Protocolo PCR

 

Optimizing DNA Amplification Protocols

Abstract

The Polymerase Chain Reaction (PCR*) is a molecular-biological process that, during recent years, has been developed into a method used in virtually every area of medicine and natural sciences. This method was first described in 1985 (1) and enables selective in vitro amplification of a special DNA fragment, thereby emulating the cellular in vivo DNA replication. In spite of the method's basically simple operation, often enough it is not possible to achieve optimum results without optimizing the protocols. This summary therefore discusses a series of critical PCR parameters and feasible strategies for optimization.

Primer length and primer sequence

Prerequisite for amplification of a specific product is the selection of suitable primers. In this context, the following criteria are of essential importance:

  1. The length of the primers;
  2. the melting temperature (Tm);
  3. the composition of the sequence and its physical properties; and
  4. primer-primer interaction (2,3,4).

Ideal primers are between 18 and 30 bp long. The influence of the primer's melting temperature on the annealing temperature in PCR is discussed hereafter.

The sequence plays a very important part. The ideal G/C content generally lies in the range of 45-55%. In order to insure stable annealing, the primer should, as far as possible, be complementary to the desired DNA sequence and, in addition, be able to form “G/C clamps”, i.e. several consecutive G/C or C/G base pairs, between the 3' end of the primer and the template DNA (5).

Self-complementarity within a primer by virtue of palindromes or long segments of polypurines and polypyrimides, or areas complementary to the sequence of the second primer, enhance the formation of primer dimers and should therefore be avoided.

Selection of primer sequences and the calculation of annealing temperatures (see below) can be computer-aided using a range of software.

Annealing temperatures

Both primers should have similar Tm values, which can be most easily calculated according to the Wallace formula [(A/T)x2 plus (G/C)x4] (6). This simple rule, however, is restricted to primers with a maximum length of 20 nucleotides. Longer primers require more complex formulas (7).

Ideally, the Tm values should lie in the range of 55-65°C. PCR optimization often begins at an annealing temperature of 5°C below the primers' Tm.

The optimum annealing temperature, however, often enough is considerably higher than the calculated Tm. One optimization strategy is therefore to run additional PCRs, gradually increasing the annealing temperature each time by 2-5°C.

The touch down method

The so-called “touch down” PCR is a method that uses variable annealing temperatures (8). Initially, annealing takes place at approx. 15°C above the calculated Tm. During the following cycles, the annealing temperature is then gradually reduced by 1-2°C until it has reached a level of approx. 5°C below Tm. This method is useful in avoiding nonspecific PCR products, especially in the case of complex genomic DNA, where nonspecific annealing is more probable (9).

Length of amplificate

Ideally, the amplificate should be between 100 and 400 bp long, but recently even considerably longer PCR fragments have been amplified (long PCR, up to 40 kbp) (10).

Hot-start method

Nonspecific primer-template complexes may be generated during sample preparation at room temperature. To prevent extension of these complexes in the first PCR cycles they have to be denaturated. In principle this is done using the hot-start method (11). Taq polymerase is added to the reaction mixture after an initial denaturation step at 95°C. This step is followed by the annealing process in the first cycle.

Cycle times

The PCR's temperature/time profile is a particularly critical parameter. The required times selected are often too long. Standard protocols with, for example, 60 seconds for each of the denaturation, annealing and extension phases, can be run on modern devices

Denaturation

In general, an initial denaturation process of a few minutes duration is carried out in order to insure, primarily in the case of genomic DNA, complete denaturation of the target regions on the DNA. After the first PCR products have been synthesized, considerably shorter denaturation times can be selected in the cycles, since only a very short denaturation time is actually required. These short denaturation times, however, may only be adequate if the thermocycler calculates the expired denaturation time on the basis of the temperature in the sample and not in the thermoblock

Amplification of a DNA fragment (length 117 base pairs) located on a plasmid (10.4 kbp) using a standard protocol. Every parameter - except the denaturation time - was maintained at a constant level.

Volume 20 µl.
L: 100 bp ladder
1: Denaturation time 30 sec.
2: Denaturation time 20 sec.
3: Denaturation time 10 sec.
4: Denaturation time 5 sec.
5: Denaturation time 1 sec.

This allows shortening the cycle times considerably. In addition, there is evidence that template DNA and PCR products are degraded by extensive denaturation times (13). Such heat damage in the DNA can, during PCR, result in a higher rate of error for nucleotide insertion (14). Furthermore, extensive denaturation times result in a substantial loss of polymerase activity (see below).

Annealing

During the annealing phase, the primers are rapidly hybridized. This operation is completed within a few seconds. Therefore, annealing times of 10-20 seconds are usually fully adequate (4).

Very long annealing times normally do not improve yield, but rather produce an increase in spurious priming and, thus, greater amounts of nonspecific PCR products.

Extension

During primer extension (at approx. 72°C), the insertion rate of commonly used DNA polymerases is at least 50 nucleotides per second (7). It is therefore possible to keep extension times shorter 15 seconds for PCR products less than 400 bp long. In addition, nucleotides are already inserted during the annealing phase, in particular, if it is possible to carry out annealing at relatively high temperatures. At 55°C, for example, Taq polymerase has an insertion rate of approx. 24 nucleotides per second (7). Of course, very long PCR products demand correspondingly longer extension times. In these cases, it may be advisable to provide for longer extension times in each PCR cycle (delay function) in order to compensate for increasing viscosity in the sample.

