Primer Design
description
Transcript of Primer Design
Primer DesignPrimer Design
http://frodo.wi.mit.edu/
Put in your sequencePut in your sequence
Primer sizeAnnealing temperature
% GC
Your sequenceYour sequence
Left primer Right primer
Pick primers
Left primer
Right primer
Product size
Search for RE siteSearch for RE site
BioEditBioEdit
Cloning & Expression Cloning & Expression VectorVector
Clone Cloning
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Drug Resistance Gene Transferred by Plasmid
Plasmid gets out and into the host cell
Resistant Strain
New Resistance Strain
Non-resistant Strain
Plasmid
EnzymeHydrolyzingAntibiotics
Drug Resistant Gene
mRNA
Juang RH (2004) BCbasics
Target Genes Carried by Plasmid
1 plasmid1 cellRecombinant
PlasmidTransformation
Target GeneRecombination
Restriction
Enzyme
Restriction
Enzyme
Ch
rom
oso
mal
DN
ATarget Genes
DNA Recombination
TransformationHost Cells
Juang RH (2004) BCbasics
Amplification and Screening of Target Gene
1
1 cell line, 1 colonyX100
X1,000
PlasmidDuplicationBacteria
Duplication
Plating
Pick the colonycontaining target gene
=100,000Juang RH (2004) BCbasics
• Once you have your restriction enzymes chosen, it is time to design the final complete gene
• The multiple cloning site (or whatever plasmid you are cloning into) should already have the 5’ portion of the gene intact (i.e. RBS, spacer, Met)
• Sequences must be in frame
NcoI BtgI51 CTTTAATAAG GAGATATACC ATGGGCAGCA GCCATCACCA TCATCACCAC M G S S H H H H H H
SacI AscI SbfI SalI NotI BamHI EcoRI EcoICRI BssHII PstI AccI HindIII101AGCCAGGATC CGAATTCGAG CTCGGCGCGC CTGCAGGTCG ACAAGCTTGC S Q D P N S S S A R L Q V D K L A
Design of the Insert
Design of the Insert71 ATGGGCAGCAGCCATCACCATCATCACCAC M G S S H H H H H H SacI AscI SbfI SalI BamHI EcoRI EcoICRI PstI AccI HindIII101AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC S Q D P N S S S A R L Q V D K L A
The gene we want:ggctgcgacagggcgagcccgtactgcggttaa G C D R A S P Y C G *
BamHI PstI AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC S Q D P N S S S A R L Q V D K L A G C D R A S P Y C G * ggctgcgacagggcgagcccgtactgcggttaa
AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA
Be aware of the amber stop codon: TAG
Multiple cloning site
Design of the InsertAlways check and re-check your sequence!
ATGGGCAGCA GCCATCACCA TCATCACCACAGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA
atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa P Y C G - L Q V D
Everything looks good: in frame the whole way!
Translate the whole gene
The wrong way to do it:AGCCAGGATCC ggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAAGCTT
atgggcagcagccatcaccatcatcaccacagccaggatccggctgcgacagggcgagccM G S S H H H H H H S Q D P A A T G R A cgtactgcggttaactgcaggtcgacaagcttR T A V N C R S T S
Frame shifted = garbage!
Design of the Insert
The gene is just inserted after the restriction site, which is out of frame with the plasmid-encoded start-codon/His-tag
**Some plasmids, for whatever reason, have restriction sites out of frame with the translated
gene**
Finishing Touches
atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa P Y C G - L Q V D
•Restriction enzymes need 5’ and 3’ base pairs to cut properly
•NEB has a reference guide for specific enzymes (see link below)
•A good rule of thumb is 6 base pairs after the recognition site
•Inserting a GC “clamp” at the end and beginning of the sequence is also a good idea
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D
Final gene, polished and ready to go:
Once the insert is designed correctly, the next step is designing primers to order from IDT, based on insert synthesis strategyOnce the insert is designed correctly, the next step is designing primers to order from IDT, based on insert synthesis strategy
Design of the Primers
Three main strategies towards insert synthesis:
• PCR amplification
• Klenow extension of overlapping primers
• Complimentary full-length primers
Three main strategies towards insert synthesis:
• PCR amplification
• Klenow extension of overlapping primers
• Complimentary full-length primers
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InsertVector
The most common method of insert synthesis
• Necessitates a pre-existing construct
• Extra restriction sites and/or amino acid residues can be added on each side of the gene
• Internal mutations are more difficult
PCR Amplification of Insert from an Existing Gene
Insert
PCR amplification from overlapping primers
•No pre-existing construct is needed
•PCR products messy, possibly making subsequent rxns difficult
•Good for inserts >150 bp
PCR amplification from overlapping primers
•No pre-existing construct is needed
•PCR products messy, possibly making subsequent rxns difficult
•Good for inserts >150 bp
PCR Synthesis of Insert
F1: 10xF2: 1x
R1: 1xR2: 10x
5’3’
5’ 3’
5’3’
5’ 3’
