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DNA Technology and Genomics Recombinant DNA    Definition: DNA in which genes from 2 different sources are linked Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products Bacterial genetics    Nucleoid: – region in bacterium densely packed with DNA (no membrane) Plasmids: – small circles of DNA Reproduction by binary fission (asexual) Bacterial DNA-transfer processes    Transformation – genotype alteration by the uptake of naked, foreign DNA from the environment (Griffith expt.) Transduction – phages that carry bacterial genes from 1 host cell to another – generalized~ random transfer of host cell chromosome – specialized~ incorporation of prophage DNA into host chromosome Conjugation – direct transfer of genetic material; cytoplasmic bridges – pili; sexual Bacterial Plasmids    Small, circular, self-replicating DNA separate from the bacterial chromosome F (fertility) Plasmid: codes for the production of sex pili (F+ or F-) R (resistance) Plasmid: codes for antibiotic drug resistance Recombination of E. coli Bacterial plasmids in gene cloning Clone genes for insertion into organisms  Clone proteins for medical/ pharmaceutical purposes  DNA Cloning  Restriction enzymes (endonucleases) – – –  Restriction site –  short extensions of restriction fragments DNA ligase –  segments of DNA cut by restriction enzymes in a reproducable way Sticky end –  recognition sequence for a particular restriction enzyme Restriction fragments –  in nature, these enzymes protect bacteria from intruding DNA they cut up the DNA (restriction) very specific enzyme that can join the sticky ends of DNA fragments Cloning vector – DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids) Eukaryotic Gene Cloning      Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest) Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind fragmented DNA w/ DNA ligase Introduction of cloning vector into cells (transformation by bacterial cells) Cloning of cells (and foreign genes) Identification of cell clones carrying the gene of interest Genomic Libraries    Cloned genes from a genome are stored in a “genomic library” Recombinant fragments in bacteria or phages Complimentary DNA (cDNA) Library – mRNA extracted – Reverse transcriptase makes a complimentary strand of gene DNA Analysis      PCR (polymerase chain reaction) Gel electrophoresis Restriction fragment analysis (RFLPs) Southern blotting DNA sequencing Practical DNA Technology Uses    Diagnosis of disease Human gene therapy Pharmaceutical products – Vaccines – Hormones  Forensics – Crime scene analysis of DNA  Animal husbandry (transgenic organisms) – “Pharm” animals  Genetic engineering in plants – Disease/ pest resistance Polymerase chain reaction (PCR)    Amplification of any piece of DNA without cells (in vitro) Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers Applications: fossils, forensics, prenatal diagnosis, etc. DNA Analysis  Gel electrophoresis: – separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of the same length Restriction fragment analysis  Restriction fragment length polymorphisms (RFLPs) – Differences in restriction fragment patterns on homologous chromosomes – Occur in noncoding DNA sequences – Serve as inheritable genetic markers   Southern blotting: process that reveals sequences and the RFLPs in a DNA sequence DNA Fingerprinting Southern Blotting DNA Sequencing  Determination of nucleotide sequences – Dideoxy Chain-Termination Method (Sanger Method) – Whole-genome approach (Venter and Celera Genomics)   Genomics: the study of genomes based on DNA sequences Human Genome Project – Begun in 1990; largely completed by 2003 Genomics  The National Center for Biotechnology Information (NCBI) – Created a database of gene sequences created by the Human Genome Project and other sequencing endeavors – Genbank – BLAST software allows for comparison of sequences Analyzing Gene Expression  Northern Blotting – Gel electrophoresis done with labeling probes to determine function  RT-PCR – Uses reverse transcriptase and PCR – Compares gene expression between different samples Studying Gene Interaction  DNA Microassay – Many DNA fragments on a glass slide or chip – Can be tested for interaction with other genes marked with fluorescent markers Determining Gene Function  In vitro mugagenasis – Disable certain genes and observe consequences – Mutations “knock out” certain genes  RNA interference (RNAi) – RNA used to block translation of certain genes Transposons  transposable genetic element; piece of DNA that can move from location to another in a cell’s genome – chromosome to plasmid, plasmid to plasmid, etc.) – “jumping genes” Eukaryotic Genes 98.5% of all DNA does not code for proteins, rRNA, or tRNA  Most is repetitive DNA  44% is made of transposable elements  Transposable Elements  Transposons – Move w/in a genome by DNA intermediate  Barbara McClintock (1940’s and 50’s) – “Jumping genes” – Researched the location of colored kernels in maize  Retrotransposons – Move by means of a RNA intermediate Multigene Families  In the human genome, ½ of coding DNA is in multigene families – Collections of identical or very similar genes – Identical- ribosomal RNA molecules – Similar- α-globin and β-globin  Pseudogenes- nonfunctional nucleotide sequences (very similar to functional genes) Genome Evolution  Duplications of chromosomes – Unequal crossing over  Duplication and divergence of DNA segments – Ancestral globin gene  present day α-globin and β-globin genes  Rearranging genes – Exon duplication/ exon shuffling – Transposable elements
 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
									 
                                             
                                             
                                             
                                             
                                             
                                             
                                             
                                             
                                            