Mapping,Primer,andPlasmidDesign

ShifraBen‐Dor

So,youhaveaDNAsequence.Nowwhat?

SequenceEdi;ng

Itiscri;caltohaveanaccuratecopyofthesequenceyouplantoworkwith.

Whetheryouarecloningaknowngene,designingafusionprotein,orplanningPCR,youshouldhaveyouridealsequencein‐silicobeforeyoustartinthelab.

Thiscansavemuch;me,troubleandheartache.

SequenceEdi;ng

Therearevariousprogramsavailableforsimplesequenceedi;ng:

‐ GCG‐ EMBOSS‐ DNAstrider‐ VectorNTI‐ MacVector‐ LaserGene‐ ApE(aplasmideditor)‐ Word(MicrosoQOffice)‐ …..

SequenceEdi;ng

Theimportantthingstorememberwhenchoosingaprogram:

‐Doesitletmejumparoundthesequencebasedoncoordinates?Seqeunce?

‐ Howeasyisittocombinetwoexis;ngfiles?

‐Filestorage?

SequenceEdi;ng

Don’tforgettobeverycarefulwithsequence“joins”

‐ Ifyouarepu]ngsequenceintoamul;plecloningsite,erasewhatisin‐between!

‐ Ifyouarejoiningatanenzymesite,besureyouknowwhateachsequenceiscontribu;ng

Restric;onMaps

1 2 1 2 3

A`ributesofmappingprograms

•  Choiceofenzymes

– Singlecu`ers– xbasecu`ers(6base)

– minimum/maximumofsites

•  Linear/circular•  Simula;onofdoubledigests

A`ributesofmappingprograms

•  Silentmuta;ons

•  Output

– Annotatedsequence– Tableofsites(sortedbyenzymenameorposi;on)

– Tableoffragmentsizes(sortedbysizeorposi;on)

– Restric;onsite(theactualsequence)– Thosethatdo/don’tcut

Mappingprogramsontheweb

•  Webcu`er•  Seqcu`er•  TACG4•  NetPlasmid

h`p://bip.weizmann.ac.il

UnderToolbox;Seq.AnalysisbyTarget;DNA;MappingandPrimers

Transla;onPrograms

•  Translatesnucleo;desequencesintopep;desequences

•  Somedoallreadingframes,somehavechoiceofframes

•  Somedofulltransla;ons,othersonlyORFs

•  Usuallyhaveop;ontoreversesequence•  Cansome;mesaddmul;pleexonsfromoneparentsequence

Transla;onPrograms

•  Thedefini;onofanORF– StarttoStop– StoptoStop

•  MinimumsequencetobeconsideredanORF

•  Alternatestartcodons(mainlymicrobialL)

•  Mul;pleATGs

Transla;onProgramsontheweb

•  ORFfinder(NCBI)

•  Translate(Expasy)

•  Transeq(EBI‐EMBOSS)

REVERSE

•  Canreverse,complementorreverseandcomplementanucleo;desequence

•  Fileremainsnucleo;desequence,doesnottranslate

PrimerDesign

Whendoweneedprimers?

•  Sequencing(oneprimer)

•  PCR(two,oneforeachstrand)– Exact(cloning,addtags,addenzymesites,sitedirectedmutagenesis,…)

– Degenerate•  Real;mequan;ta;vePCR(qPCR)

•  RNAi– Oneprimer(synthe;c)

– Hairpin(plasmid)

PrimerDesign

•  Thingstokeepinmind:

– Primerlength

– AT/GCra;oshouldbearound50%– 3’endshouldbeG/C– mel;ng/annealingtemperature

– secondarystructure– primerdimers

PrimerLength

•  Primershavetobelongenoughtobespecific,butshortenoughtodetachefficientlyfromthetemplate

•  Ideallengthsarefrom18‐24bplong

•  Forsomeapplica;ons,weuselongerones(addingenzymesites,tags,changingtheendofasequence…)

•  Werarelyuseshorterones

GCra;o

•  IftherearetoomanyGsandCs,itwillbehardtoseparatetheprimerfromthetemplate(GandChave3hydrogenbonds)

•  WegenerallytrytokeeptheG/Cpercentageascloseto50%aspossible,witharangeof45%‐55%

•  Ifnothingisfound,expandtherange

3’clamp

•  Thereisarunningargumentintheliteratureastowhatbaseispreferableatthe3’end.SomemaintainthatanSclamp(GorC)makesforbe`erpriming,otherssayitmakesitworse.WegenerallyrecommendusinganSclamp(unlessyou’redoingqPCR,inwhichcaseanAisrecommended)

Mel;ngtemperature

•  Themel;ngtemperatureoftheprimersdirectlyeffectsthetemperatureoftheannealingstepofPCR.

•  Currentlyacceptednorms:primermel;ngtemperaturesinthe58oC‐60oCrange

•  Thedifferenceinmel;ngtemperaturesofprimersshouldbeasli`leaspossible,butcanbeupto5oC

Annealingtemperature

•  The“ruleofthumb”forannealingtemperature:itshouldbe5oClessthanthemel;ngtemperature

•  Op;mally,itshouldbedeterminedforeachsetofprimersonagradientcycler

•  Currentlyaccepted:aminimumof50oC

•  Itworksdownto37oC,butspecificitymaybecomeanissue

•  Ifyou’reworkingwithdegenerateprimers,youneedlowertemperatures,thoughyoucanusethemforafewini;alcycles

Secondarystructure

•  Internalcomplementarity:Thereshouldbenoselfmatchingstretchesof3basesormore,ortheprimerwillbindtoitselfinahairpin,andnotbeabletoprime

