* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DNA Technology
Oncogenomics wikipedia , lookup
DNA profiling wikipedia , lookup
DNA sequencing wikipedia , lookup
Pathogenomics wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
DNA polymerase wikipedia , lookup
Whole genome sequencing wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Minimal genome wikipedia , lookup
SNP genotyping wikipedia , lookup
Genome (book) wikipedia , lookup
Primary transcript wikipedia , lookup
Cancer epigenetics wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Transposable element wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Point mutation wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Metagenomics wikipedia , lookup
DNA vaccination wikipedia , lookup
Human genome wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Epigenomics wikipedia , lookup
DNA supercoil wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Molecular cloning wikipedia , lookup
Microsatellite wikipedia , lookup
Genome evolution wikipedia , lookup
Genetic engineering wikipedia , lookup
Designer baby wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Microevolution wikipedia , lookup
Genomic library wikipedia , lookup
Genome editing wikipedia , lookup
Helitron (biology) wikipedia , lookup
Chapter 17 – 18 Biotechnology: Genomics & DNA Technology DNA Sequencing Sanger method determine the base sequence of DNA dideoxynucleotides ddATP, ddGTP, ddTTP, ddCTP missing O for bonding of next nucleotide terminates chain DNA Sequencing Sanger method synthesize complementary DNA strand in vitro in each tube: 1 “normal” N-bases dideoxy N-bases ddA, ddC, ddG, ddT DNA polymerase primer buffers & salt 2 3 4 2 Fred Sanger This was his 2nd Nobel Prize!! 1st was in 1958 for the structure of insulin 1978 | 1980 Advancements to Sequencing Fluorescent tagging no more radioactivity all 4 bases in 1 lane each base a different color Automated reading Advancements to Sequencing Fluorescent tagging sequence data Computer read & analyzed Raw Genome Data Automated Sequencing Machines Really BIG labs! Human Genome Project U.S government project begun in 1990 estimated to be a 15 year project DOE & NIH initiated by Jim Watson led by Francis Collins goal was to sequence entire human genome 3 billion base pairs Celera Genomics Craig Venter challenged gov’t would do it faster, cheaper private company Different Approaches gov’t method Craig Venter’s method “map-based method” “shotgun method” 1. Cut chromosomal DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. 2. Cut and clone into smaller fragments. 3. Assemble DNA sequence using overlapping sequences. 1. Cut DNA from entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. Human Genome Project On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome. Historic Event! blueprint of a human the potential to change science & medicine How does our genome stack up? Organism Human (Homo sapiens) Laboratory mouse (M. musculus) Mustard weed (A. thaliana) Roundworm (C. elegans) Fruit fly (D. melanogaster) Yeast (S. cerevisiae) Bacterium (E. coli) Human Immunodeficiency Virus (HIV) Genome Size (bases) Estimated Genes 3 billion 30,000 2.6 billion 30,000 100 million 25,000 97 million 19,000 137 million 13,000 12.1 million 6,000 4.6 million 3,200 9700 9 GenBank Database of genetic sequences gathered from research Publicly available! Organizing the Data And we didn’t stop there… Interspersed Repetitive DNA Repetitive DNA is spread throughout genome interspersed repetitive DNA (SINEs Short INterspersed Elements) make up 25-40% of mammalian genome in humans, at least 5% of genome is made of a family of similar sequences called, Alu elements (PV92 anyone?!) 300 bases long Alu is an example of a "jumping gene" called a transposon; a DNA sequence that "reproduces" by copying itself & inserting into new chromosome locations Rearrangements in the Genome Transposons transposable genetic element piece of DNA that can move from one location to another in cell’s genome One gene of an insertion sequence codes for transposase, which catalyzes the transposon’s movement. The inverted repeats, about 20 to 40 nucleotide pairs long, are backward, upside-down versions of each other. In transposition, transposase molecules bind to the inverted repeats & catalyze the cutting & resealing of DNA required for insertion of the transposon at a target site. Transposons Insertion of transposon sequence in new position in genome Insertion sequences cause mutations when they happen to land within the coding sequence of a gene or within a DNA region that regulates gene expression. Transposons Barbara McClintock 1947 | 1983 discovered 1st transposons in Zea mays (corn) in 1947 Retrotransposons Transposons actually make up over 50% of the corn (maize) genome & 10% of the human genome. Most of these transposons are retrotransposons, transposable elements that move within a genome by means of RNA intermediate, transcript of the retrotransposon DNA. Families of Genes Human globin gene family evolved from duplication of common ancestral globin gene Different versions are expressed at different times in development allowing hemoglobin to function throughout life of developing animal Hemoglobin Differential expression of different beta globin