1 Recombinant DNA Technology CHMI 4226 E Tools of genetic engineering 1. Enzymes Week of January 5, 2009

2 Outline WEEK TOPIC Jan 5 Review + Tools of genetic engineering - 1. Enzymes Jan 12 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 2Protein expression Mar 9/16 Gene cloning and characterization Mar 23Transgenic and knock-out mice Mar 30 High-throughput techniques Suggested textbook: Recombinant DNA, 2 nd edition, Watson et al. W.H. Freeman and co.New-York

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4 DNA – quick refresher Fig RNADNA O NH 2 Fig RNA only

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8 Central dogma DNARNAprotein Transcription Translation Replication Reverse Transcription Replication

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10 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

11 Tools of Genetic Engineering 1. Enzymes –Restriction enzymes –Modification enzymes (polymerases, kinases, ligase, etc) 2. Vectors 3. Polymerase chain reaction (PCR) 4. Your imagination….

12 Type II restriction enzymes Type II restriction enzymes: –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. Nomenclature: EcoR I –Eco: isolated from E. coli –R: strain R –I: first restriction enzyme isolated from E. coli Why? –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.

13 Type II restriction enzymes EcoR I bound to DNA

14 Type II restriction enzymes Blunt end-generating RE (e.g. EcoR V): Protruding end-generating RE 5’ GATATC 3’ 3’ CTATAG 5’ 5’ GAT 3’ 5’ ATC 3’ 3’ CTA 5’ 3’ TAG 5’ + 5’ GAATTC 3’ 3’ CTTAAG 5’ 5’ G 3’ 5’ AATTC 3’ 3’ CTTAA 5’ 3’ G 5’ + EcoR V generates 5’ phosphate and 3’ OH ends 1) EcoR I: Generates 5’ protruding ends with 5’ phosphate and 3’ OH ends 5’ CTGCAG 3’ 3’ GACGTC 5’ 5’ CTGCA 3’ 5’ G 3’ 3’ G 5’ 3’ ACGTC 5’ + 2) Pst I: Generates 3’ protruding (also called 5’ recessed) ends with 5’ phosphate and 3’ OH ends

15 If RE cuts here: generates 5’ phosphate and 3’ OH ends If RE cuts here: generates 3’ phosphate and 5’ OH ends 5’ 3’

16 Agarose gel electrophoresis Ethidium bromide staining of DNA DNA length marker

17 DNA length markers

18 Restriction enzymes -Hind III

19 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! Restriction enzymes Reaction conditions

20 Restriction enzymes Reaction conditions Isoschizomers: different restriction enzymes cutting the same sequence.

21 Restriction enzymes Reaction conditions

22 Restriction enzymes Reaction conditions

23 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 Restriction enzymes Star activity

24 Dam: GA me TC (methylation on N6) Dcm: C me C me AGG et C me C me TGG (methylation on C5) Restriction enzymes Inhibition by DNA methylases

25 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

26 Getting DNA sequences from public databases - NCBI NCBI: National Center for Biotechnology Information Link:

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28 Entrez Gene - Input In this example: gadd153 is YFG  your favorite gene

29 Entrez Gene - Output

30 Entrez Gene – What’s in the output? Gene/mRNA/protein sequence; Gene structure (exon/intron); Bibliography Interactions involving your favorite protein (YFP) Sequence homology Phenotype (mutations, hereditary diseases, etc) Gene Ontology: –Cellular function –Cellular processes influenced by YFP –Sub-cellular compartment where YFP is found Signaling pathways Sequences –mRNA (RefSeq) –Protein

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32 Size of the piece of DNA featured here Coding sequence Origin of the molecule, type (DNA, RNA) Lots of goodies…

33 Amino acid sequence Nucleotide sequence

34 Genebank:Fasta: So what? Some softwares/algorithms recognize only one sequence format; Useful sequence converter: –ReadSeq Biosequence Format Converter – Sequence formats

35 Restriction mapping Link: 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!

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38 FIRST ASSIGNMENT!

39 Modification enzymes DNA modifying enzymes: –DNA polymerases –DNA kinases –DNA phosphatases –DNA ligases –DNAses RNA modifying enzymes: –Reverse transcriptases –RNAses

40 Modification enzymes

41 Modification enzymes

42 End-modification enzymes

43 DNA Kinases Add 1 phosphate from ATP (the  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 ([  32 P]-ATP) in the reaction.

44 DNA phosphatases Removes phosphate from the 5’ end of DNA molecules. Used to create DNA molecules without 5’ phosphate.

45 DNA phosphatases

46 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.

47 Ligases

48 Ligases

49 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.

50 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.

51 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.

52 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

53 Reverse transcriptases Make a DNA molecule from an RNA template. Require (in addition to all 4 dNTPs) a DNA primer to initiate DNA synthesis.

54 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.

55 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

56 DNA polymerases 5’-3’ exonucleases activity

57 DNA polymerases 3’-5’ exonucleases activity

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