Full-length insert should still be the major product
Insert
Klenow Extension of Overlapping Primers
•Two primers that are complimentary in their 3’ region are designed (overlap 15bp)
•Extended to full length by the Klenow fragment of DNA Polymerase I
•Useful if insert is 50 to 150 bp
•Two primers that are complimentary in their 3’ region are designed (overlap 15bp)
•Extended to full length by the Klenow fragment of DNA Polymerase I
•Useful if insert is 50 to 150 bp
Insert
5’3’
5’ 3’
Klenow
Klenow fragment: retains 3’ to 5’ polymerase activity, but does not have exonuclease activityKlenow fragment: retains 3’ to 5’ polymerase activity, but does not have exonuclease activity
•The simplest approach
• Order two primers that compliment each other
• Mix the two primers, heat, and anneal slowly (to ensure proper base-pairing)
•Feasible if the total insert size is < 60 bp
Complimentary Full-Length Primers
Insert5’3’
5’ 3’ Anneal
Designing Primers to OrderOnce the insert synthesis technique is decided, primer design is fairly straight-forward
Forward primers:
•Assess necessary overlap and copy the sequence from your designed gene, along with extra 5’ sequence
Reverse primers:
•First, design exactly as if it were a forward primer: Copy necessary overlap and extra 3’ sequence from your designed gene
•Once all this is in place, use pDRAW32 sequence manipulator to calculate the reverse compliment
•Order the pDRAW32 calculated sequence directly
Cloning Out an Existing GeneIn the example mentioned previously, we would normally use full length overlapping primers, but let’s look at the more common case of having a preexisting gene:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D
tgcggcccagccggccatgggctgcgacagggcgagcccgtactgcggtggaggcggtgctgcagcgc A A Q P A M G C D R A S P Y C G G G G A A A
Preexisting gene:
Goal gene:
gccagccaggatccgggctgcgacagg ccgtactgcggttaactgcaggtcgacgc
Forward Primer: Design of Reverse Primer:
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Overlap
Extra sequence from gene design
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc S Q D P G C D R A S P Y C G - L Q V D
Ordering Primers
Forward primer to order:gccagccaggatccgggctgcgacagg
Reverse primer to order:GCGTCGACCTGCAGTTAACCGCAGTACGG
http://www.idtdna.com/Home/Home.aspxNow we can order the primers:
Design of Reverse Primer: ccgtactgcggttaactgcaggtcgacgc
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•Anti-biotic resistance (working concentration)
•Ampicillin (100g/mL)
•Kanamycin (35g/mL)
•Tetracycline HCl (10g/mL)
•Chloramphenicol (170g/mL in ethanol)
Purification Tags and Selection (Anti-biotic Resistance)
Digestion of Insert and Vector
•Digest with the same restriction endonucleases
•Optional (recommended) step:
•Treat the plasmid DNA with Antarctic phosphatase
•Decreases the background by stopping self-ligation of singly cut plasmid and background re-ligation
Ligation of the Insert into the Vector
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•Ligation covalently attaches the vector and the insert via a phosphodiester bond (5’phosphate and 3’ hydroxyl of the next base)
Transformation
•The functional construct is now ready to be transformed into new E. coli and grown up
•The new DNA isolated from the E. coli must then be sequenced to make sure that everything worked
•Once the sequence is confirmed, we are ready to go!
pBluescrippBluescripttpBluescrippBluescriptt
MCS
MCS, Multiple Cloning Site
ampicillinresistance
gene
A widely used plasmid cloning vector
origin ofreplication
• select for transformants with antibiotic• electroporation = 109-1010
colonies/g DNA• heat-shock = 105-109 colonies/g
DNA)
Identifying Recombinants• based on interruption of a gene
• eg., lacZ gene = -galactosidase• intact -galactosidase produces blue color in
presence of X-gal
-complementation or blue-white screening
Blue white screeningBlue white screening
Ampr
ori
pUC18(3 kb)
MCS (Multiple cloning sites)
Lac promoter
lacZ’
Screening by insertional inactivation of the lacZ gene
The insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.
Recreated vector: blue transformantsRecombinant plasmid containing inserted DNA: white transformants
Recreated vector (no insert)
Recombinant plasmid (contain insert)
back
Multiple cloning sitesMultiple cloning sitesMultiple restriction sites enable the convenient insertion of target DNA into a vector
Ampr
ori
pUC18(3 kb)
MCS (Multiple cloning sites)
Lac promoter
lacZ’
…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…
. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…
EcoRI SacI KpnISmaIXmaI BamHI
XbaI
SalIHincIIAccI PstI SphI
Lac Z
Recombinant DNA / Genetic EngineeringRecombinant DNA / Genetic Engineering
The advent of recombinant DNA technology (gene cloning or manipulation) has dramatically broaden the spectrum of microbial genetic manipulation.
