General Biology/Genetics/Recombinant DNA Technology

Recombinant DNA technology Revolutionized modern biology Ability to manipulate genes in vitro Hybrid genes, including combining genes of different species Detailed study of gene function Determine nucleotide sequences of genes and their regulators (deduce amino acid sequences of proteins) Genome projects: complete nucleotide sequence of >40 genomes, including human Made possible by convergence of: discovery of restriction enzymes genetics of bacteria and their plasmids Recombinant DNA technology Uses Detailed study of gene function Homeostasis, response to stress Development (birth defects) Evolution of genes informs on evolution of life Human betterment Medicine Identification, treatment of genetic disorders Molecular medicine: from deduced amino acid sequences, design better drugs Foods Improve crop yield, resistance to disease Improve nutritional value Forensics DNA fingerprinting: guilt or innocence Restriction endonucleases Originally found in bacteria to prevent invasion of viral DNA, cuts double stranded DNA that is unmethylated, will not cut newly synthesized DNA since hemi-methylated, a product of semi-conservative replication of DNA sever phosphodiester bonds of both polynucleotide strands in order to combine foreign DNA create restriction fragments (restriction digestion) 5’ phosphate and 3’ –OH at ends usually nucleotide specific target sequence 4-6 bp most common, the more bases, then the more specific for recombination cuts in or near sequence ends sticky=overhanging ends, 5’ or 3’ blunt ends - straight cut, will anneal with any other blunt end in the presence of high ligase Hundreds of know restriction endonucleases, usually named after the bacteria that it was found in e.g. EcoR1, Alu1, BamHI, HindIII Restriction endonucleases Gene cloning Cloning: Restriction digestion of DNA insertion of restriction fragment into cloning vector Bacterial plasmid Bacterial virus Yeast artificial chromosomes Transformation of bacteria with recombinant plasmid, virus Screening for clone of interest by using reporter genes or resistance upon exposure to anti-biotic Uses of cloned gene Determine nucleotide sequence and deduce amino acid sequence from genetic code Submit to GenBank (available on WWW) Manipulate gene to study function In vitro In vivo Transgenic (recombinant) organisms Knockout organisms Medical and commercial uses Other molecular procedures Polymerase chain reaction (Mullis) Amplifies target DNA without cloning Target amount can be single molecule Amplified DNA can be sequenced, cloned, etc. Southern blotting Used to identify restriction fragments carrying particular gene Also used for DNA fingerprinting and RFLP analysis cDNA construction Reverse transcription from mRNA template RFLP(restriction fragment length polymorphism) analysis Basis of DNA fingerprinting using SNP - single nucleotide polymorphisms and repeats of DNA sequence Many uses Criminal cases using multiple probes Parentage Species identification Gene evolution Species evolution Sanger DNA sequencing Uses dideoxynucleotides (ddNTP), a template strand, DNA polymerase 1 (Also known as Kornberg enzymes) and dNTPs Missing 3’-OH for nulceopjilic attack for elongation DNA synthesis stops after one is incorporated into DNA fragment ratio of ddNTP to dNTP determines likelihood of termination Manual method with 32P-labeled ddATP and 4 test tubes - ddATP, ddCTP, ddGTP, ddTTP Automated method using ddNTPs labeled with fluorescent dyes in capillary tube Often done commercially Automated sequencing Typical machine 2 hour sequencing run 600-1000 bases per sample multiple samples Up to 500,000 bases per day (12 hr) Data processed by computer In big labs, sequencing reactions also are automated Genome projects Determine entire nucleotide sequence of genome >40 genomes sequenced Helicobacter pylori Escherichia coli Saccharomyces cerevisiae Caenorhabditis elegans Drosophila melanogaster Homo sapiens (first rough draft) Computer identifies all genes, based on properties of genes (e.g., start/stop codons, introns, etc.). Biochips Microarray of DNA fragments, size of postage stamp; can be expensive, but has decreased in cost Microarray chips contain wells of DNA that code for specific genes that uses the concept of hybridization with the gene of interest to see if a gene is expressed or is present. Designed to detect: mutated genes (SNPs) expressed genes Instant DNA profile (“GATTACA”) DNA chip controversies Medicine Risks and informed consent for gene replacement therapy Alteration of human gene pool Parental choice Privacy Genetically modified foods Safety Labeling Forensics Mandatory tests Reliability standards Gene patenting Techniques to study and manipulate genes are patented (e.g., cloning and PCR) Should genes be patented? Are they the intellectual property of the discoverer? Don’t they belong to all of us? Should indigenous peoples be compensated for useful genes extracted from their local plants and fungi? Stem cells Totipotent cells from early embryo grow into any tissue or cell type Recombinant genes can be introduced Considerable use in analyzing gene expression in mice Possible therapeutic use in humans Very controversial This text is based on notes very generously donated by Paul Doerder, Ph.D., of the Cleveland State University.