Polymerase chain reaction optimization

The PCR optimization includes the targeted modification of the reaction conditions a polymerase chain reaction.

• 9.1 contamination
• 9.2 Hot-Start
• 9.3 Substrate Specificity

Optimization parameters

Optimization of PCR - related methods involves the selective modification of various parameters.

• Choice of a suitable thermostable DNA polymerase
• Draft sequence of the primer (Primer Design)
• Varying the concentrations of substrates ( DNA template, primer template, dNTP concentration)
• Varying the concentrations of products ( more copied DNA, correct product, less pyrophosphate )
• Avoid interfering substances (eg T. substrate - like inhibitors )
• Control of the environmental conditions (temperature, thermal stability, ionic strength, osmolarity, cofactors ).

Polymerase

The selection of a suitable DNA polymerase has an influence on the amount of synthesis.

Primer

Primer design includes the selection of appropriate DNA sequences for the primers and the primer based on hybridization temperature ( synonymous annealing temperature ) in the PCR. The annealing temperature of the PCR is usually two degrees Celsius below the melting temperature of the primers, which represents a compromise between the fullest possible primer hybridization and the highest possible specificity of primer binding.

Substrate concentration

The final concentration of the primers during PCR is between 0.1 uM and 1 micron (usually 0.4 uM ), the concentration of dNTPs is for each of the four nucleotide bases 100 - 1000 uM (usually per 400 uM ) and the mass concentration of the DNA of 1 to 20 ng per microliter of the PCR mixture (usually 200 ng per reaction ).

Temperature cycle

The dissociation ( melting ) of the double-stranded DNA is usually performed at 95 ° C. Subsequently, the hybridization takes place ( annealing ) at a primer-dependent temperature, last ( Type A polymerases ) or 68 ° C, the synthesis of DNA at the respective optimum of each thermostable DNA polymerase at 72 ° C ( Type B polymerases ).

Error rate

Error rates of different polymerases (English fidelity ) are known and have been described. Bacterial thermostable DNA polymerases ( type A) usually have a higher error rate than Archean (type B), which is due to the proofreading exonuclease activity of the B- type polymerases. The error rate of Taq polymerase is 8 · 10-6 errors per base pair that the KOD polymerase 3.5 x 10-6 errors per base pair, which of Tli polymerase and the Herculase 2.8 × 10-6 errors per base pair that the Pfu polymerase 1.3 x 10-6 errors per base pair and the Pfu Ultra 4.3 x 10-7 errors per base pair.

Synthesis rate

The overall rate of synthesis of a DNA polymerase depends inter alia on the basis of rate of synthesis and the processivity of the polymerase, and the presence of inhibitors (e.g., inflammatory products, PCR inhibitors).

The base rates of synthesis of various polymerases ( engl. productivity ) have been compared. The rate of synthesis of the Taq polymerase is about 60 base pairs per second. Under the unmodified thermostable DNA polymerases only the rate of synthesis of KOD polymerase is greater than 100 base pairs per second (about 120 bp / s). Among the modified thermostable DNA polymerases, mutations have been described which enhance the rate of synthesis. The KOD polymerase and some modified thermostable DNA polymerases ( iProof, Pfu Ultra, Phusion, Velocity or Z Taq ) are used because of their high rate of synthesis to a PCR variant with shorter cycles of amplification (Fast PCR, High-speed PCR).

The processivity (English processivity ) describes the average number of base pairs before a polymerase from the DNA template ( template Sheet ) drops. The processivity of the polymerase used limits the maximum distance of the primer to the probe in real time quantitative PCR. The processivity of Taq polymerase is approximately 200 base pairs.

When heated, the dNTPs in aqueous solutions, the cytosine residues deaminate in dCTP continuously to uracil residues, which reduce the rate of synthesis of DNA polymerases. In order to counter these so-called dUTP poisoning reduction, is sometimes added to products of about 5 kilobases in a PCR with Archean polymerase, a thermostable dUTPase. Analog parallel deaminate the adenosine residues in the dATP to deoxyinosine triphosphate ( dITP ), which can also lead to inhibition of Archaean DNA polymerases. Therefore, a thermostable dITPase for PCR may be added with archaic DNA polymerases.

Since the DNA polymerase catalyzes the DNA chain extension with the elimination of pyrophosphate, the concentration of product can be increased by the addition of a thermostable pyrophosphatase. The pyrophosphatase catalyzes the hydrolysis of pyrophosphate to phosphate, which reduces the product inhibition occurs because the equilibrium constant of the reaction in favor of the synthesized DNA is altered.

False negative results

If a PCR was performed and demonstrated in the results no duplicated DNA, although the sequence to be detected was actually exists, it is called a false negative result.

Substrate refusal

The occasionally occurring mainly in the Archean polymerases "refusal " difficult substrates (English fussiness ) can lead to false negative results, such as in ancient DNA, DNA with a high GC content, genomic DNA or DNA with secondary structures. By adding weak chaotropic molecules such as dimethyl sulfoxide, formamide, betaine, trehalose, or the inhibition of the reaction can be reduced. The use of chaotropic compounds can lower the melting temperature of the primer is up to 5 ° C, so the annealing temperature of the PCR has to be adapted accordingly. Increasing the concentration of magnesium ions, as a cofactor of the thermostable DNA polymerase, 1.5 mM also increases the product concentration at the expense of error rate.

