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Recombinant DNA Technology CHMI 4226 E Tools of genetic engineering 1. Enzymes Week of January 5, 2009 1 Outline WEEK • • • • • • • • • • • TOPIC Jan 5 Jan 12 Review + Tools of genetic engineering - 1. Enzymes Tools of genetic engineering – 2. Vectors and basic cloning strategies Jan 19 Tools of genetic engineering – 3. Polymerase chain reaction Jan 26/Feb 2 cDNA libraries Feb 9 Analysis of gene expression – Northern blots, RNAse protection, PCR Feb 12 – Mid-term examination Feb 23 Mutagenesis Mar 2 Protein expression Mar 9/16 Gene cloning and characterization Mar 23 Transgenic and knock-out mice Mar 30 High-throughput techniques Suggested textbook: Recombinant DNA, 2nd edition, Watson et al. W.H. Freeman and co.New-York. 1992. 2 3 DNA – quick refresher NH2 O RNA DNA Fig. 2.11 Fig. 2.10 RNA only 4 5 6 7 Central dogma Transcription DNA Translation RNA protein Reverse Transcription Replication Replication 8 Replication 9 Transcription/Translation NOTE: The sequence of the mRNA is complementary to the template (non-sense, transcribed) strand and is identical to the non-template (sense, non-transcribed) strand on the DNA 10 Tools of Genetic Engineering • 1. Enzymes – Restriction enzymes – Modification enzymes (polymerases, kinases, ligase, etc) • 2. Vectors • 3. Polymerase chain reaction (PCR) • 4. Your imagination…. 11 Type II restriction enzymes • Type II restriction enzymes: – – – – – • Nomenclature: EcoR I – – – • Eco: isolated from E. coli R: strain R I: first restriction enzyme isolated from E. coli Why? – • homodimers. Recognize a short, specific DNA sequence (generally 4 to 8 nt). Cut DNA at their binding site. THEREFORE, THE PRESENCE OF A RE BINDING SITE ON A PIECE OF DNA IMMEDIATELY TELLS YOU THAT THIS SITE WILL BE CLEAVED IN THE PRESENCE OF THIS RE. Cut palindromic sequences Generate either blunt or protruding ends. Restriction enzymes allow bacteria to defend themselves against foreign DNA (e.g. viral DNA) Type I and type III RE also exist, but they do not cut aat their DNA binding site. Therefore, they are used only very rarely in genetic engineering applications. 12 Type II restriction enzymes EcoR I bound to DNA 13 Type II restriction enzymes Blunt end-generating RE (e.g. EcoR V): EcoR V generates 5’ phosphate and 3’ OH ends 5’GATATC3’ 5’GAT3’ 3’CTATAG5’ 3’CTA5’ + 5’ATC3’ 3’TAG5’ Protruding end-generating RE 1) EcoR I: Generates 5’ protruding ends with 5’ phosphate and 3’ OH ends 5’GAATTC3’ 5’G3’ 3’CTTAAG5’ 3’CTTAA5’ + 5’AATTC3’ 3’G5’ 2) Pst I: Generates 3’ protruding (also called 5’ recessed) ends with 5’ phosphate and 3’ OH ends 5’CTGCAG3’ 5’CTGCA3’ 3’GACGTC5’ 3’G5’ + 5’G3’ 3’ACGTC5’ 14 If RE cuts here: generates 5’ phosphate and 3’ OH ends 5’ 3’ 5’ 3’ 5’ If RE cuts here: generates 3’ phosphate and 5’ OH ends 3’ 15 Agarose gel electrophoresis DNA length marker Ethidium bromide staining of DNA 16 DNA length markers 17 Restriction enzymes l-Hind III 18 Restriction enzymes Reaction conditions • The activity of restriction enzymes can be affected by several parameters: – – – – Temperature Ionic strength (salt concentration) Type of salt Reducing agents (DTT, 2-ME) • Buffers with optimal conditions of salt, pH, etc are provided upon purchase of any RE enzyme. • Non-optimal conditions can lead to non-specific cutting, a phenomenon called star activity. • SO: Care should be taken when digesting a DNA molecule with 2 different RE – make sure the digestion conditions are compatible! 19 Restriction enzymes Reaction conditions Isoschizomers: different restriction enzymes cutting the same sequence. 