Successful PCR Guide - Separations

1 SuccessfulPCR GuideRoutine PCRReal Time PCR (qPCR) High Fidelity PCRHigh Performance PCRHot Start PCRRT-PCRPCR Cloning3rd EditionHow to Select The Best PCR Enzyme for Your Application takara Taq *Hot Start VersionTaKaRa Taq takara Ex Taq takara Ex Taq takara e2 TAK Premix Taq PrimeSTAR HS DNA Polymerase PrimeSTAR with GC Buffers PrimeSTAR Premix PrimeSTAR Premix SpeedSTAR HS DNA PolymerasePerfectShot Ex Taq One Shot LA PCR Mix LA PCR Kit Version PrimeSTAR with GC BuffersLA PCR Kit Version takara Ex Taq Hot Start Version takara LA Taq Hot Start VersionSYBR Premix Ex Taq (Perfect Real Time) Premix Ex Taq (Perfect Real Time) takara LA Taq with GC Buffers takara LA Taq takara LA Taq Premix Ex Taq Routine PCR Convenient PremixesHigh Performance PCRHot Start PCR orMultiplex PCR*High GC Content orSecondary StructuresHighFidelity PCRLong PCRHigh Speed PCRReal Time PCRFor Longer PCRHigh Sensitivity PCRFor Direct ElectrophoresisRoutine PCRHigh Fidelity PCRP remix Ex Taq HSPremix Taq HSHot Start PCR *Hot start enzymes contain an anti-Taq antibody to minimize non-specific amplification Successful PCR GuideTakara Bio USA1 Table of ContentsTable of ContentsChapter 1:Points to Consider.

2 3 Chapter 2:Routine PCR ..7 Chapter 3:Real Time PCR (qPCR) ..11 SYBR Detection Method Probe Detection Method Various Other MethodsChapter 4:High Fidelity 5:High Performance High Speed PCR High Yield PCR Long PCRC hapter 6:Hot Start Multiplex PCRC hapter 7:Reverse Transcriptase PCR ..31 Chapter 8:PCR Cloning ..33 PCR Related Products ..34 Appendix I:Frequently Asked Questions ..35 Appendix II:PCR Nomenclature ..39 Appendix III:Troubleshooting ..40 Appendix IV:PCR Protocols ..47 Appendix V:Technical Fact Sheet ..50 Appendix VII: Guide to takara PCR VIII:Technical Articles ..54 Appendix IX:Licensing ..56 Ordering Back CoverTakara Bio USA is a wholly owned subsidiary of takara Bio Inc. andserves as the North and South American base for takara Bio sales,marketing and support activites in those a complete description of takara Bio USA s product offering,please visit our website at BioUSATaKaRa BioEuropeTaKaRa Biotechnology(Dalian) takara Korea BiomedicalTaKaRa Bio Bio Inc.

3 , Otsu Shiga, JapanABOUT takara BIO USAT akara Bio Inc. is a world class supplier of life science researchproducts headquartered in Otsu, Shiga, Japan. takara Bio was thefirst domestic manufacturer to introduce restriction enzymes tothe Japanese market in 1979, and has consistently developednovel, cutting edge life science technologies and products. Thistalent for innovation, combined with takara Bio s unwaveringcommitment to quality, has resulted in an outstanding line ofunique, dependable products for life science research. takara Bio holds worldwide patents on Long and Accurate (LA)PCR, and has built a portfolio of PCR licensed high-performancePCR reagents and kits, including Ex Taq , LA Taq , PrimeSTAR ,SpeedSTAR , e2 TAK , SYBR Premix Ex Taq (Perfect Real Time)and PremixEx Taq (Perfect Real Time) .Table of ContentsSuccessful PCR GuideTakara Bio USA2 Profile of PCR Reactions1 2 3 4 5 Time (min)Temperature ( C)947255221 CycleStep 1 Initial DenaturationStepStep 2 Step 3 Begin Step 1 After 30 cycles hold at 4 CRepeat Step 1 3for 25-30 cycles105-fold amplification of target DNA fragmentPrimerannealingDenaturationStepS ynthesis of complementary chainExponential PhaseLinear PhasePlateau PhaseLag PhaseCtRn (Reporter Fluorescence)Cycle NumberThreshold RnBaselineNo TemplateProfile of Routine PCR ReactionStep 1:Denaturation.

4 Double-stranded DNA fragment isdenatured in a reaction mixture containing primers,dNTP and polymerase. Step 2:Annealing. Primers are annealed to denatured sin-gle-stranded DNA. Step 3:Extension. Annealed primers are extended withDNA polymerase. Cycling parameters must be empirically determinedas optimum conditions for PCR vary depending onthe DNA template and primers of qPCR Reaction RN= change in reporter fluorescenceCt= Cycle ThresholdBaseline= a linear function subtractedfrom the data to eliminate #100% efficiency90% efficiency80% efficiency011112222443387641613105322519 66447347128996182561701109512323198101,0 24613357112,0481,166643124,0962,2131,157 138,1924,2052,0821416,3847,9903,7481532, 76815,1816,7471665,53628,84412,14417131, 07254,80421,85918262,144104,12739,346195 24,288197,84270,824201,048,576376,900127 ,482212,097,152714,209229,468224,194,304 1,355,998413,043238,388,6082,578,296743, 4772416,777,2164,898,7631,338,2592533,55 4,4329,307,6502,408,8662667,108,86417,68 4,5344,335,96927134,217,72833,600,6157,8 04,72628268,435,456638,941,16814,048,505 29536,870,912121,298,22025,287,311301,07 3.

