Chapter5Powerpoint 11.45.23 PM

Chapter5Powerpoint 11.45.23 PM - HOUR EXAM 1: September 29,...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: HOUR EXAM 1: September 29, 2009 (Tuesday) EXAM WILL COVER: CHAPTER 25 CHAPER 4 CHAPTER 5 TO END of Sept 24 Lecture EXAM 1 REVIEW: Monday, Sept. 28, 2008, 5-6:00 PM, BSW208 Sept 18, 2008 CHAPTER 5 Exploring Genes and Genomes Dangers of Genetic Engineering? The Bubble Boy Gene Therapy of Human Severe Combined Immunodeficiency (SCID)-X1 Disease. (2000) [Bubble boy disease] Cured by introducing engineered DNA coding for cytokine receptor γ-chain [bone-marrow transplant] Problems with X-SCID gene therapy will be discussed near end of Chapter 5. CHAPTER 5: EXPLORING GENES and GENOMES LECTURE TOPICS (4 Groups) 1) RESTRICTION ENZYMES CUT DNA 2) GEL ELECTROPHORESIS OF DNA 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS Recombinant DNA technology (started in mid-late 1970's) • An incredibly powerful set of tools for gene manipulation. • Methods associated with this "technology" make genetic engineering a reality. • DNA (genes), RNA, and protein structure and function can be altered by design for beneficial (or detrimental – biological warfare/terrorism?) results. KEY TOOLS and METHODS OF GENE EXPLORATION 1. ENZYMES to cut, join and replicate DNA in test tubes (in vitro) a) restriction enzymes are DNA cutters b) DNA ligases are DNA joiners c) DNA polymerases for DNA replication 2. GEL ELECTROPHORESIS to separate and isolate specific DNAs 3. BLOTTING METHODS based on hybridization (BASE-PAIRING) of complementary DNA and/or RNA 4. SOLID PHASE methods to sequence and synthesize DNA 5. POLYMERASE CHAIN REACTION (PCR) for gene detection and amplification Sac II Restriction Enzyme Recognition Site 180 degree rotation (2-fold axis of symmetry Ex: Eco RI Cut 5' G AATT C 3' 3' C TTAA G 5' Cut 5' G 3`OH 3' C TTAA 5`P 5` P AATT C 3' G 5' 3` OH Symmetry axis single strand overhang Blunt ends 4 bp site 6 bp site Cut DNA cut with three different restriction enzymes (-) Longer DNA Agarose gel electrophoresis of DNA DNA(-) moves to (+) electrode Shorter DNA (+) Detect fluorescence of a dye (Ethidium Bromide) that binds to DNA Polyacrylamide Gel Electrophoresis for DNA Sequencing ELECTROPHORESIS (-) Longer ALL adjacent bands differ in length by only ONE base (+) Shorter DNA Blotting: “Southern blot” method devised in 1975 At First rejected, Ed Southern’s paper has since been cited about 50,000 times. DNA Southern found that: 1) DNA transfers by diffusion from the gel to the Millipore (nitrocellulose) filter. 2) DNA on filter can be denatured and detected by hybridizing to a radioactive probe. [“Probe” is a DNA or RNA that is identical or related enough to form stable base- paired regions – i.e, has complementary sequences to the DNA.] DNA Blotting and Hybridization to a “probe” “Probe” is either identical or related complementary DNA (or RNA) (X-Ray film) Restriction fragment length polymorphism (RFLP): Ex: MstII RFLP for Sickle-Cell detection Normal Sickle-Cell Parent Carriers Parent carriers * 1.1 kb 1.3 kb Normal Sickle cell CHAPTER 5: EXPLORING GENES LECTURE TOPICS 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS DNA Sequencing: Landmark genomes completed 75 bases tRNA - (1964) [slow, complicated method] 5386 bases NX174 DNA (1977) [fast] 155,844 bases tobacco chloroplast DNA (1986) 1.8 million bases H. influenzae (1995) 3 million bases E. coli (1997) 3 billion bases human (2000!!) [fastest!!!!] Haemophilus influenzae genome (1995) 1.8x106 bp Year 2000 Human DNA sequence completed!! (sort of…. mostly) (3x109 bp) Gene sequences are now precisely located on human chromosomes Gene sequencing shows how similar chromosomal location of related genes are in different organisms My First DNA Sequence The same tech megatrends that are reshaping grown-up gadgets are revolutionizing kids' toys. Nowadays, youngsters can race nitropowered remote control trucks, fiddle with programmable robots, and guest-star in the latest sitcoms. If those aren't sophisticated enough for your brainiac tykes, the Discovery Kids DNA Explorer helps