set of DNA fragments from the same DNA molecule. The family of DNA fragments generated by a single enzyme can be detected easily gel electrophoresis.
Preparing a restriction mapApplications of of DNARestrictionendonucleases Fragmenting genomic DNA(RE ases) prior to Southern Blotting. Generating DNA fragments RE ases catalyze that can be sub-cloned in sequence – appropriate vectors. dependent double- stranded breaks in Generating DNA fragments DNA yielding a for labeled probes homogeneous population of DNA fragments.
Vector DNA or Plasmid DNAVehicle DNA Bacterial Bacteriophage DNAs DNAThe DNA which Vectoracts as a carrier is DNAa vehicle DNA Viral DNAs Yeast DNAs
Types of VectorsTypes of Bacterial plasmid vectorsvectors Bacteriophage vectors Cosmid vectors Expression vectors Bacterial Artificial Chromosomes (BAC) Yeast Artificial Chromosomes (YAC) Ti and Ri vectors
Capable of autonomous replication independent of theCharacteristics main bacterial chromosomeof anprokaryotic Easy to isolate, i.e. small.vector Non -toxic to host cells. Have space for foreign inserts. Have unique restriction sites for common restriction enzymes. Have convenient markers for selection of transformants, e.g. antibiotic resistance genes Be relaxed, i.e. multiple copies in a host cell.
Size Origin of replication (ori) Multiple cloning site (MCS)Practical Selectable marker genesFeatures of RNA polymerase promoterDNA Cloning sequencesVectors DNA sequencing primers
Plasmid DNA – small circular DNA found inPlasmids bacteria They replicate autonomously. Easily purified Confer antibiotic resistance to host bacteria –allow easy identification. First type of cloning vector developed.
Two types of phage vectorsPhage Vectors have been extensively developed-λ and M13. phage vectors have engineered phage genomes previously genetically modified to include restriction sites. after insertion of foreign DNA, the recombinant phage genome is packaged into the capsid and used to infect host cells
23 Hybrid vector constructed to contain features from bothCosmids phages and plasmids. Cosmids have a selectable marker, multiple cloning sites from plasmids and a cos site from l phage
24 large fragments of DNA canArtificial be cloned.Chromosomes Mapping of genes is easier. One copy of YAC is present per cell. yeast artificial chromosomes (YACs) bacterial artificial chromosomes (BACs) played important role in the human genome project
Two types of host-vectors Cloning • Propagation of vector DNA insertsExpression • Production of vector proteins
Isolate desired DNAMaking of Cut with a suitabler DNA REase Ligate into a suitable cloning vector Transform r DNA into a suitable host cell
Step 1– fragmentation - breaking apart a strand of DNAMolecularCloning /DNA cloning Step 2 – ligation-gluing together pieces of DNA in a desired sequence.Molecular cloningrefers to the Step 3 –Transfection -process of making inserting the newly formed DNA into cells.multiple DNAmolecules. Step 4-Screening / selection – selecting out the cells that were successfully tranfected with the new DNA
Recombinant DNA cloning procedure Screening of FineForeign Transfection transformants chemicals DNA Hormones Host Desired r DNA Enzymes cell products vaccinesPlasmid Antibiotics DNA Antibodies Blood factors
1. Choice of host organisms and cloning vectorDNA 2. Preparation of vector DNAcloning 3. Preparation of DNA to beprotocol – cloned7 steps 4. Creation rDNA. 5. Introduction of rDNA into the host organism. 6. Selection of organisms containing rDNA. 7. Screening for clones with desired DNA inserts and biological properties.
