PCR – diagnostics. Aizhan Zhussupova

      Questions for self-control

      1. What components are essential for PCR?

      2. The first stage of the PCR process is carried out at 90-95 °C for 30 seconds. What happens to the DNA at this temperature? What factors assist the DNA denaturation?

      3. What is the optimum temperature for the enzyme DNA polymerase used in the PCR process? The extension step usually occurs at a lower temperature than the annealing step? Do all primers have the same melting temperature?

      4. What would be the effect on the PCR reaction if any of the following circumstances arose: a) there are no primers in the reaction, b) there are no dNTPs in the reaction, c) there is no Taq polymerase in the reaction?

      5. What would the generally expected effect on the PCR reaction be of adjustments that increase the temperature of the annealing phase and the length of the elongation phase?

      6. In principle, what outcome would be least expected in a failure to separate pre-PCR and post-PCR activities?

      7. What outcome would you least expect if the amount of template in a multiplex PCR fell significantly below the optimal amount?

      8. What would the expected effect be on a PCR reaction if the primers used were slightly shorter and more variable than the intended oligonucleotide sequences?

      9. What are the positive and negative controls are usually used for?

      10. Why would a scientist perform processing of specimens, reaction mixture preparation, amplification and detection steps in separate rooms? What is a cross-contamination?

      Chapter 3

      COMPONENTS OF THE PCR

      Primers

      Typical primers are 18-28 nucleotides with 50-60 % G+C content. Typical concentration in a PCR reaction is 0.1-0.5 mM; higher concentrations may give nonspecific products or primer dimers. Additionally, 3 or more C’s or G’s at 3’ ends of primers may promote mispriming at G+C rich sequences.

      Furthermore, palindromic sequences within primers should be avoided; as should any internal inverted repeats that would cause primer to self anneal. Primers with a similar melting temperature that are completely complementary are recommended. The optimal melting temperature range for primers is 55 °C to 80 °C. An approximate melting value for your primer can be calculated before it is synthesized using the following equation: T_m (^oC) = 2(N_A+N_T) + 4(N_G+N_C).

      DNA polymerase

      There are 2 common polymerases used for PCR, Taq and Pfu. The typical concentration is 2.5-5.0 units of enzyme per 100 pL reaction for targets below 10 kb. Larger targets may require up to 10 units of enzyme per 100 pL of reaction volume. Note: 1 mL 1000 jL, 1 jg – 1000 ng. The most critical parameter affecting yield of PCR product is the extension time. Taq polymerase can amplify DNA faster than Pfu polymerase but it is not nearly as efficient. Normal extension time for Pfu polymerase is 2 min/kb of template whereas Taq polymerase can be as low as 0.5-1 min/kb.

      However, the mutated PCR product percentage per 1 kb fragment is 2.6 % for Pfu polymerase and 16 % for Taq polymerase. 72 °C is the optimal temperature for extension, as Pfu is most active and efficient under this condition. On December 22, 1989 the journal Science awarded Taq polymerase (and PCR) its first «Molecule of the Year». The Saiki R.K., Gelfand D.H., Stoffel S. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase // Science 1988; 239 (4839): 487-491. Paper became the most cited publication in biology for several years.

      Reaction buffer

      Generally a 10-50 mM Tris/HCl buffer with a pH above 8.0 (typically 8.3-8.8). KCl can be added to facilitate primer annealing, but shouldn’t be higher than 50 mM as this may inhibit polymerase. The deoxynucleotide triphosphates should have a total concentration of 0.4-1.0 mM, meaning each dNTP is present in an equal amount (100-250 pM). Magnesium is also required and should have a higher concentration than the total dNTP concentration (0.5-2.5 mM Mg²+). Magnesium affects primer annealing and template denaturation, as well as enzyme activity and fidelity. An excess of magnesium gives nonspecific amplification products, while low magnesium yields lesser amount of desired product.

      Adjuncts and cosolvents

      Effects of additives in the polymerase chain reaction are reviewed by Ernest J. Mueller in the third edition of PCR Technology: Current Innovations by Tania Nolan, Stephen A. Bustin by Taylor and Francis Group CRC Press, 2013. Assuming that the reaction is suitably formulated, PCR amplifications should be achieved if a suitable template (substrate) is available. Much of this literature is focused on methods for effective amplification of less-than-perfect substrates, whether the cause lies with high-melting DNA or template that carries inhibiting contaminants.

      Examples of poorly amplified templates have been reported throughout the 20 years of literature, and a variety of additives have been applied with the claim of melting DNA template and facilitating polymerase amplification.

      As such dimethyl sulfoxide (DMSO; 1-10 %) can improve dena- turation of GC-rich DNA and help overcome difficulties of polymerase extension through secondary structure. Lastly, ammonium sulfate increases the ionic strength of reaction mixture, which alters denaturing and annealing temperatures of DNA and enzyme activity. Enhancement also appears to depend on template structure, and thus the literature evidence suggests that adjunct-mediated PCR enhancement is template- and adjunct-specific.

      In particular terms, this implies that each PCR enhancer must be optimized for each amplified DNA locus and the adjunct of choice. Another application of PCR additives is to combat sample-introduced inhibitors. The phenomenon was initially counteracted by diluting samples past the inhibition threshold of the polymerase.

      This tactic is not always possible, especially for clinical, environmental, and plant-derived nucleic acid samples. Bovine serum albumin (BSA) in fact can bind certain PCR inhibitors. The concentration range used can vary from 10-100 pg/ml. Formamide (1.2510 %) facilitates primer-template annealing reactions and lowers the denaturing temperatures of melt resistant DNA.

      Strategies to relieve inhibition depend on the inhibitor, and the lengthy listings of PCR inhibitors include many of the compounds used to enhance amplification efficiency as well as reagents required in the purification of nucleic acids. In cases where inhibitors cannot be purified or dilutes away, it appears that the solution, if possible at all, is empirical and as specific and difficult as the amplification of high- melting templates.

      Reaction conditions and experimental protocol

      Temperature and length of time required for annealing depend upon base composition, length and concentration of amplification primers. Generally, the annealing temperature is 5 °C below the true melting temperature of the primers. Primers will anneal in a few seconds, for efficiency, higher annealing temperatures can be used, which enhance the discrimination against incorrectly annealed primers. The annealing conditions need to be more stringent in the first 3 cycles in order to increase specificity. If the temperature is lower than optimum additional DNA fragments are commonly observed. Denaturing conditions are best at 94-95 °C for 30-60 seconds. Lower temperatures may result in incomplete denaturation of target template and PCR products.

      Higher temperatures and a longer amount of time can lead to loss of enzyme activity. Diluting sample after first few rounds of PCR can be used to enhance PCR efficiency. This dilution may dilute potential inhibitors and the next round can use same primers or nested primers. In addition, the lowest number of cycles possible to achieve sufficient product should be used to assure a low number of errors.

      The order of addition of reaction mixture components is also of importance. Pfu polymerase has exonuclease activity and must be added last (i.e. after dNTP’s), otherwise