20 Restriction enzymes Reaction conditions 21 Restriction enzymes Reaction conditions 22 Restriction enzymes Star activity • A = High enzyme concentration • B = high glycerol concentration (>10%) • C = Low ionic strength • D = elevated pH (> 8) • E = presence of organic solvents (e.g. ethanol, DMSO, DMF) • F = replacing Mg+2 by Mn+2, Cu+2, Zn+2 or Co+2 23 Restriction enzymes Inhibition by DNA methylases Dam: GAmeTC (methylation on N6) Dcm: CmeCmeAGG et CmeCmeTGG (methylation on C5) 24 How to know which RE cuts your favorite DNA molecule? • 1) Get the sequence of the DNA molecule • 2) Plug the sequence in a program which will find the RE sites of interest 25 Getting DNA sequences from public databases - NCBI • NCBI: National Center for Biotechnology Information • Link: http://www.ncbi.nlm.nih.gov/Sitemap/index.html 26 27 Entrez Gene - Input In this example: gadd153 is YFG your favorite gene 28 Entrez Gene - Output 29 Entrez Gene – What’s in the output? • Gene/mRNA/protein • Gene Ontology: sequence; – Cellular function • Gene structure – Cellular processes influenced by YFP (exon/intron); – Sub-cellular • Bibliography compartment where YFP • Interactions involving is found your favorite protein • Signaling pathways (YFP) • Sequences • Sequence homology – mRNA (RefSeq) • Phenotype (mutations, – Protein hereditary diseases, etc) 30 31 Origin of the molecule, type (DNA, RNA) Size of the piece of DNA featured here Coding sequence Lots of goodies… 32 Amino acid sequence Nucleotide sequence 33 Sequence formats • Genebank: • Fasta: • So what? Some softwares/algorithms recognize only one sequence format; • Useful sequence converter: – ReadSeq Biosequence Format Converter – http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi 34 Restriction mapping • Link: http://www.restrictionmapper.org/ • Instruction: – – – – Select sequence of interest Copy Paste in window in Restriction Mapper site Select the enzymes for which you want the location of the cutting sites (I selected BamH I, EcoR I and Pst I) – Press « Map Sites » – Bingo! 35 36 37 FIRST ASSIGNMENT! 38 Modification enzymes • DNA modifying enzymes: – – – – – DNA polymerases DNA kinases DNA phosphatases DNA ligases DNAses • RNA modifying enzymes: – Reverse transcriptases – RNAses 39 Modification enzymes 40 Modification enzymes 41 End-modification enzymes 42 DNA Kinases • Add 1 phosphate from ATP (the g phosphate) to the 5’ end of a DNA molecule. • Used to: – Phosphorylate DNA molecules which do not possess a 5’ phosphate – Label DNA molecules at both 5’ ends: requires the use of radioactive ATP ([g32P]-ATP) in the reaction. 43 DNA phosphatases • Removes phosphate from the 5’ end of DNA molecules. • Used to create DNA molecules without 5’ phosphate. 44 DNA phosphatases 45 DNA ligases • Catalyses the formation of a phosphodiester bond between the 3’ OH of a DNA molecule and the 5’ phosphate of another. • Used to create a covalent bond between 2 DNA fragments. 46 Ligases 47 Ligases 48 DNA nucleases (DNAses) • Enzymes which catalyse the depolymerization (degradation) of DNA molecules. • Can work from the ends of the DNA (exonuclease) or directly in the molecule (endonuclease). • Used to: – Get rid of unwanted DNA. – Modify the ends of DNA molecules. 49 DNAses • Exonucleases can degrade DNA in two possible ways – From the 3’ end towards the 5’ end – From the 5’ end towards to 3’ end. 50 RNA nucleases (RNAses) • Enzymes which degrade RNA molecules. – RNAse A degrades both single stranded and double stranded RNA – RNAse H degrades only the RNA in an RNA-DNA hybrid. 51 RNA polymerases • Synthesize a RNA molecule from a DNA template (transcription). • Require promoter sequences upstream of the DNA sequence to be transcribed. Also needs all 4 NTPs 52 Reverse transcriptases • Make a DNA molecule from an RNA template. • Require (in addition to all 4 dNTPs) a DNA primer to initiate DNA synthesis. 53 DNA polymerases • Catalyze the synthesis of a DNA molecule. • Require (in addition to all 4 dNTPs) – a DNA primer to initiate DNA synthesis – a DNA template. 54 DNA polymerases • Can possess 3 types of activities: – Polymerase: always in the 5’ to 3’ direction – Exonuclease: can be in the 5’-3’ or 3’-5’ direction. – Whether a DNA polymerase will exhibit polymerase or exonuclease activity depends on the abundance of free dNTPs: • Presence of dNTPs: polymerase activity is on. • No dNTPs in the reaction: exonuclease activity is on. • Klenow enzyme: – Very widely used in genetic engineering – A modified a E. coli DNA polymerase – Possesses: • 5’-3’ polymerase activity • 3’-5’ exonuclease activity 55 DNA polymerases 5’-3’ exonucleases activity 56 DNA polymerases 3’-5’ exonucleases activity 57 58