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 in nature, these enzymes protect bacteria from intruding DNA they cut up the DNA (restriction) very specific Restriction site recognition sequence for a particular restriction enzyme Restriction fragments segments of DNA cut by restriction enzymes in a reproducable way Sticky end short extensions of restriction fragments DNA ligase 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