5 741,824230,466,61845,517,160 PCR Reaction EfficiencySuccessful PCR GuideTakara Bio USA3 Although PCR has become routine in many laboratories,careful experimental design is still critical for a successfuloutcome. Preliminary experiments to optimize reaction con-ditions are essential (including determination of reactionbuffer pH, cycling parameters, concentrations of key compo-nents such as Mg2+, dNTP, primers and DNA polymerase). PCRsuccess also depends upon individual template-primer combi-nations for Endpoint PCR and template, primer, probe anddetection method for following chapter discusses the most common issueswhich should be addressed when designing a PCR experiment. ENDPOINT PCR USING REGULAR TaqPCR (Polymerase Chain Reaction) is a simple and powerful toolfor amplification of DNA in vitro. The PCR method is performed ina thermocycler which repeats three incubation steps at differenttemperatures.

6 The three steps include: Step: The double-stranded target DNA isheat denatured. 94 C for 30 Step:The two primers complementary to thetarget segment are annealed to the template DNA at lowtemperature. 55 C for 30 Step:The annealed primers are then extendedat an intermediate temperature by a DNA polymerase. Thetarget copy number doubles upon each cycle, resulting inexponential amplification and potentially billions of copiesof the original DNA fragment (see PCR reaction efficiencytable). 72 C for 1 StartedIt is ideal to have a room dedicated for PCR use only. However, thisis not possible in many research labs. Use of barrier filter tips anddedicated pipettes are imperative. Contamination from dirtypipettes is one of the most common causes of experimental PCR bench area used should be decontaminated frequentlyusing a product which removes DNA, such as DNA-OFF (TAK9036), as well as cleaned with ethanol (70%) before and after theassembly of the reaction.

7 Care should be taken to avoid carelesscontamination from the outside DNA Successful PCR of a target DNA depends on the purity and/or quali-ty of the DNA template and the quantity of template DNA used. Many common DNA purification protocols utilize reagents (suchas organic solvents, detergents, salts, etc.) which are inhibitors ofDNA polymerases. These reagents must be removed (generallyby ethanol precipitation) before inclusion of the template in aPCR removal of these reagents, the DNA should be ready for usein PCR. However, special care must be taken when working withlonger targets (>10 kb) during the DNA preparation to avoidshearing of the intact DNA purification, it is important to use the appropriateamount of template. The use of either excessive plasmid DNA orinsufficient genomic DNA template are two of the most commonPCR mistakes. A minimum of 104copies of target sequence mustbe used to obtain a signal in 25 30 cycles for a final concentra-tion of DNA at 10 ng/ PreparationThe melting temperature (Tm is defined as the temperature atwhich half of the primer binding sites are occupied) of a DNAhybrid depends somewhat upon its length, and the primersequence should be designed with the recommended primerlength in mind ( , primers that are too long and, therefore, toostable, are problematic).

8 Recommended PCR primer lengths range from 18 25 bases forfragments smaller than 5 kb, and 20 30 bases for fragmentsgreater than 5 kb. These parameters allow the Tmdifferencesbetween the template and the unstable primer to be minimized,allowing for more efficient following list provides additional guidelines for primer sequence:1. Primers should end (3') in a G or C, or CG or GC. This designincreases the efficiency of priming by forming a tight G/C Design primers with balanced melting temperatures (within2 3 C of each other). Temperatures between 65 70 C are pre-ferred, as higher annealing temperatures increase Avoid complementarity in the 3'-ends of primers, as primerdimers will be preferentially synthesized because of shortlengths in a Avoid primer self-complementarity (ability to form secondarystructures, such as hairpins) which effectively reduce Avoid runs of three or more C s or G s at the 3' ends of primers,which may promote mispriming at G or C-rich sequences(because of the stability of annealing).

9 Primer Annealing TemperatureMany formulas are available to determine the theoretical Tmofnucleic acids. The following commonly-used formula can be usedto estimate the melting temperature for any oligonucleotide:Tm= 2 C x (number of A+T) + 4 C x (number of G+C) A more technical formula is (Tm = + (log10[Na+]) + (%G+C) 675/n)where [Na+] is the molar concentration of monovalent cations ( [Na+] = [K+] ) and n = number of bases in the oligonucleotide.(1)For example, to calculate the melting temperature of a 22meroligonucleotide with 60% G+C in 50 mM KCl:Tm= + (log10[ ]) + (60) 675/22= + ( ) + C Polymerase AmountThe optimal amount of polymerase for use in a given reaction isdependent upon the template size and the type of template. Forgenomic or plasmid templates <5 kb in length, use the followingenzyme concentrations:UnitsRxn U50 LEx Taq U50 LLA Taq U50 LPrimeSTAR U50 Le2 TAK Excess enzyme may facilitate non-specific amplification which canresult in a diffuse smear of bands.

10 In contrast, insufficient enzymelowers the efficiency of amplification which may result in low orno product to ConsiderPoints to ConsiderCycle NumbersFor most PCR reactions, the optimum cycle number is 25 30cycles. The exact number should be determined by consideringthe quantity or complexity of template DNA and the length ofthe target DNA fragment. Insufficient cycles may result in lowproduct yield, whereas excess cycles may encourage amplificationof secondary products or contaminants, resulting in spuriousbands or a diffuse smear upon ConditionsWhen takara Ex Taq or Ta k a r a L A Ta q are used, denaturationfor 10 seconds at 98 C is generally recommended. There may beapplications that require a lower temperature for a longer using thin-walled tubes, a shorter denaturation time ( seconds at 94 C) is recommended. takara se2 TAK DNAP olymerase and PrimeSTAR HS DNA Polymerase both have 98 Cdenaturation times for 10 sec.