cloning and restriction enzymes

8/8/2019 Cloning and Restriction Enzymes

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Cloning and Restriction EnzymesJennifer M. Anderson, PhD


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DNA Modifying Enzymes

AKA: Restriction EnzymesThe restriction/modification system functions as a

type of immune system for individual bacterialstrains, protecting them from infection by foreignDNA (e.g. viruses).W. Arber and S. Linn (1969)


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W. Arber and S. Linn (1969)Plating efficiencies of bacteriophage lambda (l phage)grown on E. coli strains C , K-12 and B: K-12 and Bwere protected from bacteriophage.The DNA of phage which had been grown on strains K-12 and B were found to have chemically modified bases

which were methylated .Additional studies with other strains indicate thatdifferent strains had specific methylated bases.Typical sites of methylation include the N6 position of adenine, the N4 position of cytosine, or the C5 positionof cytosine.Methylation occurres at very

specific sites in the DNA


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(EcoR1 methylase specificity. Rubin and Modrich, 1977)In addition to possessing a particular methylase, individual

bacterial strains also contained accompanying specificendonuclease activities.

The endonucleases cleaved at or near the methylationrecognition site .

These specific nucleases, however, would not cleave at thesespecific palindromic sequences if the DNA was methylated .

A characteristic feature of the sites of methylation, was that they involved palindromic

DNA sequences .


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Restriction Enzymes

In the bacterial strain EcoR1, the sequence GAATTCwill be methylated at the internal adenine base (by theEcoR1 methylase).

The EcoR1 endonuclease within the same bacteria willnot cleave the methylated DNA.Foreign viral DNA, which is not methylated at thesequence "GAATTC" will therefore be recognized as

"foreign" DNA and will be cleaved by the EcoR1endonuclease.Cleavage of the viral DNA renders it non-functional.


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R estriction endonucleases

They are one of the primary tools in modern molecular biology for the manipulation and identification of DNAsequences.Restriction endonucleases are commonly named after the bacterium from which it was isolated.The utility of restriction endonucleases lies in their specificity and the frequency with which their recognition sites occur within any given DNA sample.

If there is a 25% probability for a specific base at any given site, then the frequency with which different restriction endonuclease sites will occur can be easilycalculated (0.25n):


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NucleotideSpecificity Example

Frequency of Occurrence

Four Alu I 256 (0.25 Kb)Five Nci I 1024 (1.0 Kb)Six EcoR I 4096 (4.1 Kb)Seven EcoO109I 16384 (16.4 Kb)Eight Not I 65536 (65.5 Kb)

On average, any given DNA will contain an Alu I siteevery 0.25 kilobases, whereas a Not I site occurs onceabout every 65.5 kilobases.

Not I is therefore a very useful enzyme for isolatinglarge regions of DNA, typically in research involvinggenomic DNA manipulations.Alu I would be expected to digest a DNA sample into

lots of little pieces.


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Examples of RENName Source Recognition Sequence Comments

Alu I Arthrobacter luteus| 5' A G C T 3' 3' T C G A 5' |

" Four cutter " . Leaves bluntends to the DNA.

Bfa IB acteroidesfragilis

| 5' C T A G 3' 3' G A T C 5' |

" Four cutter " . Leaves 5'overhang.

EcoR1

E scherichiacoli

| 5' G A A T T C 3' 3' C T T A A G 5' |

" Six cutter " . Leaves 5'

overhang. Behaves like a"

four cutter " ('star' activity) in highsalt buffer. $44 for 10,000units.

Hae IIH aemophilus

aegyptius

| 5' Pu G C G C Py 3' 3' Py C G C G Pu 5' |

" Six cutter " . Pu is any purine,Py is any pyrimidine. Leaves 3'overhang.

Bgl IB acillusglobigii

| 5' GCCN NNNNGGC 3' 3' CGGNNNNNCCG 5' |

" Six cutter with interruptedpalindrome " . Leaves 5'overhang. Different recognitionsites may have non-complementary sequences.


