Genetic Engineering

Genetic Engineering

ByAliyu, Habibu1Biotechnology All inclusive term for several technologies including but not limited to recombinant DNA. Refers to the use of technology in applications for solving fundamental problems in biology. the application of organisms/Biological systems/ processes in the manufacturing and Service industries.Genetic Engineering Use of techniques involving recombinant DNA technology to produce molecules and/or organisms with new properties/heritable, directed alteration of an organisms DNA.

2Recombinant DNA technologySet of molecular techniques for locating, isolating, altering, and studying DNA segments. The term recombinant is used because frequently the goal is to combine DNA from two distinct sources. Genes from two different bacteria might be joined, for example, or a human gene might be inserted into a viral chromosome.Used to create a number of commercial products, including drugs, hormones, enzymes, and cropsRecombinant DNADNA molecule produced artificially and containing sequences from unrelated organisms.

History

1966 The genetic code is deciphered when biochemical analysis reveals which codons determine which amino acids.

1970 Hamilton Smith, at Johns Hopkins Medical School, isolates the first restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide sequence. Over the next few years, several more restriction enzymes will be isolated.

1972 Stanley Cohen and Herbert Boyer combine their efforts to create recombinant DNA. This technology will be the beginning of the biotechnology industry.

1976 Herbert Boyer cofounds Genentech, the first firm founded in the United States to apply recombinant DNA technology

1978 Somatostatin, which regulates human growth hormones, is the first human protein made using recombinant technology.

1983 Kary Mullis does PCR. 1985 Kary Mullis publishes method. Patents follow.2000? Gateway cloning5In 1973, a group of scientists produced the rst organisms with recombinant DNA molecules. Stanley Cohen at Stanford University and Herbert Boyer at the University of California School of Medicine at San Francisco and their colleagues inserted a piece of DNA from one plasmid into another, creating an entirely new, recombinant DNA molecule. They then introduced the recombinant plasmid into E. coli cells. Within a short time, they used the same methods to stitch together genes from two different types of bacteria, as well as to transfer genes from a frog to a bacterium.They called the hybrid DNA molecules chimeras, after the mythological Chimera, a creature with the head of a lion, the body of a goat, and the tail of a serpent. These experiments ushered in one of the most momentous revolutions in the history of science.

Needles in HaystacksHow 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 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.Polymerase Chain Reaction Based on DNA polymerase creating a second strand of DNA. Needs template DNA and two primers that flank the region to be amplified. Primers are short (generally 18-30 bases) DNA oligonucleotides complementary to the ends of the region being amplified.DNA polymerase adds new bases to the 3' ends of the primers to create the new second strand.go from 1 DNA to 2, then 4, 8, etc: exponential growth of DNA from this region A key element in PCR is a special form of DNA polymerase from Thermus aquaticus, a bacterium that lives in nearly boiling water in the Yellowstone National Park hot springs. This enzyme, Taq polymerase, can withstand the temperature cycle of PCR, which would kill DNA polymerase from E. coli. advantages:rapid, sensitive, lots of useful variations, robust (works even with partly degraded DNA)disadvantages:Only short regions (up to 2 kbp) can be amplified. limited amount of product made

Restriction endonucleasesAlso called restriction enzymes: digest DNA at specific sequences

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 species3. 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.

Plasmid is cut open with a restriction enzyme that leaves an overhang: a sticky endForeign DNA is cut with the same enzyme.The two DNAs are mixed. The sticky ends anneal together, and DNA ligase joins them into one recombinant molecule.The recombinant plasmids are transformed into E. coli using heat plus calcium chloride.Cells carrying the plasmid are selected by adding an antibiotic: the plasmid carries a gene for antibiotic resistance.9Sequence Recognition -R.E.Restriction endonucleases -- cut double stranded DNA at specific sequences, protection against viruses in bacteria. Sequences often palindromes: a sequence which is the same when read in either direction. A man a plan a canal: Panama

