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Chapter 3 Methods in Molecular Biology and Genetic Engineering.

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Presentation on theme: "Chapter 3 Methods in Molecular Biology and Genetic Engineering."— Presentation transcript:

1 Chapter 3 Methods in Molecular Biology and Genetic Engineering

2 1953. Double helix structure, Noble Prize for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material".

3 3.1 Introduction restriction 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 same Type I: cleavage site can be up to 1000 bp away from recognition site Type III: have closer cleavage sites, usually 20 to 30 bp

4 3.1 Introduction cloning 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 3.2 Nucleases 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 Nucleases endonuclease – 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 3.2 Nucleases Restriction endonucleases can be used to cleave DNA into defined fragments. FIGURE 02: Restriction endonuclease 1.Recognize a specific sequence 2.Cut, or restrict, that sequence sticky endblunt end

8

9 The Nobel Prize in Medicine 1978 Discovery of restriction enzymes and their application to problems of molecular genetics Biozentrum der Universität, Basel, Switzerland Johns Hopkins University School of Medicine, Baltimore, MD, USA

10 3.2 Nucleases A 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 Cloning Cloning 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 Cloning subclone – 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 3.3 Cloning FIGURE 04: Plasmid transformation Three site in plasmid: ori amp r lacZ with MCS

14 Action of DNA ligase O P O O O CH 2 Base DNA ligase Restriction Enzyme O Base O OH P O O O CH 2 Base O O O _ _ _ 5’5’ 3’3’

15 Enhance of the cloning efficiency: Dephosphorylation of the 5’-phophate in the vector by alkaline phosphatase to prevent self-ligation From Dr. Yu

16 3.3 Cloning transformation – 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-gal 1.LacZ gene encodes β-galactoside (β-gal) 2.β-gal can cleave X-gal into blue compound 3.IPTG 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 Shuttle Vector FIGURE 07: A vector can be used in both yeast and bacteria (shuttle vector)

20 Expression vector RBS: Ribosome Binding Site

21 FIGURE 08: Luciferase graph/Firefly Photo © Cathy Keifer/Dreamstime.com Expression 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. Photo courtesy of Robb Krumlauf, Stowers Institute for Medical Research FIGURE 09: A mouse promoter controls tissue-specific expression of lacZ FIGURE 10: Fluorescent proteins are powerful research tools Courtesy of Joachim Goedhart, Molecular Cytology, SILS, University of Amsterdam.

23 Fluorescent proteins are powerful research tools 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 GFP(Green), YFP(Yellow), CFP(Cyan), BFP(Blue)

24 The Fly brain

25 GAL4/UAS Binary System Targeted gene expression in fly Brand & Perrimon 1993

26 Shih & Wu unpublished GFP expression in subset of fly mushroom body

27 Wu et al., 2011 GFP express in single neuron in fly brain

28 The Nobel Prize in Chemistry 2008 For the discovery and development of the green fluorescent protein, GFP Woods Hole & Boston University Medical School Columbia UniversityHoward Hughes Medical Institute

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 nm  Protein absorb light at 280 nm  260/280 ratios to quantify the amount of nucleic acid  DNA, 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) technique Adapted from an illustration by Darryl Leja, National Human Genome Research Institute (www.genome.gov).

34 In situ Hybridization: Locating genes in chromosomes FISH: Fluorescence in situ hybridization From 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. Adapted from an illustration by Michael Blaber, Florida State University. FIGURE 14: DNA sizes can be determined by gel electrophoresis

36 FIGURE 15: Agarose gel electrophoresis pattern of SV40 DNA Reproduced from W. Keller, Proc. Natl. Acad. Sci. USA 72 (1975): Photo courtesy of Walter Keller, University of Basel. Type I topoisomerase: Relaxes negative supercoiled DNA to relaxed or linear DNA

37 3.6 DNA Separation Techniques DNA can also be isolated using density gradient centrifugation. DNA density dependents on G-C content An AT base pair has a lower molecular weight than a GC base pair

38 3.7 DNA Sequencing Chain 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 Sequencing Chain 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 Inset photo courtesy of Jan Kieleczawa FIGURE 17: DideoxyNTP sequencing using fluorescent tags 1.Different fluorescents label for each ddNTP 2.Hit with a laser and pass by an optical sensor 3.Glass capillary tube dissipate heat rapidly

41 Thermus aquaticus ( Taq DNA polymerase) 3.8 PCR and RT-PCR

42 Polymerase chain reaction (PCR) permits the exponential amplification of a desired sequence, using primers that anneal to the sequence of interest. *Kary Mullis in the 1980s *Taq polymerase from Thermus aquaticus *Thermal cycler 95 o C 40 o C 72 o C 20x T m : annealing temperature for primer/template pair

43 Exponential produce the primer to primer- defined sequence

44 Press Release 13 October 1993 The Royal Swedish Academy of SciencesThe 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 chain reaction (PCR) method, and half to Professor Michael Smith, University of British Columbia, Vancouver, Canada, for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies. Decisive progress in gene technology through two new methods: the polymerase chain reaction (PCR) method and site-directed mutagenesis Kary B. MullisMichael Smith The Nobel Prize in Chemistry 1993

45 3.8 PCR and RT-PCR RT-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-PCR fluorescence 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 probe – Fluorescent tag at 5’-end – Fluorescence quenching tag at 3’-end With PCR rounds the 5’ tag is separated from the 3’ tag Fluorescence increases with incorporation into DNA product

49 TeqMan probe wikipedia 1.Primer binding 2.Probe hybridization 3.PCR conditions

50 SYBR Green I Dye 1.Primer binding 2.PCR conditions SYBR Green I Dye binds to the minor groove of ds DNA Reporter dye are not sequence specific, spurious products produced by the reaction may lead to false positive signals.


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