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H indIII

H aemophilus influenzae

C os

5 overhanging

Sticky ends

Pst 1 Providencia stuartii

3 overhanging


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U sing REN

One unit of activity is typically defined as the amount of enzymerequired to digest (or "restrict") one microgram of reference DNA in onehour at 37 C.The endonuclease hydrolysis is a spontaneous reaction and does not, for example, require addition of ATP. Reaction buffers for restrictionendonucleases usually contain a buffer component (typically 10 mM

TRIS buffer around pH 8.0), magnesium salt (often 10 mM MgCl2), areducing agent (usually 1mM dithiothreitol, or DTT), a protective carrier protein (typically 100 ug/ml bovine serum albumin, or BSA), and salt(sodium chloride).T he biggest determinant of enzyme activity is typically the ionicconcentration (Na C l content) of the buffer . Although there are

hundreds of different restriction endonucleases, the majority of them canexhibit between 30-100% activity using a simple system of three buffers, containing either low (20 mM), medium (100 mM) or high (250mM) salt (NaCl) concentrations in the above described buffer.Enzyme digests are typically performed for 1-2 hours at 37 C.However, quantitative digestion can sometimes only be achieved after

extended incubation (i.e. overnight).


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U ses of REN

RFLP Restriction Fragment Length Polymorphism

DNA finger printing

Molecular diagnosticsCloning

cDNA library constructionPopulation studies


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Example of RFLP

From Santolamazza et al. 2004

Targets the rDNA IGS


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16s+1 16s-1

~460 bp

16sAmR

5 3

16sDvF2

~306bp

~154bp

16S

D ermacentor

Ixodes, Rhipicephalus, H aemaphysalis

Amblyomma

500

200

L L

1 2 3 4 5 6 7 8 9 1 0 11 1 2 1 3 1 4 1 5 1 6 1 7 1 8 19 2 0 2 1 N C

Dermacentor Amblyomma Hae Rhip IxodesL L

D U D U D U Haemaphysa li s

Rhi picepha lu s Ixod es

PCR Amplification

TauI digestion

Example of PCR-RFLP: Tick Diagnostic

Anderson et al. 2004


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Cloning Applications

cDNA libraryGenomic DNA libraryRecombinant DNA vaccinesRecombinant gene protein expressionSequencingGene amplificationFinding Polymorphisms (one clone, one template)Etc.


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Cloning

Template Choice: Genomic DNA cDNA (from mRNA)

Vector Choice: Virus- Bacteriophage- high replication efficieny (up

to 200 fold) Plasmids good for preparation of pure DNS Phagmids element of both phages and plamsids


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Cloning Basics

DNA

Vector

B asic Elements :Piece of DNAAppropriate vector Insertion SiteCompetent Cells


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Gene Cloning Methods contdT he vectors

PlasmidsC apacity : 5-10 kbORI (origin of replication)

Gene for antibioticresistance (Tet R ,Amp R )Polylinker with RENsites for ligationpU C19pSMART-LC KAN

http://www.agen.ufl.edu/~chyn/age2062/lect/lect_09/FG10_001.GIF


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Phage Advantage over Plasmids :

infect cells more efficiently

all phages derive from a single phage => allidenticalaccommodate more foreign DNA (~ 20kb).Therefore better for making genomic libraries

Lambda phage (replacement vector)Possess complementary cohesive (cos) ends giant sticky ends (similar to REN overhangs)



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C osmidsHave the cos sites of lambda and the ori of plasmid hence the name

cosmid. Packaged in lambda head but replicate like a plasmid.

- large inserts (40-50kb )

M 13 phagessingle stranded and allow replication to get ssDNA. U seful for

that and for sequencing.



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PhagemidsLike cosmids, have characteristics of both phages and plasmidshence the name. => ssDNA. E.g. pBluescript (lacZ)



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Introduction of Restriction Sites U sing PCR It is possible to introduce restriction site sequences into PCR products by havingthese sequences incorporated into the 5' end of the PCR primer(s).The short restriction site sequence on the 5' end of the PCR primer will nothybridize, but as long as the 3' hybridizing region is long enough (i.e. its Tm ishigh enough; ~20 mer) the primer will specifically bind to the appropriate site.The PCR product will thus have an additional DNA squence at the 5' end whichwill contain the endonuclease restriction site.