OtherPrimerIssues

•  PrimerDimers

Whenthe3’endoftheoneprimeriscomplementarytotheotherprimer,theprimerscanannealtoeachotherandcreateanewtemplate

•  PrimerComplementarity

Iftheprimersarecomplementaryanywhereelse,itcaninterferewithhybridiza;on

•  Primer/Template:

Avoidstretchesof3basesormoreinarowofthesamebase‐itcanleadtomispriming(G,C)orbreathing(A,T)

PrimerDesign

•  Ifyouarechangingthebeginningofacodingregion:

– ATGstartcodon– Kozaksequence(GCC)GCC(A/G)CCATGG

– signalsequence(secreted,membranebound)

Reverse (not complement) 3’ primer

GATAAGCTTGATATCGAATTGCCATGTTGAAGCCATCATTACCATT CTATTCGAACTATAGCTTAACGGTACAACTTCGGTAGTAATGGTAA

5’ 3’

GATAAGC CTATTCGAACTATAGCTTAACGGTACAACTTCGGTAGTAATGGTAA

5’ 3’

GATAAGCTTGATATCGAATTGCCATGTTGAAGCCATCATTACCATT ATGGTAA

5’ 3’

Primer = GATAAGC

Primer = AATGGTA 5’ 3’

PrimerDesign

•  Alwaysmakesurethatyouareinframe!

•  Doublechecktheorienta;onofthesequencebeforeyousubmititforsynthesis!

PrimerDesign

AlwayssequencePCRproducts!!!!

(preferablyaQersubcloning,unlessyouarejustcheckingforpresenceofproduct)

1 60 233 GATAAGCTTG ATATCGAATT GCCA.GTTGA AGCCATCATT ACCATTCACA TCCCTCTTGT 240 GATAAGCTTG ATATCGAATT GCCATGTTGA AGCCATCATT ACCATTCACA TCCCTCTTAT 239 GATAAGCTTG ATATCGAATT GCCATGTTGA AGCCATCATT ACCATTCACA TCCCTCTTAT gamma G AAGAGCAAGC GCCATGTTGA AGCCATCATT ACCATTCACA TCCCTCTTAT

61 120 233 TCCTGCAGCT GCCCCTGCTG GGAGTGGGGC TGAACACGAC AATTCTGACG CCCAATGGGA 240 TCCTGCAGCT GCCCCTGCTG GGAGTGGGGC TGAACACGAC AATTCTGACG CCCAATGGGA 239 TCCTGCAGCT GCCCCTGCTG GGAGTGGGGC TGAACACGAC AATTCTGACG CCCAATGGGA gamma TCCTGCAGCT GCCCCTGCTG GGAGTGGGGC TGAACACGAC AATTCTGACG CCCAATGGGA

121 180 233 ATGAAGACAC CACAGCTG.. .......... .......... ......ATTT CTTCCTGACC 240 ATGAAGACAC CACAGCTGGT GGGAAATCTG GGACTGGAGG GGGCTGATTT CTTCCTGACC 239 ATGAAGACAC CACAGCTG.. .......... .......... ......ATTT CTTCCTGACC gamma ATGAAGACAC CACAGCTG.. .......... .......... ......ATTT CTTCCTGACC

181 210 233 ACTATGCCCA CTGACTCCCT CAGTGTTTCC 240 ACTATGCCCA CTGACTCCCT CAGTGTTTCC 239 ACTATGCCCA CTGACTCCCT CAGTGTTTCC gamma ACTATGCCCA CTGACTCCCT CAGCGTTTCC

GenomicPrimers

•  Thereareafewaddi;onalfactorstokeepinmindwhenplanningprimerstogenomicDNA:

– Genomicrepeats

– Pseudogenes

– Genefamilies

PrimerPredic;onontheWeb

h`p://bip.weizmann.ac.il/toolbox/target/dna/dna_primers.html

PrimerPredic;onwithlocalprograms

Oligo7

LaserGene

Amplify

PlasmidDesign

Thingstorememberwhendesigningplasmids

•  Whatisyourtargetcellline?– Eukaryo;c/Prokaryo;c– Promoter,Originofreplica;on….

•  Howareyougoingtoreplicatethisplasmid?– Bacterialoriginofreplica;on– Copynumbercontrol

•  Whatisyourtargetcell“space”–  Intracellular,extracellular,vesicular– Leadersequence

Whattodowhenyou’restuck

•  3‐way,4‐way,5‐way…liga;ons•  “PlasmidShuffle”

•  Linkers•  AddviaPCR

•  ALWAYSREMEMBERTOCHECKYOURREADINGFRAME!!!!

Problem: cloning site is SalI, but only have Sal on one side of the gene

SalI

XbaI

PstI

Cloning Vector

SalI

SalI PstI

Ready to go!

PstI

SalI

Original Vector

blunt blunt

Cloning Tricks: Sal +Xba = Sal

GTCGAC CAGCTG

G TCGAC CAGCT G

GTCGA TCGAC CAGCT ACGTG

SalI Klenow

TCTAGA AGATCT

T CTAGA AGATC T

TCTAG CTAGA AGATC GATCT

Klenow XbaI

GTCGA TCGAC CAGCT ACGTG

TCTAG CTAGA AGATC GATCT

GTCGACTAGA CAGCTGATCT

ligate SalI !

Cloning Tricks: Sal +Xho

GTCGAC CAGCTG

G TCGAC CAGCT G

SalI

CTCGAG GAGCTC

C TCGAG GAGCT C

XhoI

GTCGAG CAGCTC

CTCGAC GAGCTG

Can also be done with BamHI and BglII