junior scientists extract and map real deoxyribonucleic acid. As third-grade science projects go, this is light-years beyond the ol' baking soda volcano. Next step: cloning Fido. [ DNA Explorer (ages 10 and up): $80, http://www.discovery.com/ ] DNA sequencing by chain termination of DNA synthesis method: (Sanger method) Reaction Conditions that must be satisified: • Reactions specific for each base (A,T,G,C) • Controlled random reactions on all elongating DNA chains • Equimolar collection of reaction products (same frequency of DNA chain stopping for all 4 bases and all elongating chains) DNA sequencing by chain termination of DNA synthesis Chain terminating dideoxynucleotide DNA sequencing by Chain Termination [Primer] * [Template] Example: for A dNTP’s too Reaction products For A : * * * * 5` • Do same for C, G, T • Denature and separate by electrophoresis • Detect fluorescence ddA ddA ddA ddA 3` (etc.,etc. for as many T’s as there are in template) DNA Sequencing Movie Dideoxy_Sequencing_of_DNA.mov [From Berg et al. textbook website] DNA Sequencing Gel Electrophoresis (-) A C G T (from Lehninger) Longer (3` end of new chain) (+) 32P, 35S radioactivity Now use fluorescence llll A C G T Shorter (5` end, nearer primer) DNA Sequencing: Automated Detection by Fluorescence • Key to human genome project • • Read 400-1000 (even more!) bases at a time. [1 x 106bases/day/machine]. • All 4 detected at same time. All 4 dNTP reactions run in same tube at the same time with 4 fluorescent ddNTPs A T Shorter (near 5`end of primer) G C (Time) Longer (3`end of growing chain) CHEMICAL SYNTHESIS OF DNA (SOLID PHASE, AUTOMATED METHODS) Make oligodeoxynucleotide chain from 3’ to 5’ end (short DNA, parts of genes for probes and primers) [oligo means 2-20 bases] Use in DNA/RNA sequencing, cloning, and gene probing by hybridization Easy to make DNA 100 nucleotides long (18-20 used most often) Chemically synthesized DNAs are key to protein engineering by site-directed mutagenesis. Start synthesis with blocked nucleotide linked to a solid support (glass bead). Blocked nucleotide for DNA Synthesis: Phosphite triester method 5`-block (A,G,C,T) Modified 3`-end protected by NH2 groups Solid Phase DNA Synthesis 3` 5` v 5` 3` Last steps: 1. Remove βCE 2. “deprotect” bases DMT off NH3 removes all CHAPTER 5: EXPLORING GENES LECTURE TOPICS 3) DNA SYNTHESIS – LARGE ARRAYS OF DNAs 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS Synthesizing Oligonucleotides Movie SynthesizingOligonucleotide.mov [From Berg et al. textbook website] A DNA chip DNA Chip: 256 Octanucleotides • Ink jet printer head robots deposit DNA on chips • Hybridize with 8-mer test sequence GCGGCGGC All Octanucleotides (8 bases): 48 = 65,536 Need chip of 1.6 cm2 with 50 μm sites Only 32 steps of synthesis (8 hours) DNA chips to identify BCRA1 mutants (breast cancer gene) Use a DNA chip with 48,300 spots Green: normal CT Red: test gene (mutant) Yellow: red and green superimposed (red is mutation) DNA chip study of gene expression in 84 human breast tumor samples. Red: Genes with higher than normal activity in tumor. Green: genes with lower than normal activity in tumor. DNA chips identify changes in yeast gene expression under different conditions. [Red: higher than normal] [Green: lower than normal] Modern human flu strain 1918 pandemic Flu virus induces altered mouse gene expression: Studied by DNA chip analysis. Data gives clues about what may be gene targets in humans of H5N1 bird flu 1918 pandemic flu strain (overexpressed) Connectivity Concept: Using gene expression profiles (DNA chips) to connect perturbed gene expression (like in diseases) with “correcting” drug treatments. [Lamb, et al, Science 313, Sept. 29, 2006 p.1929] CHAPTER 5: EXPLORING GENES LECTURE TOPICS 2) GEL ELECTROPHORESIS OF DNA (continued) 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS DNA polymerase chain reaction: Concept (starting with only one of two strands of DNA) Original DNA 3` 5` 3` (n=0) primer1 1 Copy (n=1) 5` 3` Denature (separate strands), then cool mixture Heat 5` 3` 5` 5` 3` template (Taq) DNA polymerase + dNTPs 3` 5` 3` 