[Methods for handling PCR with mixed bands]

—— ——

PCR (polymerase chain reaction) and gel electrophoresis are commonly used techniques in molecular biology research. They are usually used together to identify and analyze PCR amplification products. However, when PCR gel electrophoresis is carried out, heterobands are often found in the electrophoretic map. These heterobands may interfere with the experimental results, or even lead to experimental failure.

This article will discuss the causes of heterobands in PCR gel electrophoresis and provide some possible solutions.

●●●

PCR condition optimization:Adjusting the PCR reaction conditions and annealing at too low a temperature can lead to non-specific binding between primers and templates, resulting in non-specific amplification; If the annealing temperature is too high, it may inhibit the normal binding of primers and templates, affecting amplification efficiency. The optimal annealing temperature can be gradually determined through gradient PCR experiments. Generally speaking, the temperature of the annealing step in PCR reaction is usually between 50 ° C and 68 ° C, depending on the base sequence of the primer and the characteristics of the target DNA. Some primers require higher annealing temperatures to ensure specific binding in specific regions, but in general, the highest annealing temperature will not exceed 68 ° C.

●●●

Unreasonable primer design:If the primer length is too short, the sequence is repetitive, or contains high GC regions, it may lead to non-specific binding between the primer and the template, resulting in the generation of heterozygous bands. Primers need to be redesigned to ensure appropriate length, non repetitive sequence, moderate GC content, and avoid high GC regions within the primer or in the binding area between the primer and the template.

●●●

Improper primer dosage:Excessive primer usage or low specificity may lead to non-specific amplification, resulting in the production of mixed bands. Suggest changing primers or reducing the amount of primers used.

●●●

Template impurity:Template DNA contains impurities such as proteins, RNA, or other non target DNA fragments, which may serve as templates for PCR amplification, leading to the appearance of additional bands.

●●●

Template degradation:Template DNA undergoes degradation during extraction or storage, and the resulting small fragments of DNA may become templates for non-specific amplification.

●●●

Purification template DNA:The template DNA used in PCR experiments can come from various sources, including genomic DNA (gDNA), complementary DNA (cDNA), and plasmid DNA. However, DNA from different sources may vary in composition and complexity, which can affect the optimal starting amount for PCR amplification. For example, in a 50 µ L PCR reaction, plasmid DNA only requires 0.1-1 ng, while gDNA requires 5-50 ng. In addition, the type of DNA polymerase used also affects the optimal template starting amount. The modified DNA polymerase has a stronger affinity and higher sensitivity to the template, therefore requiring a relatively smaller initial amount of DNA. Optimizing the starting amount of DNA is crucial, as a high starting amount may increase the risk of non-specific amplification, while a low starting amount may reduce the yield of PCR. Sometimes, PCR experimental protocols use copy number to represent the initial amount of DNA, especially for gDNA. The calculation of copy number involves Avogadro constant and molar mass, with the specific formula being: copy number=L × molar number=L × (total mass/molar mass).

●●●

Mg ² ⁺ concentration too high:Mg ² ⁺ is an important component in PCR reactions, but its high concentration can reduce the specificity of PCR amplification and increase the risk of non-specific amplification. The optimal concentration of Mg ² ⁺ needs to be determined through experiments.

●●●

Poor dosage or quality of enzymes:Excessive enzyme dosage or poor enzyme quality can affect the PCR reaction. Suggest reducing the enzyme amount or replacing it with another source of enzyme.

●●●

Using hot start PCR:Hot start PCR can reduce non-specific amplification that may occur during reactions at low temperatures.

Hot start PCR is a technique used to reduce non-specific amplification. In conventional PCR, the primer may have already bound to the template DNA before the reaction temperature reaches the amplification temperature. This may lead to non-specific amplification at the beginning of PCR, resulting in mixed bands or false positive results. Hot start PCR solves this problem by adding thermally stable DNA polymerase to the PCR reaction.

The key to hot start PCR is to use a polymerase that requires high-temperature activation. Such enzymes become active only at higher temperatures, so they only begin to amplify the target DNA sequence at the beginning of the reaction, without triggering non-specific reactions at low temperatures.

Some commonly used hot start polymerases include improved versions of thermally stable Taq polymerases, such as Taq DNA polymerases with thermally activated domains, as well as high fidelity polymerases such as Pfu or Phusion.

So nowadays, most enzymes meet the requirements.

●●●

Check the laboratory environment:Avoid DNA contamination and contamination of experimental instruments, use sterile techniques and processed reagents, and maintain laboratory cleanliness.

●●●

Gradient optimized PCR:Use temperature gradient for PCR reaction to find the temperature for specific amplification.

Baiheng Technology Intelligent Two Dimensional Gradient PCR InstrumentThe temperature gradient for denaturation and annealing can be optimized simultaneously in the same reaction. Denaturation can be set with 8 horizontal denaturation gradients and 12 vertical annealing temperatures to optimize the optimal annealing temperature, thereby improving the specificity and efficiency of PCR reactions.

This technology is very useful for optimizing primers, templates, and PCR conditions to ensure high-quality and specific PCR products are obtained.

These methods can be used alone or in combination to help reduce or eliminate PCR aliasing issues.