A similar or different restriction site sequence can be added via the other PCR primer.If the other primer has a different restriction sequence then the PCR fragment can

be inserted in a directional dependent manner in a host plasmid.T he potential problems with this method include :

There is no easy way to prevent internal sites containing similar restrictionsequences from being cut when the end of the PCR product are cutRestriction sequences are inverse repeat sequences, thus the potential exists for

primer dimer association and resultant non-productive annealing


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Blunt End Ligation

Thermostable DNA polymerases add a single dA residue onto the 3' end of thePCR product.There are three choices to be made when attempting to subclone without the useof added restriction sites within the primers: U se a DNA polymerase which leaves the 3' strand blunt (e.g. Vent) and do a blunt

end ligation (i.e. host vector was opened up with blunt cutting restrictionendonuclease)

"Fix" the 3' A overhang by chewing back with Pol I, dNTP's. U se the 3' A overhang to anneal and ligate to a "T" vector - a vector which has a

single dT overhang on its 3' ends."T" vectors can be made by opening up a vector, via a blunt cutting restrictionendonuclease and ligating in a specific linker.The linker contains a restriction sequence for a restriction endonuclease whichrecognizes an interrupted palindrome and cuts in the internal region with asingle 3' overhang.The linker will contain two copies of the restriction recognition sequence, thefirst with a sequence in the interrupted palindrome which leaves a 3' T overhangand the second with a sequence in the interrupted palindrome which leavesanother 3' T overhang

This may sound confusing, so here's an example of how to make a T vector:


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Cloning Cont.

You can use PCR to add: Promoters

Ribosome binding sites Start codons Stop codons Etc


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Why go through all of that effort??

C loing K its : Topo TA cloning (Invitrogen)U se the A at the ends of the PCR product to ligate tothe T on the vector Easy ligation of PCR products No need for Restriction enzyme digestion of plasmid No need for complimentary REN ends on PCR product

Topoisomerase I: both a REN and a ligase, biological

role is to cleave and rejoin DNA during replication.


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Topo TA Cloning


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Topo-TA Cloning: Fast and Easy!

U p to 99% efficiency


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Another useful Plasmid: pGEM-ZBlue/White Screening (Promega)

Plasmid vector with coding region for the enzyme beta-galactosidase (LacZ gene)Disruption of this gene (by your insert) will allow the

clones to be identified by color screeningVectors w/o an insert will express beta-galactosidaseenzyme when expressed in bacteria (JM109, etc) and

plated with 0.5 IPTG and 40 ug/ml X-gal. The enzyme

will convert the X-gal to a colored product, thus bluecolonies are productedClones with an insert will appear white (no enzymeexpression).


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Cloning Protocol

PCR amplificationRun gel of product: single band

Clean PCR product (gel purification)

Ligate PCR product (TOPO TA cloning)

Transform Bacteria

Plate Bacteria (antibiotic resistance)

Pick 8-16 clones for screening

PCR with vector primers*

Clean PCR

Sequence PCR product

Analyze sequences Pick clone with proper orientation


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Some Basic Problems with Cloning

Clean PCR products before cloning Check for single band; consider gel purification of

PCR product for optimal cloning

PCR product should be used immediatelyotherwise your cloning efficiency will decreasesignificantly (you will loose the As).Directionality: insert can be ligated in bothdirections Pick several clones and screen each for proper

orientation (PCR, clean, sequence, analyze)


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Restriction Enzyme Practical

Go to website for PubMed: http://www.ncbi.nlm.nih.gov/entrez/ Change search field to Nucleotide Look for DQ826526 Should be a salivary protein for P . dubo scqi


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Change the display to FASTA and thencopy the sequence (ctrl c)


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Restriction Enzyme ExerciseGo to: http://www.restrictionmapper.org/Paste (cntrl v) your sequence into the window and click Map Sites

How many REN sitesare found?

Will EcoRI cut your sequence?

How many times doesApoI cut?

I have amplified thisgene and the PCR

product is ~500 nt. Iwant to digest it into2 aproxiamtely equalfragments, which

enzyme would I use?


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Restriction Enzyme Exercise

You have the following enzymes in your freezer (DraII, AleI, BamHI, Hsp921, KpnI, SbfI, VspI) andyou want to know if any of them cut your pcr

product so you can save your lab money!This time paste your sequence into the window andselect the following REN form the Includewindow (highlight the following enzymes, you willneed the hold the shift button down to include more

then one enzyme: DraII, AleI, BamHI, Hsp921,KpnI, SbfI, VspIDo any of the enzymes cut your sequence? If yes,which one? How many times does it cut? Where?


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Questions?????

cloning and restriction enzymes

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