phosphate chains of the double helix. Since both chains are negatively charged, they have a natural tendency to repel each other. In fact, if DNA is placed in water free of any ions, the two strands of DNA are very likely to come apart. Positive ions such as Na+ and Mg++ (found in sodium chloride and magnesium chloride), however, can interact with the negatively charged DNA strands to mask the forces of repulsion. The higher the salt concentration, the more likely DNA will remain double-stranded.
In addition, at higher salt concentrations, two strands of DNA can be made to anneal to each other even if there is no perfect complementarily between them. Under conditions of very high salt concentrations, the double helix structure for some DNA segments can be quite stable, so much so that an even higher temperature is required to denature it. But the magnesium salts is not the only important part of the PCR (see Fig. 3.1).
Figure 3.1. Comics on PCR: Please… just send the Taq. No more little tubes of magnesium!
Popular blog from Promega Connections (http://promega.word- press.com) top ten tips for successful PCR:
– Modify reaction buffer composition to adjust pH and salt concentration.
– Titrate the amount of DNA polymerase.
– Add PCR enhancers such as BSA, betaine, DMSO, nonionic detergents, formamide or (NH4)2S04.
– Switch to hot-start PCR.
– Optimize cycle number and parameters like denaturation and extension times.
– Choose PCR primer sequences wisely.
– Determine optimal DNA template quantity.
– Clean up your DNA template to remove PCR inhibitors.
– Determine the optimal annealing temperature of your PCR primer pair.
And if you want to, you can even build a custom PCR protocol using their iOS and Android device apps at: http://worldwide.promega. com/resources/mobile-apps/.
Saying it shortly, the hot start PCR is a technique that reduces non-specific amplification and offers the convenience of PCR set up at room temperature, avoiding a non-specific amplification of DNA by inactivating the Taq polymerase at lower temperature (see Fig. 3.2).
Figurе 3.2. HоtStart-IT®mеthоdhttp://www.affymetrix.com/catalog/131145/USB/HotStart-IT+Taq+DNA+Polymerase
Polymerases used in Hot Start PCR are unreactive at ambient temperatures. Polymerase activity can be inhibited at these temperatures through different mechanisms, including antibody interaction, chemical modification and aptamer technology. At permissive reaction temperatures reached during PCR cycling, the polymerase dissociates from its inhibitor and commences polymerization. Use of hot start DNA polymerases is most often recommended for high-throughput applications, experiments requiring a high degree of specificity, or even routine PCR where the added security offered by a hot start enzyme is desired.
Questions for self-control
1. Name the effect of Mg2+ ions and pH on PCR.
2. Name the software for designing primers and probes.
3. Explain the concept of internal control sample.
4. Explain the basic stages of the PCR laboratory design.
5. Name factors affecting the achievement of «plateau effect» during the PCR.
6. Draw a simple model of PCR. Name basic cycles. Explain specificity.
7. What is the normalization of the data? How would you perform it?
8. Name the properties of oligonucleotides (primers and probes).
9. Name basic criteria used for the choice of primers.
10. Name settings that affect the interaction of the oligonucleotide and DNA.
11. Draw a chart for PCR laboratory.
12. Name basic equipment and materials for PCR.
Chapter 4
REAL-TIME PCR AND ITS THERMAL CYCLER SYSTEMS
Real-time PCR (or qPCR), first introduced by Higuchi and coworkers in 1992 is a laboratory technique of molecular biology, used to amplify and simultaneously detect or quantify a targeted DNA molecule. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further study of its products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. Since the first practical demonstration of the concept realtime PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. With the correct kits, reagents and experimental design it is an exceptionally powerful research tool quick and easy to generate high quality meaningful data in no time; flexible, as many alternative instruments and fluorescent probe systems have been developed and are currently available; its assays can be completed rapidly since no manipulations are required for post-amplification.
Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels. The probe is labeled at the 5’-end with the fluorescence donor (e.g. Fluorescein), a few bases downstream or on the 3’-end with a quencher (e.g. TAMRA). With no complementary sequence available the fluorescence of the donor is quenched (see Fig. 4.1).
Figure 4.1. Basic principles of real-time PCR
Real-time PCR thermocycler follows a protocol that alternates through temperatures that are optimal for denaturing, annealing, and extension (see Fig. 4.2).
Figure 4.2. Three steps of one real-time PCR cycle
Figure 4.3. Steps and variables of a successful mRNA quantification using real-time RT-PCR; From: http://www.gene-quantification.de/optimization.html
During PCR the labeled oligonucleotide hybridizes to the target sequence and the 5’-dye is removed by the 5’ to 3’-exonuclease activity of Taq.
No longer quenched fluorescence of the donor can be measured. Its intensity is proportional to the amount of product formed during the exponential phase (threshold value; see Fig. 4.4).
Figure 4.4. Exponential increase of fluorescent signal in q-PCR
An increase in the product targeted by the reporter probe at each PCR cycle therefore