5` primer1 3` 2 Copies (n=2) 5` 5` + 3` + primer2 3` 5` 5` 3` 3` 5` Polymerase Chain Reaction (PCR): Concept 1 Copy (D.S.) One (n=1) “cycle” Primers & 2 copies (D.S.) Many cycles DNA Amplification cycle 1 cycle 2 cycle 3 n cycles Short products (target sequence) Short products predominate. They are amplified as 2n Polymerase Chain Reaction Movie Polymerase_Chain_Reaction.mov [From Berg et al. textbook website] PCR: # Copies of DNA = 2n 4 8 16 32 64 Copies 1 21 22 3 23 4 24 5 25 6 26 n 1 0 2 Start with one copy (D.S.) Copies 1 2 Cycle 27 After 45 cycles: 245 = 3.5 x 1013 = 3,500 billion copies PCR in CRIMINOLOGY The CSI Young Investigators DNA Kit This official forensic kit contains realistic experiments that allow your child to solve a mystery in the style of the investigators on the popular television series. Item 72523 ................... $59.95 Available for Immediate Shipment. Children learn the basics of forensic science such as dusting for fingerprints and extracting DNA. What you Get Clear step-by-step instructions are written as if the child were taking part in the investigation, analyzing clues alongside the CSI team members. DNA Lab Kit. Includes miniature centrifuge, electrophoresis chamber, beakers, chromatography ring, lab glasses, and more equipment to examine DNA code (DNA samples and instructions are provided). Requires three 9-volt and four D batteries. For ages 10 and up. You may also want: The CSI Young Investigators Forensic Lab Kit USE OF PCR in CRIMINOLOGY D = Defendant (D) * (D) * V = Victim * * Data shows that defendant’s clothes have victim’s DNA (in bloodstains) Probability of a random DNA match is 1 in 33 billion!! CHAPTER 5: EXPLORING GENES LECTURE TOPICS 2) GEL ELECTROPHORESIS OF DNA (continued) 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS CONSTRUCTION, CLONING AND EXPRESSION OF DNA Novel combinations of genes can be cloned, amplified and expressed in foreign environments KEY STEPS / PROCEDURES 1. Construct recombinant DNA molecule. • DNA inserted in a vector 2. Clone and amplify DNA in vector`s host cells 3. Select individuals that have recombinant gene • ***Antibiotic resistance*** • Gene probing • Antibody reaction CLONING DNA [cut both with same enzyme] Foreign DNA (insert) Plasmid vector [Join with DNA ligase] Construct recombinant DNA molecule Introduce DNA into host cells by transformation or viral infection Select cells that have recombinant DNA Cloning Cutting and joining DNA molecules “Cut” * “Join” Linkers- to clone blunt-ended DNA Synthetic Linker DNA Step 1: “Join” Step 2 Clone Plasmid Cloning Movie Plasmid_Cloning.mov [From Berg et al. textbook website] All Recombinant DNA Vectors: Properties must allow to: 1. Clone in specific sites 2. Select [antibiotic, β-galactosidase] • Insertional inactivation 3. Replicate in host cells Ex: Prokaryotic (E.coli) Plasmid Vector: Example that satisfies requirements 1 Select 2 Specific cloning sites 3 2 Replication 1 2 1 2 3 pUC18 prokaryotic plasmid vector: Polylinker with many cloning sites 2 1 Select 2 Specific cloning sites 3 Replication 1 3 1 (An E.coli vector) SOME CLONING VECTORS • Plasmids (Autonomously replicating) • - [2-6 kbp inserts – prokaryotic gene sizes] • - Insertional inactivation of antibiotic resistance or β-galactosidase activity • Lambda phages (good for libraries of eukaryotic cDNA’s) [10-23 kbp inserts – a few eukaryotic genes] • YAC’s (yeast artificial chromosomes) – For big pieces of chromosomes [1 Mb inserts (106 base pairs; about 1000 genes)] • Ti plasmid of Agrobacterium tumefaciens (for plant genetic engineering [one or a few genes insert into chromosomes] Bacteriophage lambda (λ) lifecycle * Bacteriophage lambda (λ) as a cloning vector Delete this piece L L R R L R Recombinant λ DNA in new phage particle Yeast Artificial Chromosome (YAC) To clone large chromosomal pieces Up to 106 bp PLANT GENETIC ENGINEERING (With Ti plasmid of Agrobacterium tumefaciens) Crown Gall Disease Caused by Agrobacterium Delete and insert genes Cells are transformed T-DNA inserts in chromosomes Delete some T-DNA * Delete these genes T-DNA Ti plasmid Transformation