1. ©2003/04 Alessandro Bogliolo Primer design

2. ©2003/04 Alessandro Bogliolo Outline 1.Polymerase Chain Reaction 2.Primer design

3. ©2003/04 Alessandro Bogliolo 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

4. ©2003/04 Alessandro Bogliolo PCR Primers 3’5’ 3’ 5’3’ 5’ 3’ 5’ Primers Target DNA segment

5. ©2003/04 Alessandro Bogliolo 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 dNTP's from 5' to 3', reading the template from 3' to 5' side, complementary to the template) PCR cycling

6. ©2003/04 Alessandro Bogliolo 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 2 n, where n is the number of cycles performed (1 million after 20 cycles)

7. ©2003/04 Alessandro Bogliolo template 1 st cycle 2nd cycle 3rd cycle target PCR efficiency Increasing the cycle number above ~40 has little positive effect The plateau occurs when: –The reagents are depleted –The products re-anneal –The polymerase is damaged Unwanted products accumulate

8. ©2003/04 Alessandro Bogliolo PCR Fidelity Taq DNA polymerase lacks the 3´  5´ proof- reading activity commonly present in other polymerases. Taq mis-incorporates 1 base in 10 4. 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.

9. ©2003/04 Alessandro Bogliolo PCR Optimization Target length Concentrations PCR cycle parameters –times –temperatures Primer design

10. ©2003/04 Alessandro Bogliolo Primer Design Specificity Specific for the intended target sequence (avoid nonspecific hybridization) Stability Form stable duplex with template under PCR conditions Compatibility Primers used as a pair shall work under the same PCR condition Uniqueness Length Annealing Temperature Primer Pair Matching Internal Structure Base Composition Internal Stability Characteristics of primers:Thoughts on primer design: Melting Temperature

11. ©2003/04 Alessandro Bogliolo Uniqueness There shall be one and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA. There shall be no annealing site in possible contaminant sources, such as human, rat, mouse, etc. (BLAST search against corresponding genome)

12. ©2003/04 Alessandro Bogliolo Length Primer length has effects on uniqueness and melting/annealing temperature: 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 15 bases to ensure uniqueness. Usually, we pick primers of 17-28 bases long. This range varies based on if you can find unique primers with appropriate annealing temperature within this range.

13. ©2003/04 Alessandro Bogliolo Base Composition Base 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 us the right melting/annealing temperature for ordinary PCR reactions, and will give appropriate hybridization stability. However, melting/annealing temperature and hybridization stability are affected by other factors, which we’ll discuss later. Therefore, (G+C) content is allowed to change.

14. ©2003/04 Alessandro Bogliolo Internal Stability Stability Profile: Internal stability is calculated with entropy values of neighbor nucleotides. Usually, We draw a graph of ΔG for all nucleotides of the primers. This is known as the 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.

15. ©2003/04 Alessandro Bogliolo 3’ Stability & 5’ Stability Primer 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 brings out 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 3 complement 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.

16. ©2003/04 Alessandro Bogliolo Melting Temperature Melting Temperature, Tm – the temperature at which half the DNA strands are single stranded and half are double-stranded.. Tm is characteristics of the DNA composition; Higher G+C content DNA has a higher Tm due to more H bonds. Calculation Method 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  [log 10 [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(log 10 [K+]) where  H is the sum of enthalpy of the nearest neighbors,  S is the sum of entropy of the nearest neighbors, c is the molar concentration of primer, and R is the gas constant (1.987). As you can tell from the equation – higher G+C, higher Tm; Higher [probe], higher Tm; higher [K+], higher Tm Differences are sometimes significant, like 8 degrees, and sometimes trivial, like 0.1 degree. Try different software to calculate T m. Pick the common value.

17. ©2003/04 Alessandro Bogliolo Annealing Temperature T anneal = T m_primer – 4  C or 0.3  T m_primer + 0.7  T m_product – 14.9 Where T m_primer is the melting temperature for primer and T m_product is the melting temperature for product. Annealing Temperature, T anneal – the temperature at which primers anneal to the template DNA. It can be calculated from T m. To ensure that primers anneal to the template before the two strands of template anneal to each other, it’ required that the T m_product – T anneal ≥ 30  C

18. ©2003/04 Alessandro Bogliolo Stringency in Primer Annealing Stringency determines the specificity of the amplified DNA product. T anneal is the most significant factor affecting the stringency in primer annealing. T anneal : too low  less stringent  primer matches elsewhere too high  more stringent  primer may fail to match Other factors: GC%: GC pairs are more stringent than AT paris Salt & Buffer

19. ©2003/04 Alessandro Bogliolo Internal Structure These 2  structures are harmless when the annealing temperature does not allow 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. If primers can anneal to themselves, or anneal to each other rather than anneal to the template, the PCR efficiency will be decreased dramatically. They shall be avoided.

20. ©2003/04 Alessandro Bogliolo Primer Pair Matching Primers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them. One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3  C. The closer their T anneal, the better.

21. ©2003/04 Alessandro Bogliolo Summary 1.Uniqueness: ensure correct priming site; 2.Length: 17-28 bases.This range varies; 3.Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible; 4.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. 5.Melting Tms between 55-80  C are preferred; 6.Assure that primers at a set have annealing Tm within 2 – 3  C of each other. 7.Minimize internal secondary structure: hairpins and dimers shall be avoided.