* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Principles_of_Genetic_engineering
Epigenetics in learning and memory wikipedia , lookup
Genetically modified food wikipedia , lookup
Saethre–Chotzen syndrome wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Public health genomics wikipedia , lookup
DNA supercoil wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Neuronal ceroid lipofuscinosis wikipedia , lookup
Primary transcript wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Gene expression programming wikipedia , lookup
Non-coding DNA wikipedia , lookup
Gene expression profiling wikipedia , lookup
Gene desert wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Epigenomics wikipedia , lookup
Genome (book) wikipedia , lookup
Genome evolution wikipedia , lookup
DNA vaccination wikipedia , lookup
Gene nomenclature wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Gene therapy wikipedia , lookup
Point mutation wikipedia , lookup
Molecular cloning wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Genomic library wikipedia , lookup
Genome editing wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Designer baby wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Helitron (biology) wikipedia , lookup
Microevolution wikipedia , lookup
Genetic engineering wikipedia , lookup
Principles of Genetic engineering To describe the main stages in genetic engineering Genetic engineering: recombinant DNA technology, – altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. • inserting a foreign gene from one species into another, forming a transgenic organism • altering an existing gene so that its product is changed • changing gene expression so that it is translated more often or not at all. Purpose of GMOs • Improved feature – Herbicide resistance gene inserted into a plants’ genome • For synthesis of useful products – For synthesis of useful products – Human hormones mass-produced in bacteria • insulin, growth hormone – -carotene in rice grains • turns into vitamin A when eaten Basic steps in genetic engineering 1. Isolate the gene 2. Insert into a vector 3. Insert modified vector into host cell (Transformation & selection) 4. Allow host to multiply and to synthesise protein 5. Separate and purify the product of the gene Step 1: Isolating the gene A. Using restriction enzymes 1. A probe is used to locate the gene 2. Restriction enzymes recognise restriction sites and cut out gene • Restriction sites are palindromic – The same sequence when read left to right (5’ to 3’) on one strand and right to left on complementary strand • Originally obtained from bacteria – Cuts up phage DNA – DNA of virus that infect bacteria – Why is this useful? – Why are the staggered cuts called sticky ends? 3. Terminal transferase enzyme can add sticky ends if restriction enzyme generates blunt ends B. Using Reverse transcriptase • Gene for this enzyme originally found in retroviruses (contain RNA instead of DNA) • why is the enzyme useful for these? • Converts mRNA into single-stranded cDNA – E.g. insulin mRNA from B-cells of islets of Langerhans • Then DNA polymerase produces complementary strand to form double stranded DNA • Advantage – more mRNA in cell than DNA • Why is it an advantage to use cDNA if you are inserting a eukaryotic gene into a prokaryote? C. Using an automated sequencer • Amino acid sequence of protein analysed • Gene for protein synthesised • Using triplet code Step 2: Inserting gene into a vector • Vector – molecule of DNA which is used to carry a foreign gene into a host cell, e.g. – Bacterial plasmids • double stranded circular DNA – Virus genomes • Can carry large fragments – Yeast cell chromosomes • • Recombinant DNA DNA from different sources That have been combined Plasmid and foreign DNA are cut with the same enzyme – Generates complementary sticky ends that can anneal Ligase enzyme seals gaps/nicks in S-P backbone – forms phosphodiester bonds between inserted gene and the plasmid Step 3: Transformation (bacteria taking up plasmid) 1. Soak E.coli in CaCl, mix with plasmid, mild heat shock – Makes membrane more permeable to plasmids 2. Electroporation – High voltage pulse disrupts membrane 3. Microinjection – using fine micropipette 4. Viral infection – using virus’ own mechanism to insert DNA 5. Bacterial infection – Agrobacterium tumefaciens which naturally insert Ti plasmids into plant genome 6. Liposomes – containing DNA easily cross lipid membrane Selection Colonies of bacteria will grow from each bacterial cell which – Did not take up plasmid – Took up plasmid which re-sealed without inserted gene – Transformed bacteria : Those that took up recombinant plasmids (i.e. have inserted gene) How can we tell the difference? Using antibodies for protein produced Adding a fluorescent marker gene to plasmid o Glowing bacteria have plasmid Replica plating Replica plating Using plasmids with 2 genes for antibiotic resistance Plasmid used carries – ampicillin resistance gene (amp) Allows only transformed bacteria to grow in agar plates with ampicillin antibiotic (‘amp plates)’ – Tetracyclin resistance gene (tet) Gene is inserted within this gene Tet gene inactivated Transformed bacteria cannot grow in ‘Tet plates’ Those that grow in amp plates but not in tet plates are transformed bacteria Step 4: Multiplication of the host cells by cloning Large scale fermenters Bacteria undergo binary fission o Large numbers produced quickly o E. coli divide every 20 min o All genetically identical because of asexual reproduction Transcription and translation Step 5: Extraction and purification of protein Bacteria killed and separated from proteins by centrifugation Protein of interest separated from others by – Large scale chromatography – ultrafiltration