Magnesium ion concentration

A very simple but significant strategy for optimization is titration of the magnesium ion concentration (4). In addition to Mg2+ ions bound by the template DNA, the nucleotides (dNTPs) and the primers, Taq DNA polymerase also requires free Mg2+ ions. Their concentration has an influence on primer annealing, the melting temperature of the PCR product and product specificity. An excessively high concentration leads to a reduction in stringency, i.e. reaction specificity (7). The concentration of free Mg2+ ions should exceed that of the total dNTP concentration by 0.5-2.5 mM.

Primer and nucleotide concentration

The choice of primer and nucleotide concentration has significant influence on PCR. A high primer concentration increases the probability of spurious priming and leads to the generation of nonspecific products. At the same time, it enhances the generation of primer dimers (see above) (9). A substantial surplus of primer can therefore even result in a reduction of the amplification yield from the PCR target.

Thus, the rate of primer concentration should lie in the range of 0.1-1.0 µM.

The dNTP concentration should be titrated together with the primers. It should lie in the range of 20-200 µM. Reduction in primer and dNTP concentration can result in a dramatic improvement in stringency, since at low dNTP concentration, Taq polymerase catalyzes the polymerization at a higher degree of accuracy .

 

Result:

Evidence of 2 different large rRNA operons in the genome of Heterorhabditis was not provided until the primer concentration, the dNTP concentration and the amount of polymerase had been optimized.

Polymerase concentration

The concentration of Taq polymerase is a critical factor in determining the stringency of a PCR. A high concentration results in a reduction in specificity and incurs unnecessary costs.

Amplification of a DNA fragment (length 117 base pairs) on a plasmid (10.4 kbp) using a standard protocol. Every parameter - except the amount of Taq polymerase - was maintained at a constant level. Volume 50 µl.

1: Taq polymerase: 0.25 U
2: Taq polymerase: 0.5 U
3: Taq polymerase: 1 U
4: Taq polymerase: 2 U
L: 100-bp ladder

Buffers and reaction supplements

Current literature includes discussions on various PCR buffers and supplements, such as DMSO, PEG 6000, formamide, glycerol, spermidine and nonionic detergents, used to increase the reaction specificity or efficiency (15). Certain polymerases will only reach their optimum level of activity (16) in the presence of such supplements.

References

(1) Saiki, R.K., Scharf, S.J., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. & Arnheim, N. (1985). Enzymatic amplification of ß-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354.

(2) Persing, D.H. (1993). Target selection and optimization of amplification reaction. In: Diagnostic molecular microbiology, 88-102. Eds.: David H. Persing, Thomas F. Smith, Fred C. Tenover, Thomas J. White, ASM.

(3) Hayashi, K. (1994). Manipulation of DNA by PCR. In: PCR - the Polymerase chain reaction, 3-14. Eds.: K.B. Mullis, F. Ferre & R.A. Gibbs, Birkhäuser.

(4) Innis, M.A. & Gelfand, DH (1990). Optimization of PCRs. In: PCR Protocols: A guide to methods and applications, 3-11, Academic press.

(5) Charlieu, J.-P. (1994). PCR as a technique used daily in molecular biology. In: PCR Technology, current innovations, 1-4. Eds.: G. Hugh & A.M. Griffin, CRC press.

(6) McConlogue, L., Brow, M.A.D. & Innis, M.A.(1988). Structure independant DNA amplification. Nucleic Acids Res. 16: 9869.

(7) Rolfs, A., Schuller, I., Finckh, U. & Weber-Rolfs, I. (1992). PCR principles and reaction components, 1-18. In: PCR: Clinical diagnostics and research, Springer.

(8) Don, R.H. et al. (1991). "Touch down" PCR to circumvent spurious priming during gene ampification. Nucleic Acids Res. 19: 4008.

(9) Hosta, F. (1991). Enhancement of specifity and yield of PCR. United States Biochemical Corp. Editorial comments Vol. 18, No.3, 1-5.

(10) Cheng, S., Chang, S.-Y., Gravitt, P. & Respess R. (1994). Long PCR. Nature 369: 684-685.

(11) D’Aquila, R.T., Bechtel, L.J., Videler, J.A., Eron, J.J., Gorczycy, P. & Kaplan, J.C. (1991). Maximizing sensitivity and specificity of PCR by preamplification heating. Nucleic Acids Res. 19: 3749.

(12) Rolfs, A., Schuller, I., Finckh, U. & Weber-Rolfs, I. (1992). Physical features of thermocyclers and their influence on the efficiency of PCR amplification, 259-262. In: PCR: Clinical diagnostics and research, Springer.

(13) Douglas, A. & Atchinson, B. (1993). Degradation of DNA during denaturation step of PCR. In: PCR-Methods and Applications, 133-134. Vol.3, No. 2. Cold Spring Harbor.

(14) Eckert, K.A. & Kunkel, T.A. (1991). The fidelity of DNA polymerases used in the polymerase chain reactions. In: PCR A practical approach; Eds.: M.J. Mcpherson, P. Quirke & G.R. Taylor, IRL Press.

(15) Roux, K.H. (1995). Optimization and troubleshooting in PCR. In: PCR-Methods and Applications, 185-194. Vol. 4, No. 5. Cold Spring Harbor.

(16) Rolfs, A., Schuller, I., Finckh, U. & Weber-Rolfs, I. (1992). Substances affecting PCR: Inhibition and enhancement, 51-58. In: PCR: Clinical diagnostics and research, Springer.

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