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:

+

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