Chapter 20 BIOTECHNOLOGY DNA CLONING Uses recombinant DNA

Chapter 20 BIOTECHNOLOGY

DNA CLONING Uses recombinant DNA formed from two different sources One source is typically a bacterial plasmid Isolate plasmid from bacteria Insert DNA from foreign source Re-insert into bacteria (now recombinant bacterium)

RESTRICTION ENZYMES � Enzymes that cut DNA at specific sequences (restriction endonucleases) � � Bacteria use them for protection against foreign DNA Each has a specific cutting sequence – restriction site Cuts result in DNA fragments For cloning, use of restriction enzymes that

EUKARYOTIC GENES IN BACTERIA � Involves a cloning vector � � � Bacterial plasmid to copy gene Isolate plasmid and hummingbird DNA Cut both with same restriction enzyme Mix plasmid and DNA fragments Plate on bacteria on agar containing ampicillin and X-gal

GENOMIC LIBRARY Complete set of plasmid containing clones � Each carries copies of particular segments � Extract DNA from organism � Digest with restriction enzyme � Insert into plasmids � Seal with ligase � Bacteria take up plasmids and grow �

GENOMIC LIBRARY � � Sometimes interested in just the protein itself Can make DNA library from expressed m. RNA – c. DNA � Use reverse transcriptase Added to m. RNA from cell type in question � Makes single stranded DNA using d. T’s � m. RNA degraded � DNA pol III makes complimentary strand � Does not contain any introns �

GENOMIC LIBRARY � Detecting DNA sequence in genomic library Cells from wells are transferred to nylon mesh � Membrane is treated to break open the cells � Membrane is incubated in radioactive probes � Membrane is laid on photographic film �

BACTERIAL AND EUKARYOTIC EXPRESSION SYSTEMS Bacterial systems can be difficult Certain gene expression patterns differ Introns can be a problem Bacteria Eukaryotic Yeast do no have RNA splicing machinery systems works better is easier to grow than bacteria Many eukaryotic proteins will not function unless modified after transcription

AMPLIFYING DNA May need to produce additional copies if small amount or impure Polymerase chain reaction (PCR) Denature Anneal Extension 2 n = n is number of cycles

GEL ELECTROPHORESIS � Separate nucleic acids or proteins based on size N. A. /Proteins carry negative charge � Cut N. A. with restriction enzyme � Place sample in wells � � Smaller fragments will move farther than larger fragments � Stain with dye (ethidium bromide)

SOUTHERN BLOTTING Used to compare restriction fragments from different samples Cut DNA with restriction enzyme Gel electrophoresis DNA transfer Hybridization

NORTHERN BLOTTING Used to measure the amount of m. RNA in different samples Gel electrophoresis of m. RNA Transfer to nitrocellulose Allow m. RNA’s to hybridize with labeled probes for β-globin Transfer to film and see what stages present

RT-PCR Used for m. RNA expression Isolate m. RNA’s Reverse transcriptase is added to make c. DNA This is a template for PCR amplification When run on a gel only copies of the gene will be observed as bans

DNA MICROARRAYS Isolate m. RNA from two tissues Convert to c. DNA (fluorescently labeled) by reverse transcriptase Probes are single strandedtypically 20 nucleotides c. DNA are mixed and to the chip Complimentary strands will bind to the c. DNA on chip Scanner detects hybridization

DETERMINING GENE FUNCTION RNAi Use of double stranded RNA to match the sequence of a particular gene Once attached it will trigger breakdown of the gene or block transcription Used in many animals, but ethical considerations in humans

CLONING ANIMALS 1997 -Dolly Remove nucleus from unfertilized egg and replace with nucleus of differentiated cell Died at age of 6 from lung disease usually seen in much older sheep

STEM CELLS Unspecialized cells Embryonic stem cells Isolated from embryo at blastula stage Reproduce indefinitely in culture Differentiate into many different cells Adult stem cells Differentiation capabilities limited Blood cells, bone, cartilage, fat, muscle, brain nerve cells

MEDICAL USE OF DNA TESTING Identification of genes causing genetic diseases PCR and labeled probes to look for differences/pathogens Sickle cell, hemophilia, cystic fibrosis, Huntington’s disease, Duchene MD Companies now offer screening for genetic disorders

GENE THERAPY Introduce genes for therapeutic purposes Only works with disorders traceable to one defective gene Bone marrow cells make excellent vectors SCID – Severe combine immudeficieny First treated with gene therapy

PHARMACEUTICAL PRODUCTS Determining sequence/structure of proteins has led to treatments Imantinib – inhibits receptors responsible for developing a leukemia Can produce medications in culture or through transgenic animals

FORENSIC EVIDENCE Use of genetic markers to develop a genetic profile Short Tandem Repeats (STR’s) – tandemly repeated units, 3 -5 bases in specific regions Number of repeats is highly variable person to person PCR amplifies the STR and then the number of repeats can be determined with electrophoresis

BACTERIAL CLEANUP Bacteria can be modified to clean up materials Heavy metals, biodegradable materials in landfills, petroleum Was used after BP oil spill to clean up spill