and selection of transgenic plants Infect with Agrobacterium containing recombinant Ti plasmid that has a selectable marker Agar with growth hormones and kanamycin (antibiotic for selection) Kanamycin-resistant plants contain the foreign gene CHAPTER 5: EXPLORING GENES LECTURE TOPICS 2) GEL ELECTROPHORESIS OF DNA (continued) 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS MAKING GENE LIBRARIES Genomic library – from all fragments of a cell`s chromosomal DNA (large pieces) • YAC’s • Bacteriophage lambda cDNA library – DNA copies of a mix of all cellular mRNA (a few kbp cDNA’s of all expressed genes) • lambda phage • plasmids λ Genomic Library Construction 1. Cut 2. Ligate 3. Get recombinant L λ DNA R 4. reconstituted λ virus 5. Get more virus from infected cells * Making a cDNA library from mRNA [OH-] cuts RNA Primer 1st strand 2nd strand cDNA Primer 5`-------CCnCC Add linkers Clone in a vector Terminal transferase adds dG’s SCREENING GENE LIBRARIES (searching for a needle in a phagestack!!) Need to screen 500,000 clones to find a specific sequence in a genomic library • • Easier for “abundant RNA” in a cDNA library • • N.A. hybridization screening (with gene probe) • • Chromosome walking (connecting long pieces) Immunochemical (antibody) screening of an expression library Make synthetic DNA probes (can even predict DNA sequence by reverse translation of protein) map whole chromosomes (Clone in λ, YAC, or BAC) Screening gene (or cDNA) libraries: replica plating 1) λ phage plaques or 2) bacterial colonies Hybridize to DNA probe or Antibody reaction Make replica plate on filter Clone containing gene DNA/DNA hybrid or antibody Master plate Autoradiogram of replica plate Screening a lambda library (genomic or cDNA) (or cDNA) Get recombinant λ DNA Infect cells Isolate individual plaques Screening a lambda genomic library: use DNA probe Individual plaques (dead cells – clear spots) Hybridize to gene DNA probe Make replica plate λ clone containing gene DNA/DNA hybrid Master plate Autoradiogram of replica plate For cDNA library (protein expressed) * Bacterial colony or λ plaque * Immunological Screening Antibody detects cells that contain the protein of interest “Blotting” replica * Reverse translation: To find DNA clone coding for a specific protein 1. Use amino acid sequence to predict gene sequence. 2. Synthesize (and label with radioactivity) 21bp DNA of all possible gene sequences and use to “screen” a library. (need to consider degenerate codons) 1. 2. 256 possible DNA coding sequences (2x4x2x2x4x2 = 256) Screening Oligonucleotides Movie Screening_Oligonucleotide.mov [From Berg et al. textbook website] Ways to use recombinant DNA Technology From Gene to unknown protein From Protein to get unknown Gene CHAPTER 5: EXPLORING GENES LECTURE TOPICS 2) GEL ELECTROPHORESIS OF DNA (continued) 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 4) POLYMERASE CHAIN REACTION (PCR) 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING 6) DNA CLONING VECTORS 7) GENE LIBRARIES: MAKING AND SCREENING THEM 8) CHROMOSOME MAPPING 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS “Chromosome Walking” (use YACs) Start with DNA (A’): hybridize to library Order of A D deduced by successive hybridizations CHAPTER 5: EXPLORING GENES LECTURE TOPICS 9) EXPRESSION OF CLONED GENES 10) ENGINEERING NOVEL PROTEINS Dangers of Genetic Engineering? Be careful what you ask for! DNA vector delivery Gene gun Microinjection liposomes viruses electroporation DNA Delivering DNA vector to cells • Calcium phosphate precipitated DNA • Microinjection • Virus vectors (SV40, vaccinia, retroviruses) • GENE GUN (microprojectiles coated with DNA) • Liposomes (coat DNA with “cell membrane”) • Electroporation Electroporation: DNA delivery to plant cells Make holes in cell wall Protoplast CLONED GENES / VECTOR SYSTEMS: (some examples) • Human Proinsulin cDNA • Engineered mammals (giant mice – somatotropin gene) • Engineered Plants (Ti-plasmid of Agrobacterium tumefaciens) Ex: Flavr-Savr tomatoes (Calgene) • 1918 pandemic influenza virus – reconstructed from cloned pieces of its genome. [samples