Presentation on theme: "Methods in Molecular Biology and Genetic Engineering"— Presentation transcript:
1 Methods in Molecular Biology and Genetic Engineering Chapter 3Methods in Molecular Biology and Genetic Engineering
2 1953. Double helix structure, 1962. Noble Prize for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material".
3 3.1 Introductionrestriction endonuclease – An enzyme that recognizes specific short sequences of DNA and cleaves the duplex (sometimes at the target site, sometimes elsewhere, depending on type).Type II: recognition site and cleavage site are the sameType I: cleavage site can be up to 1000 bp away from recognition siteType III: have closer cleavage sites, usually 20 to 30 bp
4 3.1 Introductioncloning vector – DNA (often derived from a plasmid or a bacteriophage genome) that can be used to propagate an incorporated DNA sequence in a host cell.Vectors contain selectable markers and replication origins to allow identification and maintenance of the vector in the host.
5 nucleases hydrolyze an ester bond within a phosphodiester bond. phosphatases hydrolyze the ester bond in a phosphomonoester bond.FIGURE 01: The target of a phosphatase (a) and a nuclease (b). An endonuclease (c) and an exonuclease (d)
6 3.2 Nucleasesendonuclease – Nucleases that cleave phosphoester bonds within a nucleic acid chain.They may be specific for RNA or for single-stranded or double-stranded DNA.exonuclease – Nucleases that cleave phosphoester bonds one at a time from the end of a polynucleotide chain.They may be specific for either the 5′ or 3′ end of DNA or RNA.
7 FIGURE 02: Restriction endonuclease 3.2 Nucleasessticky endblunt endRestriction endonucleases can be used to cleave DNA into defined fragments.Recognize a specific sequenceCut, or restrict, that sequenceFIGURE 02: Restriction endonuclease
9 The Nobel Prize in Medicine 1978 Biozentrum der Universität, Basel, SwitzerlandJohns Hopkins University School of Medicine, Baltimore, MD, USAJohns Hopkins University School of Medicine, Baltimore, MD, USADiscovery of restriction enzymes and their application to problems of molecular genetics
10 3.2 NucleasesA map can be generated by using the overlaps between the fragments generated by different restriction enzymes.FIGURE 03: A restriction map is a linear sequence of sites separated by defined distances on DNA
11 3.3 CloningCloning a fragment of DNA requires a specially engineered vector.recombinant DNA – A DNA molecule that has been created by joining together two or more molecules from different sources.ligating (or ligation) – The process of joining together two DNA fragments.
12 3.3 Cloningsubclone – The process of breaking a cloned fragment into smaller fragments for further cloning.MCS (multiple cloning site) – A sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules.
13 FIGURE 04: Plasmid transformation 3.3 CloningThree site in plasmid:oriamprlacZ with MCSFIGURE 04: Plasmid transformation
14 Action of DNA ligase 5’ Base Base O O O OH _ Restriction Enzyme O P O CH2BaseOOCH2BaseO3’
15 Enhance of the cloning efficiency: Dephosphorylation of the 5’-phophate in the vector by alkaline phosphataseto prevent self-ligationFrom Dr. Yu
16 3.3 Cloningtransformation – The acquisition of new genetic material by incorporation of added exogenous, nonviral DNA.Blue/white selection allows the identification of bacteria that contain the vector plasmid and vector plasmids that contain an insert.FIGURE 05: E. coli colonies on agar plates with ampicillin, IPTG, and the color indicator X-galTransformation: CaCl2, electroporationLacZ gene encodes β-galactoside (β-gal)β-gal can cleave X-gal into blue compoundIPTG as β-gal inducer
17 3.4 Cloning Vectors Can Be Specialized for Different Purposes FIGURE 06: Several types of cloning vectors are available
18 3.4 Cloning Vectors Can Be Specialized for Different Purposes Shuttle vectors can be propagated in more than one type of host cell.Expression vectors contain promoters that allow transcription of any cloned gene.
19 FIGURE 07: A vector can be used in both yeast and bacteria Shuttle VectorFIGURE 07: A vector can be used in both yeast and bacteria(shuttle vector)
22 3.4 Cloning Vectors Can Be Specialized for Different Purposes Reporter genes can be used to measure promoter activity or tissue-specific expression.FIGURE 10: Fluorescent proteins are powerful research toolsCourtesy of Joachim Goedhart, Molecular Cytology, SILS, University of Amsterdam.FIGURE 09: A mouse promoter controls tissue-specific expression of lacZPhoto courtesy of Robb Krumlauf, Stowers Institute for Medical Research
23 Fluorescent proteins are powerful research tools GFP(Green), YFP(Yellow), CFP(Cyan), BFP(Blue)Reprinted from Vision Res., vol. 45, T. G. Wensel, et al., Rhodopsin-EGFP knock-ins..., pp Copyright 2005, with permission from Elsevier [http://www.sciencedirect.com/science/journal/ ]. Photo courtesy of Theodore G. Wensel, Baylor Col
26 GFP expression in subset of fly mushroom body Shih & Wu unpublished
27 GFP express in single neuron in fly brain Wu et al., 2011
28 The Nobel Prize in Chemistry 2008 Woods Hole & Boston University Medical SchoolColumbia UniversityHoward Hughes Medical InstituteFor the discovery and development of the green fluorescent protein, GFP
29 3.4 Cloning Vectors Can Be Specialized for Different Purposes Numerous methods exist to introduce DNA into different target cells.FIGURE 11: DNA can be introduced into cells in several ways
30 3.5 Nucleic Acid Detection DNA, RNA nucleic acids absorb light at 260 nmProtein absorb light at 280 nm260/280 ratios to quantify the amount of nucleic acidDNA, RNA can be nonspecifically stained with ethidium bromide(EtBr) or SYBR
31 3.5 Nucleic Acid Detection Hybridization of a labeled nucleic acid to complementary sequences can identify specific nucleic acids.probe – A radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment.