10Cloning Vector TypesFor different sizes of DNA:plasmids: up to 5 kbphage lambda () vectors: up to 50 kbBAC (bacterial artificial chromosome): 300 kbYAC (yeast artificial chromosome): 2000 kbPlasmidsExtrachromosomal, circular small (2-3 kb) DNA in a bacterial cell which can replicate independently but which cannot integrate into the host chromosome. Drug resistance plasmids are not essential for the cell's growth, but confer antibiotic resistance. Plasmids used for molecular cloning have been artificially created by recombining fragments of various existing plasmids. Plasmids contain multiple cloning sites with several restriction endonuclease sites.

Example of a Plasmid

Example of a plasmid + insert (DNA of interest)

DNA ligaseDNA ligase joins 5'-phosphate and 3'-hydroxyl ends of DNA Two fragments formed by EcoRI can be rejoined by ligase.Similarly, Eco RI fragments from two different pieces of DNA can be joined15Ligation

Tools of recombinant DNA - cloning

Sources of DNA to Clone Genomic DNA: cut up whole genome and clone small pieces. Advantage is, you get everything. Disadvantage is, a lot of it is junk.Two general methods:1. randomly shear DNA into small pieces, then ligate linkers to the ends: oligonucleotides that contain a useful restriction site.2. partially digest the DNA with a restriction enzyme that has a 4 base recognition site. These sites will appear at random every 256 (44) base pairs. Take long pieces.cDNA: DNA copy of mRNA, made with reverse transcriptase. Advantage: you just get the expressed genes. Disadvantages: you don't get control sequences or introns, and frequency depends on level of expression.

Synthetic DNA: synthesized de novo (for example multiple cloning sites or linkers), or made by PCR

Basic Cloning Process

Plasmid is cut open with a restriction enzyme that leaves an overhang: a sticky endForeign DNA is cut with the same enzyme.The two DNAs are mixed. The sticky ends anneal together, and DNA ligase joins them into one recombinant molecule.The recombinant plasmids are transformed into E. coli using heat plus calcium chloride.Cells carrying the plasmid are selected by adding an antibiotic: the plasmid carries a gene for antibiotic resistance.

Recombinant DNA molecule

Inserting recombinant DNA into HostTransformation cell made competent to take up DNA competent cells: electroporation poke holes in membrane and calcium chloride- make cells more permeable to DNA

Transfection when the cloning vector used has aspects of a virus, the host cell can be infected (transfected) to insert the recombinant molecule Electroporation the cell is placed in an electric field such that small pores are temporarily opened in the membrane. Added DNA can enter through these pores.

Transformation

SelectionAntibotic resistancePlasmid vector contains an ampicillin resistance gene making the cell resistant. Growth of transformed cells (cells receiving the plasmid) can be identified on agar medium containing (e.g.) ampicillin.

Transformation

Further selectionThe plasmid vector contains another identifiable gene (e.g., a second drug resistance or an enzyme activity), with the coding sequence of this gene containing the restriction site for insertion. Insertion of the foreign DNA at this site interrupts the reading frame of the gene and result in insertional mutagenesis. In the following example, the -galactosidase gene is inactivated. The substrate "X-gal" turns blue if the gene is intact, ie. makes active enzyme. White colonies in X-gal imply the presence of recombinant DNA in the plasmid.

X-gal selection

29Applications Gene products- using GMOs ie microbes to produce chemicals used for medical or industrial purposesNew phenotypes- using gene technology to alter the characteristic of an organism ie farm animals or cropsGene therapy- using gene technology on humans to treat disease Transfer a gene of interest from human into a host usually microbesProduct produced quickly cheaplyUsed in commercially important crops or farm animals to increase their yield

DisadvantagesSafety of foodAdverse effect on human health or ecosystemToxinsAllergiesDecreased nutritional valueAntibiotic resistant bacteria