1 recombinant dna technology chmi 4226 e tools of genetic engineering 1. enzymes week of january 5,...

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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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