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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 it may degrade primers. If primers and nucleotides are in the mixture at appropriate concentrations then primer degradation is minimal.

Several types of samples are known to inhibit the PCR reaction, leading to false-negative results. Including an internal control in the assay is important for the quality control of the nucleic acid extraction, to prove the absence of PCR inhibitors. These internal controls can be either a house-keeping gene, an endogenous gene, a constant basal cell-expressed gene, such a the gluceraldehyde-3-phosphatase or the P-actin, or an exogenous nucleic acid that is not present naturally in the preparation, but added at the extraction step.

A number of specialised methods for particular types of samples and tissues exist, most of which are now commercially available either as manual or automated systems for robotic workstations. The development and accessibility of the robotic extraction platforms not only minimises the risk of contamination, but also enables processing of large numbers of samples under constant reaction conditions and minimal operator manipulation.

Consequently, these platforms have contributed to the establishment of high-throughput, robust diagnostic assays, shortening the processing time required per sample from hours to minutes. These are destined to improve the reliability of nucleic acid extraction from different samples, but it still remains a challenging area.

As an alternative to nucleic acid extraction, biotechnologists are increasingly focusing on polymerases that are resistant to PCR inhibitors and several are now available on the market for direct amplification of nucleic acids from pathological specimens without any additional extraction step. Assays increasingly use an internal control to demonstrate that PCR inhibitors are not present.

General PCR protocol

Prepare following mixture in appropriately sized Eppendorf tube (0.2-0.5 mL):

81 pL of ddH₂0

10 pL of 10x polymerase buffer (for native of cloned Pfu polymerase)

pL of primer #1 (100 ng/pL)

pL of primer #2 (100 ng/pL)

1 pL of template DNA (<100 ng/pL)

1 pL of 10 mM deoxynucleotide triphosphate mixture

(2.5 mM each dNTP) 2 pL of DNA polymerase

(native or cloned Pfu polymerase)

100 pL total reaction volume

________

Tap the side of the Eppendorf tube to mix the mixture properly, overlay it with drop of mineral oil and perform PCR reaction using following cycling parameters (see Table 3.1):

Table 3.1

Common stages of PCR with their specifications

Some hints for PCR troubleshooting are shown in Table 3.2:

Table 3.2

PCR observations with possible causes and solutions

For example, let’s say that you have a stock solution of 5 mM (millimolar) magnesium chloride (MgCl₂) and need to add some volume of that stock solution to a 200 μL reaction such that that reaction has a final concentration of 100 μM MgCl₂. How much of the stock solution should you add? The two most popular approaches for tackling this problem are shown below.

Approach I: (C,)(V) = (C_f)(V_f)

The formula above states that the concentration of the reagent in the initial stock solution (C) multiplied by a certain volume (V) should equal the reagent’s final, diluted concentration (C_f) multiplied by the final volume (V_f) of the reaction containing the diluted reagent.

To solve a problem using this equation, you must first identify each term. You must also make sure that all terms are in the same units. If they are not, they must be converted prior to solving the equation.

Here, we define each term as follows: C = 5 mM MgCl₂; V = unknown; C_f = = 100 pM MgCl₂; V_f = 200 pL.

Notice that C is in units of mM and C_f is in units of pM. C can be converted to units of pM as follows: C = 5 mM MgCl₂ x 1000 pM/1 pM = 5000 pM MgCl₂

The problem can now be solved: (5000 pM) (V) = (100 pM) (200 pL), V = = (100 pM)(200 pL) / 5000 pM = 4 pL. Therefore, you would add 4 pL of 5 mM MgCl₂ to a 200 pL reaction to give a final concentration of 100 pM MgCl₂.

Approach II: Dimensional Analysis and Canceling Terms

As a variation of Approach I, an equation can be written such that all units of concentration on the left side of the equation cancel accept for the one that represents that of the final concentration written on the right side of the equation. All units accept the one desired are cancelled on the left side of the equation by ensuring that they appear as both numerator and denominator terms. Unit conversions are an integral part of the equation. For sample problem, the equation can be written as follows:

Notice that the equation begins with the initial stock concentration and describes what must be done to that stock solution to arrive at the desired final concentration on the right side of the equals sign. It asks how many pL (x pL) of 5 mM MgCl₂ should be placed in a total volume of 200 pL to give a final concentration of 100 pM MgCl₂.

The mM term is converted to gM within the equation by using the conversion factor 1000 gM/1 mM. All units on the left side of the equation cancel accept for «gM». Solving for x yields:

All terms cancel and the solution to the problem is revealed to be 4. Since x was originally associated with gL in the initial equation, you must add 4 gL of 5 mM MgCl₂ to a 200 gL reaction to bring that reaction to 100 gM MgCl₂.

Lets look at the following example: MgCl₂ will be added to a series of PCR tubes in concentrations of 0.5 mM, 1 mM, 2 mM, 3.5 mM, 5 mM, and 10 mM. The stock solution of MgCl₂ you will be given has a concentration of 25 mM. In the spaces provided, calculate the volumes of 25 mM MgCl₂ stock solution required for this reaction set to bring each 50 gL reaction to its desired magnesium concentration. Using the 1^st approach, you will obtain the result as follows in Table 3.3

Table 3.3

Volume of reaction components for magnesium titration

Notice that the volume of above components row has a variety of values. That means, at this point, that the final volume in your tube will all be different (except Tubes 1M and 2M). So a wise person may ask, «How can all the reagents added be at the same concentration if the final volumes are different?)) And they wouldn’t be.