Inhibitors

Various substances may interfere with thermostable DNA polymerases in the PCR, such as anti-virals, hemoglobin, heparin, polysaccharides, hormones, IgG, lactoferrin, myoglobin, bile acids and their salts, uric acid and its salts, phenol, polyphenols, proteases, divalent cations except magnesium, EDTA and other chelators, humic acids and clay. In general, samples with these materials require a further DNA purification prior to use in PCR. In the presence of higher concentrations of polyphenols polyvinylpyrrolidone may be added, which binds them.

False-positive results

A false positive result is a generation of PCR products that do not meet the desired and the target DNA sequence in a PCR. If necessary, lead to undesirable band in an agarose gel.

Contamination

DNA contamination and non-specific hybridization of the primers before and during PCR can lead to false positive results. In particular, contamination is a big problem. Often DNA is transported by persons who are involved in the analysis, or who have contributed to the production of components in the sample. When forensic use (see DNA analysis and genetic fingerprinting) made ​​the Phantom of Heilbronn headlines: criminologists hunted for a long time an alleged perpetrator series, until it turned out that the DNA came into reality by the employee of a subcontractor. Also problematic bacterial contamination, contamination by parasites or food components and the like may be in biological samples. Thus, the worm-like organism Xenoturbella was extended period erroneously regarded as the representative of the molluscs, because you, mistook DNA from the gut, which came from food organisms for the DNA of the worm itself. Especially with very old, as possible partially fragmented DNA, eg from fossils, special caution is required. This is especially true if the people related sequences, such as the genome of Neanderthal man, is analyzed.

It is sometimes added to the enzymatic degradation of possible DNA contamination in the reaction mixture a uracil -N- glycosylase, which is denatured in the first temperature cycle. The uracil -N- glycosylase reduces uracil -containing PCR products, which arise when the laboratory has previously converted in PCR to pure detection purposes of dTTP to dUTP and for polymerases has used the type A. However, this can at low concentrations of DNA starting material lead to false negative results.

Hot Start

The template specificity of the polymerase (English specificity ) for the prevention of unwanted binding to DNA templates or no bound primers, and the consequent production of undesirable reaction products prior to the first temperature cycle is enhanced by the use of hot-start polymerases. Examples are inhibited with an antibody hot start Pfu Turbo, Platinum Pfx as commercial KOD polymerase inhibitory antibodies, and Platinum Taq as antibody inhibited Taq polymerase. These polymerases can be inhibited by inactivation with formaldehyde, inter alia, by a complexing magnesium with phosphates or by the binding of an antibody to the active site. When heated to 95 ° C hydrolyzes the formaldehyde, alternatively, the magnesium ions are released or the antibody is denatured thereby. A fourth variant is a latex beads by hydrophobic effects adsorbed polymerase that goes with increasing temperature in solution. In the fifth, oldest variant of the reaction mixture is overlayed without polymerase with wax and given the polymerase to the cooled wax. When heated to melt the wax layer and the polymerase is mixed with the reaction mixture.

Substrate specificity

A change in the template specificity of the DNA to RNA can be used for the generation of thermostable RNA dependent DNA polymerase, also referred to as thermostable reverse transcriptases. Conventional RT-PCR for reverse transcriptases used retroviral origin, such as the AMV and MoMuLV reverse transcriptase, are not thermostable. At the lower temperatures to a reverse transcription with these enzymes, however, non-specific binding of primers to the DNA template and DNA secondary structures in the template are present which on the one hand favor unwanted products and, secondly, prevent the synthesis of the correct product.

Therefore, first, the template specificity of thermostable DNA polymerases ( divalent magnesium ions) was reduced to bivalent manganese ions by exchange of the co-factor, so that with a DNA dependent thermostable polymerase and RNA could be used in an RT-PCR as a template for the synthesis of DNA. Because the rate of synthesis of Taq polymerase was relatively low with manganese ions, the Tth polymerase was used increasingly in this variant of RT-PCR. However, the addition of manganese ions also increased the number of defective products and increased the necessary amount of template DNA, which is why these enzymes are now rarely used for reverse transcription. These problems could be avoided by using the thermostable polymerase 3173 from thermophilic bacteriophage which withstands the high temperature of a PCR for a long time and preferably as a template RNA.

The preference of individual nucleotides by a thermostable DNA polymerase is as Nukleotidspezifität (English bias, partiality ',' bias ') respectively. In the PCR - based DNA sequencing with chain-terminating substrates ( dideoxy method ) is often their uniform installation and thus a uniform production of all chain termination products desirable to allow a higher sensitivity and easier evaluation. For this purpose, a KlenTaq polymerase was generated by deletion and replaced by site-specific mutagenesis, a phenylalanine at position 667 from tyrosine (short: F667Y ), and referred to as the Thermo Sequenase. This also applies to the installation of fluorescent -labeled dideoxynucleotides.