genes ensures important physiological changes during human development. The BIG Questions… How can we use our knowledge of DNA to: diagnose disease or genetic defect? cure disease or genetic defect? change/improve organisms? What are the techniques & applications of biotechnology? direct manipulation of genes for practical purposes Biotechnology Genetic manipulation of organisms is not new humans have been doing this for thousands of years plant & animal breeding Evolution & Breeding of Food Plants artificial selection! Evolution of Zea mays from ancestral teosinte (left) to modern corn (right). The middle figure shows possible hybrids of teosinte & early corn varieties Evolution & Breeding of Food Plants “Descendants” of the wild mustard Brassica genus artificial selection! Animal Husbandry / Breeding artificial selection! Biotechnology Today Genetic Engineering direct manipulation of DNA if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with this unit is a survey of those tools… Our tool kit… Bioengineering Tool Kit Basic Tools restriction enzymes ligase plasmids for gene cloning gel electrophoresis Advanced Tools PCR DNA sequencing Southern blotting DNA libraries / probes microarrays Cut, Paste, Copy, Find… Word processing metaphor… cut (Ctrl + X) restriction enzymes paste (Ctrl + V) ligase copy (Ctrl + C) via plasmids bacteria transformation via PCR find (Ctrl + F) Southern blotting probes Cutting DNA Restriction enzymes restriction endonucleases discovered in 1960s evolved in bacteria to cut up foreign DNA (“action restricted to foreign DNA”) protection against viruses & other bacteria bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA Paste DNA Sticky ends allow: H bonds between complementary bases to anneal Ligase enzyme “seals” strands bonds sugar- phosphate bonds covalent bond of DNA backbone Biotech Use of Restriction Enzymes GAATTC CTTAAG Sticky ends (complementary single-stranded DNA tails) GAATTC CTTAAG Restriction enzyme cuts the DNA AATTC G Add DNA from another source cut with same restriction enzyme G CTTAA AATTC G G AATTC CTTAA G DNA ligase joins the strands. Recombinant DNA molecule DNA GAATTC CTTAAG Application of Recombinant DNA Combining sequences of DNA from 2 different sources into 1 DNA molecule often from different species human insulin gene in E. coli (humulin) frost resistant gene from Arctic fish in strawberries “Roundup-ready” bacterial gene in soybeans BT bacterial gene in corn jellyfish glow gene in Zebra “Glofish” – GFP! 1961, 1994 | 2008 Development of GFP Shimomura, Chalfie, Tsien discovery, isolation, and purification of GFP and many fluorescent analogs Osamu Shimomura Martin Chalfie Roger Tsien Cut, Paste, Copy, Find… Word processing metaphor… restriction enzymes paste ligase cut copy plasmids bacteria transformation PCR (chapter 11) find Southern blotting probes Plasmids Trust me… this will be important! Plasmids Plasmids small supplemental circles of DNA 5000 - 20,000 base pairs self-replicating carry extra genes 2-30 genes can be exchanged between bacteria bacterial ‘sex’!! rapid evolution antibiotic resistance can be imported from environment Transformation Bacteria are opportunists pick up naked foreign DNA wherever it may be hanging out have surface transport proteins that are specialized for the uptake of naked DNA import bits of chromosomes from other bacteria incorporate the DNA bits into their own chromosome express new gene form of recombination Who’s experiment documented this? Swapping DNA Genetic recombination by trading DNA 1 arg+ trp- minimal media 3 2 argtrp+ Plasmids & Antibiotic Resistance Resistance is futile? 1st recognized in 1950s in Japan bacterial dysentery not responding to antibiotics worldwide problem now resistant genes are on plasmids that are swapped between bacteria Copy DNA Plasmids small, self-replicating circular DNA molecules insert DNA sequence into plasmid vector = “vehicle” into organism transformation insert recombinant plasmid into bacteria bacteria make lots of copies of plasmid grow recombinant bacteria on agar plate clone of cells = lots of bacteria production of many copies of inserted gene DNA RNA protein trait Biotechnology Used to insert new genes into bacteria example: pUC18 engineered plasmid used in biotech antibiotic resistance gene on plasmid is used as a selective agent Biotechnology Used to insert new genes into bacteria example: pUC18 engineered plasmid used in biotech antibiotic resistance gene on plasmid is used as a selective agent Selection for Plasmid Uptake Ampicillin becomes a selecting agent only bacteria with the plasmid will grow on amp plate all bacteria grow only transformed bacteria grow LB plate LB/amp plate Recombinant Plasmid Antibiotic resistance genes as a selectable marker Restriction sites for splicing in gene of interest Selectable marker Plasmid has both “added” gene & antibiotic resistance gene If bacteria don’t pick up plasmid then “die” on antibiotic plates If bacteria pick up plasmid then survive on antibiotic plates selecting for successful transformation selection GFP Gene Cloning Cut, Paste, Copy, Find… Word processing metaphor… restriction enzymes paste ligase cut plasmids bacteria copy transformation PCR (chapter 11) find Southern blotting probes Any Questions?