* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download SAR_Gene_technology
Transposable element wikipedia , lookup
Gene nomenclature wikipedia , lookup
Gene expression profiling wikipedia , lookup
Epigenetics wikipedia , lookup
DNA profiling wikipedia , lookup
Genome (book) wikipedia , lookup
SNP genotyping wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Genome evolution wikipedia , lookup
DNA polymerase wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Gene therapy wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Cancer epigenetics wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Point mutation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Primary transcript wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
DNA supercoil wikipedia , lookup
Epigenomics wikipedia , lookup
DNA vaccination wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Genetic engineering wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Genomic library wikipedia , lookup
Genome editing wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Molecular cloning wikipedia , lookup
Designer baby wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Microevolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Helitron (biology) wikipedia , lookup
GENE TECHNOLOGY Objectives: •To describe how sections of DNA containing a desired gene can be extracted from a donor organism using enzymes. •To explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase. •To state other vectors into which fragments of DNA may be incorporated. What is genetic engineering? • The processes used to obtain a specific gene and place it in another organism • The recipient organism expresses the gene and is transgenic (or GMO) • The new DNA is said to be recombinant DNA because it contains DNA from another organism or source Why do we want to genetically engineer organisms? • Improving the recipient organism e.g. herbicide resistant crops, growth promotion (the myostatin gene for muscle growth) in animals • Creating organisms to make useful products e.g. insulin or GH production, production of pharmaceuticals through ewe milk, beta-carotene production in rice An overview of the process 1. 2. 3. 4. 5. Obtain copy of gene (from a donor) Place gene in a vector Introduce vector to host cell Select cells containing donor DNA Clone the gene Obtaining a copy of the Gene Three main methods: 1. make a copy of the gene from its mRNA using reverse transcriptase 2. Artificially synthesise the gene 3. Cut up the DNA with restrictions endonucleases and search the DNA containing the gene – shot gun approach or use a DNA probe to identify the DNA before cutting it Reverse transcriptase • Reverse transcriptase is a polymerase enzyme • It performs the reverse of transcription • It synthesises DNA from an mRNA template • Complementary DNA (cDNA) is DNA made from mRNA Making cDNA from mRNA The principle • mRNA is complimentary to the DNA in a specific gene • Reverse transcriptase is able to make a strand of DNA that is complimentary to the mRNA • If the mRNA for a specific gene is isolated then the gene can be synthesised using reverse transcriptase • The DNA formed is called complimentary or cDNA • Animation of the Formation of cDNA b) Synthesising a gene • The base sequence of the DNA can be worked out from the aa sequence of the required protein. • The gene can then be constructed using free nucleotides and joining them together in the right order by a polynucleotide sequencer • This is only possible for short genes at present. C) Restriction Endonucleases • Enzymes that cut DNA at specific sites • Cut the bonds in the middle of the polynucleotide chain • Most enzymes make a staggered cut in the two strands, forming sticky ends which are ssDNA • Some restriction enzymes cut straight across both chains, forming blunt ends • Artificial sticky ends need to be added to blunt ends Restriction Enzymes Animation Steps in Cloning a Gene Animation Inserting a gene into a vector Placing the Gene in the vector • The most common vectors are plasmids (other e.g.s are certain virus genomes or yeast cell chromosomes). • Plasmids are extracted from bacterial cells by centrifugation • Restriction endonuclease enzymes are used to cut the plasmid DNA and the gene from the donor DNA • Once cut the plasmids and cDNA are mixed with DNA ligase • This allows the sticky ends to form complimentary base pairs by forming covalent phosphodiester bonds • Recombinant DNA is produced DNA ligase catalyses the formation of phosphodiester bridges, splicing the DNA into the plasmid DNA. Introducing the Vector DNA into the Host Cell • The plasmid is now introduced into a bacterial host cell to multiply up • A mutant harmless form of E. coli is commonly used because it has a doubling time of 30 minutes • E. coli, the plasmids and ca2+ ions are given a brief heat shock which temporarily makes the CSM permeable to DNA • This process of adding new DNA to a host is called transformation and produces transformed or transgenic bacteria • (there are a range of alternative methods on p174 – make a list) Genetic Engineering of Human Insulin as an example There are 2 major problems with this technique. 1. Not all bacteria will take up the plasmids. 2. Not all the plasmids will have taken up the foreign DNA i.e. Be recombinant plasmids. Solution to Problem 1 • Use plasmids with antibiotic resistance genes E.g. ampicillin • Grow the bacteria on agar containing that antibiotic then only those that have taken up the antibiotic resistant plasmids will survive. BUT: • You know the bacteria have taken up the plasmid but you still don’t know if they took up RECOMBINANT plasmids. • How could we tell if the bacterium had taken up a recombinant plasmid? Solution to Problem 2 • Use a restriction enzyme will cut through a different antibiotic resistance gene e.g. tetracycline. • The recombinant plasmids will have the human insulin gene inserted in the middle of the tetracycline resistance gene, meaning that it no longer has resistance. • Use replica plating to identify the recombinant colonies Replica Plating The colonies that can grow on the tetracycline are NOT transformed, so the transformed colonies can be identified from the original ampicillin plate Homework Make notes on the advantage of bacterial conjugation for taking up recombinant DNA and the experiments on pneumonia in mice and their findings (p176-177) (note conjugation animation in scheme)