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Recombinant DNA TechnologyCHMI 4226 E
Tools of genetic engineering1. Enzymes
Week of January 5, 2009
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OutlineWEEK 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 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.
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DNA – quick refresher
Fig. 2.11
RNA DNAONH2
Fig. 2.10
RNA only
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Central dogma
DNA RNA proteinTranscription Translation
Replication
Reverse Transcription
Replication
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Replication
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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
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Tools of Genetic Engineering
• 1. Enzymes– Restriction enzymes– Modification enzymes (polymerases, kinases,
ligase, etc)
• 2. Vectors
• 3. Polymerase chain reaction (PCR)
• 4. Your imagination….
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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.
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Type II restriction enzymes
EcoR I bound to DNA
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Type II restriction enzymesBlunt end-generating RE (e.g. EcoR V):
Protruding end-generating RE
5’GATATC3’
3’CTATAG5’
5’GAT3’ 5’ATC3’
3’CTA5’ 3’TAG5’+
5’GAATTC3’
3’CTTAAG5’
5’G3’ 5’AATTC3’
3’CTTAA5’ 3’G5’+
EcoR V generates 5’ phosphate and 3’ OH ends
1) EcoR I: Generates 5’ protruding ends with 5’ phosphate and 3’ OH ends
5’CTGCAG3’
3’GACGTC5’
5’CTGCA3’ 5’G3’
3’G5’ 3’ACGTC5’+
2) Pst I: Generates 3’ protruding (also called 5’ recessed) ends with 5’ phosphate and 3’ OH ends
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If RE cuts here: generates 5’ phosphate and 3’ OH ends
If RE cuts here: generates 3’ phosphate and 5’ OH ends
5’
5’
5’
3’
3’
3’
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Agarose gel electrophoresis
Ethidium bromide staining of DNA
DNA length marker
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DNA length markers
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Restriction enzymes-Hind III
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• 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 enzymesReaction conditions
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Restriction enzymesReaction conditions
Isoschizomers: different restriction enzymes cutting the same sequence.
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Restriction enzymesReaction conditions
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Restriction enzymesReaction conditions
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• 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 enzymesStar activity
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Dam: GAmeTC (methylation on N6)
Dcm: CmeCmeAGG et CmeCmeTGG (methylation on C5)
Restriction enzymesInhibition by DNA methylases
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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
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Getting DNA sequences from public databases - NCBI
• NCBI: National Center for Biotechnology Information• Link: http://www.ncbi.nlm.nih.gov/Sitemap/index.html
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Entrez Gene - Input
In this example: gadd153 is YFG your favorite gene
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Entrez Gene - Output
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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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Size of the piece of DNA featured
here
Coding sequence
Origin of the molecule, type (DNA, RNA)
Lots of goodies…
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Amino acid sequence
Nucleotide sequence
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• 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
Sequence formats
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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!
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FIRST ASSIGNMENT!
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Modification enzymes
• DNA modifying enzymes:– DNA polymerases – DNA kinases– DNA phosphatases– DNA ligases– DNAses
• RNA modifying enzymes:– Reverse transcriptases– RNAses
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Modification enzymes
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Modification enzymes
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End-modification enzymes
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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 ([32P]-ATP) in the reaction.
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DNA phosphatases
• Removes phosphate from the 5’ end of DNA molecules.
• Used to create DNA molecules without 5’ phosphate.
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DNA phosphatases
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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.
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Ligases
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Ligases
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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.
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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.
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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.
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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
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Reverse transcriptases• Make a DNA molecule from an RNA template.• Require (in addition to all 4 dNTPs) a DNA
primer to initiate DNA synthesis.
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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.
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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
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DNA polymerases5’-3’ exonucleases activity
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DNA polymerases3’-5’ exonucleases activity
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