32 3.5 Nucleic Acid Detection autoradiography – A method of capturing an image of radioactive materials on film.FIGURE 12: An autoradiogram of a gel prepared from the colonies described in Figure 3.5
33 3.5 Nucleic Acid Detection in situ hybridization – Hybridization of a probe to intact tissue to locate its complementary strand by autoradiography.FIGURE 13: The fluorescent in situ hybridization (FISH) techniqueAdapted from an illustration by Darryl Leja, National Human Genome Research Institute (www.genome.gov).
34 In situ Hybridization: Locating genes in chromosomes FISH:Fluorescencein situ hybridizationFrom Dr. Yu
35 3.6 DNA Separation Techniques Gel electrophoresis separates DNA fragments by size, using an electric current to cause the DNA to migrate toward a positive charge.FIGURE 14: DNA sizes can be determined by gel electrophoresisAdapted from an illustration by Michael Blaber, Florida State University.
36 FIGURE 15: Agarose gel electrophoresis pattern of SV40 DNA Type I topoisomerase:Relaxes negative supercoiled DNA to relaxed or linear DNAReproduced from W. Keller, Proc. Natl. Acad. Sci. USA 72 (1975): Photo courtesy of Walter Keller, University of Basel.
37 3.6 DNA Separation Techniques DNA can also be isolated using density gradient centrifugation.DNA density dependents on G-C contentAn AT base pair has a lower molecular weight than a GC base pair
38 3.7 DNA SequencingChain termination sequencing uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis at particular nucleotides.Primer - A single stranded nucleic acid molecule with a 3′ –OH used to initiate DNA polymerase replication of a paired template strand.
39 3.7 DNA SequencingChain termination sequencing uses ddNTPs to terminate DNA synthesis at particular nucleotide.Fluorescently tagged ddNTPs and capillary gel electrophoresis allow automated, high-throughput DNA sequencing.
40 FIGURE 17: DideoxyNTP sequencing using fluorescent tags Different fluorescents label for each ddNTPHit with a laser and pass by an optical sensorGlass capillary tube dissipate heat rapidlyFIGURE 17: DideoxyNTP sequencing using fluorescent tagsInset photo courtesy of Jan Kieleczawa
41 3.8 PCR and RT-PCRThermus aquaticus （Taq DNA polymerase)
42 3.8 PCR and RT-PCRPolymerase chain reaction (PCR) permits the exponential amplification of a desired sequence, using primers that anneal to the sequence of interest.Tm: annealing temperature for primer/template pair*Kary Mullis in the 1980s*Taq polymerase from Thermus aquaticus*Thermal cycler 95oC oC oC20x
43 Exponential produce the primer to primer-defined sequence
44 The Nobel Prize in Chemistry 1993 Press Release 13 October 1993The Royal Swedish Academy of Sciences has decided to award the 1993 Nobel Prize in Chemistry for contributions to the development of methods within DNA-based chemistry, with half to Dr Kary B. Mullis, La Jolla, California, U.S.A.,for his invention of the polymerase chainreaction (PCR) method, and half to Professor Michael Smith, University of BritishColumbia, Vancouver, Canada, for his fundamentalcontributions to the establishment ofoligonucleotide-based, site-directed mutagenesisand its development for protein studies.Decisive progress in gene technology through two new methods: the polymerase chain reaction (PCR) method and site-directed mutagenesisCetus生物科技公司以三億三千五百萬美金專利金賣出(史上最貴的發明專利)Kary B. MullisMichael Smith
45 3.8 PCR and RT-PCRRT-PCR uses reverse transcriptase to convert RNA to cDNA for use in a PCR reaction.Real-time, or quantitative, PCR detects the products of PCR amplification during their synthesis, and is more sensitive and quantitative than conventional PCR.
46 3.8 PCR and RT-PCRfluorescence resonant energy transfer (FRET) – A process whereby the emission from an excited fluorophore is captured and reemitted at a longer wavelength by a nearby second fluorophore whose excitation spectrum matches the emission frequency of the first fluorophore.
47 FIGURE 20: Fluorescence Resonant Energy Transfer (FRET)
48 Fluorescent Tags in Real-Time PCR This fluorescent-tagged oligonucleotide serves as a reporter probeFluorescent tag at 5’-endFluorescence quenching tag at 3’-endWith PCR rounds the 5’ tag is separated from the 3’ tagFluorescence increases with incorporation into DNA product
49 TeqMan probe Primer binding Probe hybridization PCR conditions wikipedia
50 SYBR Green I Dye Primer binding PCR conditions SYBR Green I Dye binds to the minor groove of ds DNAReporter dye are not sequence specific, spurious products produced by the reaction may lead to false positive signals.