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Recombinant DNA Technology
CHMI 4226 E
Tools of genetic engineering
1. Enzymes
Week of January 5, 2009
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Outline
WEEK
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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.
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DNA – quick refresher
NH2
O
RNA
DNA
Fig. 2.11
Fig. 2.10
RNA only
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Central dogma
Transcription
DNA
Translation
RNA
protein
Reverse
Transcription
Replication
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
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Type II restriction enzymes:
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Nomenclature: EcoR I
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Eco: isolated from E. coli
R: strain R
I: first restriction enzyme isolated from E. coli
Why?
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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.
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Type II restriction enzymes
EcoR I bound to DNA
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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’
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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’
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Agarose gel electrophoresis
DNA length marker
Ethidium bromide staining of DNA
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DNA length markers
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Restriction enzymes
l-Hind III
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Restriction enzymes
Reaction conditions
• The activity of restriction enzymes can be affected by several
parameters:
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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!
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Restriction enzymes
Reaction conditions
Isoschizomers: different restriction enzymes cutting the same sequence.
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Restriction enzymes
Reaction conditions
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Restriction enzymes
Reaction conditions
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Restriction enzymes
Star activity
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A = High enzyme
concentration
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B = high glycerol
concentration (>10%)
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C = Low ionic strength
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D = elevated pH (> 8)
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E = presence of organic
solvents
(e.g. ethanol, DMSO, DMF)
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F = replacing Mg+2 by Mn+2,
Cu+2, Zn+2 or Co+2
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Restriction enzymes
Inhibition by DNA methylases
Dam: GAmeTC
(methylation on N6)
Dcm: CmeCmeAGG et
CmeCmeTGG (methylation
on C5)
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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
• 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)
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Origin of the
molecule, type
(DNA, RNA)
Size of the
piece of DNA
featured here
Coding
sequence
Lots of goodies…
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Amino acid
sequence
Nucleotide
sequence
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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
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Restriction mapping
• Link: http://www.restrictionmapper.org/
• Instruction:
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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:
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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 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.
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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 polymerases
5’-3’ exonucleases activity
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DNA polymerases
3’-5’ exonucleases activity
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