Chapter 20

 Uses recombinant DNA  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)

 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 leave sticky ends is useful

 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

 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

 Sometimes interested in just the protein itself  Can make DNA library from expressed mRNA – cDNA  Use reverse transcriptase  Added to mRNA from cell type in question  Makes single stranded DNA using dT’s  mRNA degraded  DNA pol III makes complimentary strand  Does not contain any introns

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

 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

 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)

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

 Used to measure the amount of mRNA in different samples  Gel electrophoresis of mRNA  Transfer to nitrocellulose  Allow mRNA’s to hybridize with labeled probes for β- globin  Transfer to film and see what stages present

 Used for mRNA expression  Isolate mRNA’s  Reverse transcriptase is added to make cDNA  This is a template for PCR amplification  When run on a gel only copies of the gene will be observed as bans

 Isolate mRNA from two tissues  Convert to cDNA (fluorescently labeled) by reverse transcriptase  Probes are single stranded- typically 20 nucleotides  cDNA are mixed and to the chip  Complimentary strands will bind to the cDNA on chip  Scanner detects hybridization

 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

 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

 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

 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  SNP’s

 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

 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

 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

 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