Gene transfer technologyTransduction • Virus mediated gene transfer Tranfection • Chemical or physical tricks to persuade cells to take DNA from the culture medium • Physically inserting the geneDirect transfer • e.g. microinjectionNatural gene • A receptor – mediated lateral binding • Fusogenic proteins used transfer
This method was described by Graham and Van der Eb in 1973.Calcium A process for inserting foreignphosphate – DNA into bacteriaco precipitate Treat bacterial cells with ice-coldmethod calcium chloride Add plasmid DNA to cells chilled on ice which form calciumTransformation of phosphate –DNA precipitate.Bacterial Cells Heat the cell and DNA mixture to 42oC Membrane becomes fluid and plasmid DNA enters bacterial cells and is replicated and expressed
It involves a brief application of high voltage electric current toElectroporation the cells resulting in the formation of transient holes in the cell membrane through which plasmid DNA can enter the cell. The transformation efficiency is high. Quick restoration of membrane fluidity and closing of pores is crucial for survival of cell after the pulse.
Selectiontechniques for DNArDNA Colony hybridizationmolecules immunoassay assay Screening by Genetic protein screening activity methods
After transformation, theProcess of bacteria are challenged withselection an antibiotic (such as ampicillin). If the E. coli have taken up and expressed an ampicillin resistance gene on aSelection is a plasmid, they will live -process designed to otherwise they will die.facilitate theidentification of This process is calledrecombinant bacteria selection because selectedwhile preventing thegrowth of non- bacteria may survive.transformedbacteria.
This technique was introduced by Grunstein andDNAhybridization Hogness (1978).assay The target DNA is denatured at 800C and bound to a nitrocellulose filter discs. Such filters are hybridized with radioactive DNA probes. The results are monitored by autoradiography.
The transformed colonies are transferred to a nitrocellulose filter.Colony The colonies are lysed and the releasedimmunoassay proteins are attached to the matrix. The matrix is treated with a primary antibody which specifically binds to the proteins encoded by the target gene. Then the matrix was washed to remove any unbound antibody. Then the matrix was treated with a second antibody which was an enzyme, alkaline phosphatase. the target protein (antigen)was treated with a colorless substrate. The colorless substrate is hydrolysed by the alkaline phosphatase into a colored complex.
contains only complementary DNA molecules synthesized from mRNA molecules in a cell.cDNA mRNA from tissue of interest isLibraries isolated Converted to a double-stranded DNA by using the enzyme reverse transcriptase Called complementary DNA (cDNA) because it is an exact copy of the mRNA
In situ hybridization Used to determine the cell type thatApplications is expressing the mRNAof Tissue of interest is preserved in aRecombinant fixative solution and embedded in a wax-like substanceDNA Tissue can be sliced into very thinTechnology - sections attached to microscopeStudying Gene slidesExpression Slides are incubated with a probe to the gene of interest Probe hybridizes with mRNA in cells Probe is detected
Studying Gene Expression Gene microarrays DNA microarray analysis Single-stranded DNA moleculesApplications of are attached onto a slide using aRecombinant robotic arrayer fitted with tiny pinsDNA Can have over 10,000 spots of DNATechnology Extract mRNA from tissue of interest, tag it with fluorescent dye, and incubate overnight with the slide mRNA will hybridize to spots on the microarray that have complimentary DNA sequences Slide is scanned with a laser that causes the spots to fluoresce
Started in 1990 by the U.S. Department of EnergyThe Human International collaborative effortGenome to identify all human genes and to sequence all the base pairs of theProject 24 human chromosomes 20 centers in 6 countries: China, France, Germany, Great Britain, Japan, and the United States. April 14, 2003, map of the human genome was completed Consists of 20,000 to 25,000 protein-coding genes
Medical products made from rDNA tech Product year treatmentHuman insulin 1982 diabetessomatotropin 1985 Pituitary dwarfismHepatitis B vaccine 1986 Immunization for hepatitis B virusErythropoietin 1988 anemiaInterleukin-2 1989 Cancer of kidneyWhooping cough vaccine 1989 Immunization for Whooping coughFactor VIII 1993 hemophilia
Dr.B.Victor is a highly experienced professor, recently retired from the reputed educational institution- St. Xavier’ sAbout the College, Palayamkottai, India-Presenter 627001. He was the dean of sciences, IQAC coordinator and assistant controller of examinations. He has more than 32 years of teaching and research experience He has taught a diversity of college courses and guided 12 PhDs. Send your comments to : firstname.lastname@example.org