So let’s take one more thing into consideration and then make the final volumes identical so that the only concentration that is changing is the MgCl₂. The final volume of each of the reactions will be made identical by the addition of water.

Remember that the volumes you have entered were all for each tube. That means you have to add 5 pL of PCR buffer to each of 7 tubes. Then add 4 pL of 10 mM dNTP stock to 7 tubes and so on. That is a lot of pipetting and each time you pipet you can introduce some error in the measurement. So we will use a way to minimize the pipetting. Now it’s time to make a Master Mix, which should contain all the reagents needed for the PCR except for the one component being titrated (in this case, magnesium) and the template DNA (which should always be added last to a PCR). The Master Mix will also contain the smallest amount of water any of the 7 tubes will need. Once prepared, an aliquot of the Master Mix will be delivered to each reaction tube. Then, different amounts of MgCl₂ will be added to each tube, and additional water will be added to each reaction such that the final volume will be 50 pL. Template DNA will be added as the final step prior to thermal cycling.

Still some part of water needs to be part of a Master Mix so that concentrated salts in the reaction are diluted. This will be the least amount of water every tube will contain. In order to ensure the proper dilutions and volumes additional water might be added. To calculate it it might be necessary to answer two questions:

A. What is the largest volume of MgCl₂ being added to any one reaction? 20 qL

B. For the reaction corresponding to this MgCl₂ amount, what is the volume of water required to bring that reaction to 50 qL? 9.75 qL

At least this amount of water will be contained in each reaction you will prepare. This amount of water will be used in calculating a Master Mix. To ensure that enough Master Mix is available, calculate the total volumes of each reagent needed assuming 8 reactions must be prepared.

Additional calculations might be necessary in order to calculate the amount of an additional water needed to add to each reaction tube (in addition to the 9.75 qL added to the Master Mix), so that the total volume of MgCl₂ plus water is equal to the largest single volume of MgCl₂ being added (see Table 3.4).

Table 3.4

Volumes of additional water to add to each reaction in the magnesium salts

No additional water will need to be added to the reaction containing the largest volume of MgCl₂. For instance, for Tube 1M the volume of diluted MgCl₂ solution to add is 2 qL. The largest single volume of MgCl₂ being added is 20 qL. Therefore, 18 qL of additional water needs to be added to tube 1M. Please note that for tube 1M you are adding 2 qL of diluted MgCl₂; all other tubes, 2M-7M receive the stock solution (25 Mm MgCl₂). Important notes to consider:

– Add reagents to the bottom of the reaction tube, not to its side.

– Add each additional reagent directly into previously-added reagent.

– Do not pipette up and down to mix, as this introduces error. This should only be done when resuspending the cell pellet and not to mix reagents.

– Make sure contents are all settled into the bottom of the tube and not on the side or cap of tube. A quick spin may be needed to bring contents down.

– Keep all the reagents and components on ice.

– Do not forget to label your tubes correct and clear.

– Prepare your Master Mix according to the previous calculations.

– Gently flick the Master Mix tube with your finger to mix the solution. Making sure to use a balance tube, spin MM tube for 5 seconds in a microcentrifuge.

– Template DNA is not added to MM, but individually to each reaction.

– Slowly pipet up and down to mix the reagents after each addition.

– To each tube, add the appropriate combination of MgCl₂ and water.

– Do not forget about the positive, negative control tubes.

– Prepare agarose gel while running the reaction.

Agarose is a gelatinous substance derived from a polysaccharide in red algae. When agarose granules are placed in a buffer solution and heated to boiling temperatures, they dissolve and the solution becomes clear. A comb is placed in the casting tray to provide a mold for the gel. The agarose is allowed to cool slightly and is then poured into the casting tray. After it solidifies the gel, in its casting tray, is placed in a buffer chamber connected to a power supply and running buffer is poured into the chamber until the gel is completely submerged. The comb can then be withdrawn to form the wells into which the PCR sample is loaded.

Before switching on the power supply and loading the wells of the gel with sample, one in each, to a small volume of total PCR reaction, it is necessary to add loading dye, which is a colored, viscous liquid containing dyes (making it easy to see) and sucrose, Ficoll, or glycerol (making it dense), mix and then pipet an aliquot of the mixture into the corresponding wells. The samples should be allowed to electrophoresis until the dye front (either yellow or blue, depending on the dye used) is 1 to 2 cm from the bottom of the gel. The gel can then be moved, stained and photographed (often using a Gel Doc system). The DNA is visualised in the gel by addition of ethidium bromide, which, when intercalated into DNA, emits fluorescence under UV-light. Other possibility for visualization, for instance like in a DNA sequencing gel, is an autoradiogram (in case if the molecules to be separated contain radioactivity).

Calculations: for instance, you will need a 2 %, mass/volume agarose gel for electrophoresis of your PCR products. If your agarose gel casting trays holds 50 mL, then how much agarose and buffer would you need? The definition of m/v % in biology is grams (mass) / 100 mL (volume). Therefore, for 2 % agarose, it will be 2 g /100 mL buffer. Step 1: Calculate the mass of agarose needed for 50 mL total volume of agarose solution. Step 2: Calculate the amount of buffer needed to bring the agarose solution to 50 mL. By standard definition, 1 gram of H₂0 = 1 mL of H₂0.

The amount of buffer for the 2 % agarose solution will be 49 mL (50 mL – 1 mL {1 gram of agarose}).

Why magnesium chloride is so important? DNA has an overall negative charge because of the negatively charged oxygen molecules along the two sugar 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.