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Primer design2


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  • 1. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONEPrimer design ©2003/04 Alessandro Bogliolo
  • 2. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Outline1. Polymerase Chain Reaction2. Primer design ©2003/04 Alessandro Bogliolo
  • 3. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Polymerase Chain Reaction (PCR)• It’s a means of selectively amplifying a particular segment of DNA, possibly representing a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene.• The reaction involves: – DNA nucleotides, the building blocks for the new DNA – Template DNA, the DNA sequence that you want to amplify – Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA – DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA ©2003/04 Alessandro Bogliolo
  • 4. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE PCR Primers Primers5’ 3’3’ 5’ Target DNA segment 5’ 3’3’ 5’5’ 3’ 3’ 5’ ©2003/04 Alessandro Bogliolo
  • 5. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE PCR cycling• 30–40 cycles each comprising: – Denaturation (95°C), 60 sec. The double strand melts to single stranded DNA and all enzymatic reactions stop – Annealing (55–60°C), 45 sec. Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached – Extension (72°C), minutes (time depends on product size) The bases (complementary to the template) are coupled to the primer on the 3 side (the polymerase adds dNTPs from 5 to 3, reading the template from 3 to 5 side, complementary to the template) ©2003/04 Alessandro Bogliolo
  • 6. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE PCR efficiency• Every cycle results in a doubling of the number of strands DNA present• After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers• The result is a dramatic amplification of the DNA segment between the primers. The amount of amplification is 2n, where n is the number of cycles performed (1 million after 20 cycles) ©2003/04 Alessandro Bogliolo
  • 7. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE PCR efficiency• Increasing the cycle number above ~40 has little positive effect 3rd cycle 2nd cycle 1st cycle• The plateau occurs when: template – The reagents are depleted target – The products re-anneal – The polymerase is damaged• Unwanted products accumulate ©2003/04 Alessandro Bogliolo
  • 8. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE PCR Fidelity• Taq DNA polymerase lacks the 3´→5´ proof- reading activity commonly present in other polymerases.• Taq mis-incorporates 1 base in 104.• Partial products containing errors are amplificated by subsequent PCR cycles• A 400 bp target will contain an error in 33% of molecules after 20 cycles.• Error distribution will be random. ©2003/04 Alessandro Bogliolo
  • 9. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE PCR Optimization• Target length• Concentrations• PCR cycle parameters – times – temperatures• Primer design ©2003/04 Alessandro Bogliolo
  • 10. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Primer DesignCharacteristics of primers: Thoughts on primer design: Specificity Uniqueness Specific for the intended Length target sequence (avoid Base Composition nonspecific hybridization) Internal Stability Stability Melting Temperature Form stable duplex with Annealing Temperature template under PCR conditions Internal Structure Compatibility Primers used as a pair shall Primer Pair Matching work under the same PCR condition ©2003/04 Alessandro Bogliolo
  • 11. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE UniquenessThere shall be one and only one target site in the templateDNA where the primer binds, which means the primersequence shall be unique in the template DNA.There shall be no annealing site in possible contaminantsources, such as human, rat, mouse, etc. (BLAST searchagainst corresponding genome) ©2003/04 Alessandro Bogliolo
  • 12. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE LengthPrimer length has effects on uniqueness and melting/annealingtemperature:• the longer the primer, the more chance that it’s unique• the longer the primer, the higher melting/annealing temp.Generally speaking, the length of primer has to be at least 15bases to ensure uniqueness. Usually, we pick primers of 17-28bases long. This range varies based on if you can find uniqueprimers with appropriate annealing temperature within thisrange. ©2003/04 Alessandro Bogliolo
  • 13. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Base CompositionBase composition affects hybridization specificity,melting/annealing temperature and internal stability.• Random base composition is preferred. We shall avoid long(A+T) and (G+C) rich region if possible.• Usually, average (G+C) content around 50-60% will give usthe right melting/annealing temperature for ordinary PCRreactions, and will give appropriate hybridization stability.However, melting/annealing temperature and hybridizationstability are affected by other factors, which we’ll discuss later.Therefore, (G+C) content is allowed to change. ©2003/04 Alessandro Bogliolo