Based on the use of restriction enzyme (endonucleases) and DNA ligases as a means to cut & paste fragments of DNA
Foreign DNA fragments can be introduced into a vector molecule (eg. plasmid or bacteriophage), enable replication of the DNA in bacteria cell.
Recombinant technology in one form or another is used in many areas of biological research today.
Ability to modify and clone genes – accelerated the rate of discovery and the development of bioindustries
Recombinant DNA TechnologyRecombinant DNA Technology
ApplicationsApplications
to study basic cellular mechanisms (eg.cell signaling pathway)to study basic cellular mechanisms (eg.cell signaling pathway)
production of recombinant vaccine for therapy (cancer, genetic disorders, immune disorders, embryonic production of recombinant vaccine for therapy (cancer, genetic disorders, immune disorders, embryonic stem cells)stem cells)
Production of recombinant proteins of medical & commercial value (eg. antibodies, insulin, RE)Production of recombinant proteins of medical & commercial value (eg. antibodies, insulin, RE)
Generate genetically modified / transgenic plants (GMOs plant) or animals with enhanced commercial and Generate genetically modified / transgenic plants (GMOs plant) or animals with enhanced commercial and health properties.health properties.
Cloning of plants and animals.Cloning of plants and animals.
What is DNA cloning?What is DNA cloning?
isolation and manipulation of isolation and manipulation of fragments of an organism’s genome fragments of an organism’s genome by replicating independently as part by replicating independently as part an autonomous vector in another host an autonomous vector in another host species. species.
DNA fragment in vector will form DNA fragment in vector will form recombinant DNA.recombinant DNA.
Applications of DNA cloningApplications of DNA cloning
DNA sequencing - genome & protein database.DNA sequencing - genome & protein database.
Isolation & analysis of gene promoters / control sequencesIsolation & analysis of gene promoters / control sequences
Investigate protein / enzyme / RNA function by large-scale Investigate protein / enzyme / RNA function by large-scale production of normal & altered formsproduction of normal & altered forms
Identification of mutations -eg. gene defects cause diseaseIdentification of mutations -eg. gene defects cause disease
Biotechnology – large-scale commercial production of Biotechnology – large-scale commercial production of proteins & other molecules of biological importance (eg. proteins & other molecules of biological importance (eg. human insulin & growth hormone)human insulin & growth hormone)
Engineering animals & plants, gene therapyEngineering animals & plants, gene therapy
Engineering proteins – altering propertiesEngineering proteins – altering properties
Basic steps in gene cloningBasic steps in gene cloning
DNA
insert
isolationVector
restriction
ligation
Recombinant DNA
Transformation/amplification
Host cells
Selection / identification of clones
Validation of clones –analyses RE, Southern blot, PCR, DNA sequencing
Positive recombinant DNA
The cloning of DNA The cloning of DNA in a plasmidin a plasmid
Recombinant DNA techniques Recombinant DNA techniques used for Insulin production in used for Insulin production in E.coliE.coli
Isolate or cut the insulin gene from human DNA. Isolate or cut the insulin gene from human DNA. Restriction enzymes used to cut vector & insert for cloningRestriction enzymes used to cut vector & insert for cloning Ligate / paste insulin gene (insert) into a vector using DNA Ligate / paste insulin gene (insert) into a vector using DNA
ligaseligase Recombinant plasmid DNA containing the insulin gene is Recombinant plasmid DNA containing the insulin gene is
transformed into transformed into E. coli E. coli host cells host cells Host cells multiply and produce one or more copies of the Host cells multiply and produce one or more copies of the
recombinant DNA. The insulin gene is now clonedrecombinant DNA. The insulin gene is now cloned E. coliE. coli colony carrying the recombinant insulin is identified. colony carrying the recombinant insulin is identified. Recombinant plasmid DNA is isolated & analyzed for DNA Recombinant plasmid DNA is isolated & analyzed for DNA
sequencingsequencing The insulin gene can be subsequently subcloned into an The insulin gene can be subsequently subcloned into an
expression vector - for production of insulin in expression vector - for production of insulin in E.coli.E.coli.
SUBCLONINGSUBCLONING Simplest cloning experiment which uses many Simplest cloning experiment which uses many
of the basic techniquesof the basic techniques
Involve the transfer of a fragment of cloned Involve the transfer of a fragment of cloned DNA from one vector into anotherDNA from one vector into another
Use to investigate a short region of a large Use to investigate a short region of a large cloned fragment or to transfer a cloned gene cloned fragment or to transfer a cloned gene into an expression vectorinto an expression vector
Steps in Sub-cloningSteps in Sub-cloning
Isolation of recombinant plasmid DNA
Digestion into discreet fragments with restriction enzymes
Separation of fragments on Agarose gel electrophoresis
Purification of desired target fragment
Ligation of fragment into a new plasmid vector
Transformation and selection for positive recombinant plasmid DNA