recovered from victims buried in Arctic permafrost] Human insulin cDNA CLONING Expression (No introns) Identify proinsulin by antibody reaction Creating a Transgenic Mouse Creating_a_Transgenic.mov [From Berg et al. textbook website] Engineering a Giant Mouse: Somatostatin gene injected into mouse male pronucleus Engineered Giant Mouse: Somatostatin Control: no Cd++ or no extra rat growth hormone (somatostatin) Add Cd++ • 2x normal weight • 500x hormone levels Microinject plasmid Somatostatin PLANT GENETIC ENGINEERING (With Ti plasmid of Agrobacterium tumefaciens) Crown Gall Disease Caused by Agrobacterium Delete and insert genes Cells are transformed T-DNA inserts in chromosomes Delete some T-DNA * Delete these genes T-DNA Ti plasmid FLAVR SAVR Tomatoes: FDA APPROVED FOR SALE from the New Yorker, 1994 “Antisense” Gene Disruption Strategy CALGENE: to market in 1995 Gene is transcribed as complementary RNA to mRNA for normal plant gene involved in fruit ripening. Ti plasmid, mouse vector chromosome insertions: relate to concept of Homologous/Non- homologous Recombination. These events result in: • Gene replacement, Gene disruption, or random chromosome insertions The Bubble Boy Gene Therapy of Human Severe Combined Immunodeficiency (SCID)-X1 Disease. (2000) [Bubble boy disease] Cured by introducing engineered DNA coding for cytokine receptor γ-chain [bone-marrow transplant] BUT: Random insertions correlated with leukemia occurrence after a few years in treated X-SCID patients. • leukemia correlates with insertion of DNA into a gene. • Gene disruption - loss of normal function leads to leukemia. Correction of ADA-SCID by Stem Cell Gene Therapy Combined with Nonmyeloablative Conditioning (2002) Cured by introducing engineered DNA coding for the enzyme ADA (Adenosine deaminase) CHAPTER 5: EXPLORING GENES LECTURE TOPICS 9) EXPRESSION OF CLONED GENES RNAi gene silencing 10) ENGINEERING NOVEL PROTEINS Gene Silencing disrupts gene expression. **2006 Nobel Prize in Medicine!! [21 bp dsRNA] RNA interference (siRNAs) A mechanism for gene disruption. RISC: RNA-induced gene silencing complex ssRNA Practical Applications of Cloning by Recombinant DNA Methods. NOT a DINOSAUR, BUT: [Fall, 2005 news] An infectious flu virus was created in the lab of from cloned pieces of its genes. • Influenza virus that caused 1918 Spanish flu pandemic. (killed 20 million people.) • Used “Jurassic Park” type DNA cloning methods – RNA cloning (cDNA’s) in a DNA vector. • 1918 virus is a bird flu strain. – That’s why H5N1 bird flu is so worrisome. Modern human flu strain 1918 pandemic flu strain 1918 pandemic Flu virus induces altered mouse gene expression: Studied by DNA chip analysis. PROTEIN ENGINEERING by SITE-SPECIFIC MUTAGENESIS PROTEIN ENGINEERING: SITE-SPECIFIC MUTAGENESIS Modify coding information to get a different amino acid sequence Change base with mismatch primer for DNA replication SITE-SPECIFIC MUTAGENESIS A mismatch changes only one codon How to do it?? 1. Hybridize mismatch primer 2. Replicate DNA (plasmid) 3. Half of daughter DNA has mutation!! In Vitro Mutagenesis Movie In_Vitro_Mutagenesis.mov [From Berg et al. textbook website] Current and Future Applications of RECOMBINANT DNA TECHNOLOGY • Complete chromosome gene maps • Whole genome sequencing by shotgun approach • Discovery of molecular bases of development, evolutionary relationships • New proteins with new functions (or old proteins with new functions!) • Human hormone synthesis in bacteria • Antiviral agents • AIDS vaccine development • New pharmacological agents (proteins, RNA, DNA) • Antisense RNA therapy (RNAi, gene silencing) Current and Future Applications of RECOMBINANT DNA TECHNOLOGY • Medical diagnostic reagents (gene probes) for detection of genetic diseases, infections and cancers • Gene therapy: delivery with disarmed viruses to alleviate diseases caused by known gene defects. • Agricultural revolution with animals having altered traits, more nutritious plants, heat/drought resistant crops, etc. • Forensics - molecular detectives ...
View Full Document

Ask a homework question - tutors are online