  • 14. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Internal StabilityStability Profile:Internal stability iscalculated with entropyvalues of neighbornucleotides. Usually, Wedraw a graph of ΔG forall nucleotides of theprimers. This is known asthe stability profile.To minimize false priming, it’s critical that the stability at 5’end be high and the stability at 3’ end be relatively low. ©2003/04 Alessandro Bogliolo
  • 15. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE 3’ Stability & 5’ StabilityPrimer elongation starts at 3’ end. Therefore, as long as 3’end hybridizes to the template stably, the elongation begins.5’ end sequence plays less important role. This feature bringsout a problem – if 3’end of the primer has 3 or more than 3 C/G, it can almost bind stably to any site where there are 3complement G/C bases.Ideal situation: stable 5’end + less stable 3’end, which eliminates false priming due to annealing of 3-half of primer only. We prefer the 5’ end has 1 or two G/C bases (GC clamp) and the 3’ end has no more than 1 G/C base. ©2003/04 Alessandro Bogliolo
  • 16. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Melting TemperatureMelting Temperature, Tm – the temperature at which half the DNA strands are single stranded andhalf are double-stranded.. Tm is characteristics of the DNA composition; Higher G+C content DNAhas a higher Tm due to more H bonds.CalculationMethod 1 (Base Composition): Tm = ( A + T ) × 2°C + ( C + G ) × 4°C (<20bp)Method 2 (Salt Adjusted): Used by GCG and Primer3 Tm = 81.5 + 16.6 × [log10[Na+]] + 0.41 × [GC%] – 0.65 × [formamide%] – 675/length – mismatch%Method 3 (Nearest Neighbor): Used by OLIGO ∆H / (10.8 + ∆S + R × ln (c / 4)) – 273.15 + 16.6(log10[K+])where ∆H is the sum of enthalpy of the nearest neighbors, ∆S is the sum of entropy of the nearestneighbors, c is the molar concentration of primer, and R is the gas constant (1.987). As you can tellfrom the equation – higher G+C, higher Tm; Higher [probe], higher Tm; higher [K+], higher TmDifferences are sometimes significant, like 8 degrees, and sometimes trivial, like 0.1 degree. Trydifferent software to calculate Tm. Pick the common value. ©2003/04 Alessandro Bogliolo
  • 17. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Annealing TemperatureAnnealing Temperature, Tanneal – the temperature at whichprimers anneal to the template DNA. It can be calculatedfrom Tm . Tanneal = Tm_primer – 4°C or 0.3 × Tm_primer + 0.7 × Tm_product – 14.9 Where Tm_primer is the melting temperature for primer and Tm_product is the melting temperature for product.To ensure that primers anneal to the template before the twostrands of template anneal to each other, it’ required that theTm_product – Tanneal ≥ 30 °C ©2003/04 Alessandro Bogliolo
  • 18. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONEStringency in Primer AnnealingStringency determines the specificity of the amplified DNAproduct. Tanneal is the most significant factor affecting thestringency in primer annealing.• Tanneal : too low  less stringent  primer matches elsewhere too high  more stringent  primer may fail to matchOther factors:• GC%: GC pairs are more stringent than AT paris• Salt & Buffer ©2003/04 Alessandro Bogliolo
  • 19. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Internal StructureIf primers can anneal to themselves, or anneal to each otherrather than anneal to the template, the PCR efficiency will bedecreased dramatically. They shall be avoided.These 2° structures are harmless when the annealing temperature does notallow them to take form. For example, some dimers or hairpins form at 30°C while during PCR cycle, the lowest temperature only drops to 60 °C. ©2003/04 Alessandro Bogliolo
  • 20. i ts ISTITUTO DI CIENZE E ECNOLOGIE DELL’ NFORMAZIONE Primer Pair MatchingPrimers work in pairs – forward primer and reverse primer.Since they are used in the same PCR reaction, it shall beensured that the PCR condition is suitable for both of them.One critical feature is their annealing temperatures, whichshall be compatible with each other. The maximumdifference allowed is 3 °C. The closer their Tanneal, the better. ©2003/04 Alessandro Bogliolo
  • 21. i ts ISTITUTO DI CIENZE E ECNOLOGIE Summary DELL’ NFORMAZIONE• Uniqueness: ensure correct priming site;• Length: 17-28 bases.This range varies;• Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible;• Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming.• Melting Tms between 55-80 °C are preferred;• Assure that primers at a set have annealing Tm within 2 – 3 °C of each other.• Minimize internal secondary structure: hairpins and dimers shall be avoided. ©2003/04 Alessandro Bogliolo