Recombinant DNA Technology Human Experimentation with DNA Modern roses are the result of centuries of breeding between wild roses All dogs belong to a single species Variation in animals teaches us about variation amongst ourselves Why Recombinant DNA Technology Cloning of the Human Growth Hormone HGH or somatropin gene permits the production of HGH It is a 191 aa protein found at it s highest levels in children and young adults By cloning the human somatotropin gene and expressing it in bacteria young children with dwarfism can achieve close to normal size before cloning this was almost impossible Biotech Industry in the U S 1 The US is the largest market and leading consumer of biotech products in the work and home to more than 1300 firms 2 Between 2001 2010 the US bioscience industry grew by 6 4 adding more in that time total employment for all private sector industries in the US fell the 96000 jobs by 2 9 3 more than 5 5 million scientists engineers and technicians in the US 4 Research and development in the US biotech sector drives the commercialization of products for domestic consumption and international trade Industry Subsectors Medical Biotechnology The largest component of the biotech industry Key product areas biological drugs vaccines and invitro diagnostics In the past 10 years research testing and medical lab jobs have increased in the US by 24 Agricultural Biotech Key products biotech crops crop seeds related products the US accounts for more than of all biotech crops in the world Industrial Biotech Key products industrial application that cut across other industry sectors primary product categories nanotech enzymes and bio fuels one of the main drivers of demand for biofuels in the US is the RFS Organismal Cloning asexual reproduction in eukaryotes Molecular Cloning replication of DNA with aid of plasmid or viral vectors a small self replicating genetic element in appropriate host cells After coming a gene it s function can be investigated by many new molecular techniques 1 The nucleotide sequence can be determined Why do we clone genes 2 The amino acid protein sequence can be predicted from the DNA sequence 3 The protein function can be explored by comparing its amino acid sequence with those of other proteins stored in large databases 4 The cloned gene can be studied using molecular biology techniques 5 Recombinant genes and proteins there from can be used in medical Steps in Constructing Recombinant DNA molecules and other applicants 1 purify DNA from organism 2 Cut DNA with restriction endonucleases that recognize DNA at specific 3 Join restricted fragments into carrier molecules to make recombinant DNA sequences rDNA molecules 4 Introduce rDNA molecules into host cell by transformation 5 rDNA molecules replicate are cloned in host cell 6 Cloned DNA purified in large amount for study 7 Cloned genes can expressed to make proteins for application KEY POINT Natural existing enzymes are the main tool of rDNA tech Different restriction enzymes recognize different palindromes palindrome nucleotide pair sequence that reads the same forward or backward from a central axis of symmetry Restriction enzymes cut DNA at specific sites As long as the single stranded tails are complementary they can be ligated back together Electrophoresis allows DNA molecules to be separated by length Sugar phosphate backbone of DNA in negatively charged and will migrate toward the positive pole anode in an electric field Bacterial Cloning Vectors Essential Features of a Cloning Vector 1 Origin of replication for bacteria oriR Centromeres and telomeres for yeast 2 Several unique restriction sites for inserting clone may be in an 3 Selectable marker to distinguish host cells with vector from those artificial polylinker site without Other non essential features 1 Small plasmid 2 high copy number 3 Screenable marker e g in pUC18 if polylinker contains an insert then the lacZ gene is disrupted and an artificial substrate X gal cannot be broken down to a blue product and the bacterial colonies are white 4 Phage vectors can carry large inserts Figure 17 5 A diagram of the plasmid pUC18 showing the polylinker region located within a lacZ gene DNA inserted into the polylinker region disrupts the lacZ gene resulting in white colonies that allow direct identification of bacterial colonies carrying cloned DNA inserts The plasmid vector pUC18 carries unique restriction enzyme cleavage sites in a polylinker MSC region within the lacZ gene Cloning DNA in host cells Inserting fragments in a plasmid vector Cleavage of the plasmid and DNA to be cloned with a restriction enzyme and insertion of a DNA fragment into the polylinker region disrupts the lacZ gene making plasmids carrying inserts unable to metabolize Xgal resulting in the formation of white colonies on nutrient plates containing Xgal Paul Berg s Exp The first recombinant DNA molecules Other types of cloning vectors Bacteriophage were amongst the first cloning vectors used Bacterial Artificial chromosomes BACs and Yeast Artificial Chromosomes YACs are used to clone very large fragments in bacteria or yeast the latter have centromeres and telomeres at their ends Expression Vectors these are designed to ensure proper expression of a cloned gene in the relevant host species there are many expression vectors that differ in the promoters that they use Transgenic Plants have been made by manipulating a naturally existing Ti Plasmid that enables transfer of genes from bacteria to plant cells Once the cells receive the new DNA they can be regenerated to make a new transgenic plant carrying the incorporated gene Recombinant DNA libraries are collections of cloned Sequences A library is a set of clones each clone contains a small piece of a genome Genomic library contains at least one copy of all the sequences in a genome e g several million 5kb clones to cover the entire human genome 3 x 109 bp cDNA library contains the genes that are actively expressed in a given tissue make complementary cDNA from the mRNA of that tissue Fig 17 11 Libraries are screened for specific clones using a radioactive probe fragment of DNA e g a radioactive cDNA used to identify a genomic clone Difference between a genomic and a cDNA library Constructing a cDNA library from mRNA Recovering specific clones from a DNA library A technique commonly used to detect a bacterial colony carrying a particular DNA clone DNA Hybridization Denaturation and renaturation hybridization of DNA