Molecular Biology Topics

PRIMER DESIGN AND PCR AMPLIFICATION

Written by MicroDok

What is a primer? A primer is a short DNA fragment that contains specific sequences which are complementary to the template DNA to be copied and amplified. Primers are short DNA fragments (usually about 18-20 bases or nucleotides long) that contain specific sequences which are complementary to the template DNA molecule to be copied and amplified. PCR primers generally range in length from 15-30 bases. They are designed to flank the region of interest during the PCR amplification process in a thermocycler. Primers for PCR reaction or amplification should contain at least 40-60 % (G+C). G+C stands for guanine (G) and cytosine (C). When designing your primers, care should be taken to avoid oligonucleotide sequences that might produce internal secondary structures during the amplification process. More so, the 3ʹ-ends of the primers should not be complementary in order to avoid the production of primer dimers during and after the PCR amplification process. Primer dimers are not good, and may affect the quality or integrity of the resulting amplified DNA bands. Primer dimers unnecessarily deplete primers from the reaction; and this may result in an unwanted polymerase chain reaction (PCR) that competes with the desired reaction. During primer design, try to also avoid three G or C nucleotides in a row near the 3ʹ-end of the primer, as this may result in nonspecific primer annealing. This may increase the synthesis of undesired reaction products at the end of the PCR amplification process. It is important that both primers (i.e. reverse and forward primers) should have nearly identical melting temperatures (Tm). This way, the two primers should anneal roughly at the same temperature. The annealing temperature of the PCR reaction is usually dependent upon the primer with the lowest melting temperature. It is important to always consult a primer design company to assist you with the design and development of your primers in case you may be facing some challenges doing so. There are many online tools that you can use to design your primers. Some primer design companies include:

  • Promega
  • Primerdesignis
  • Genesig
  • GenScript
  • Eurofinsgenomics
  • ABM Inc

GENERAL PCR AMPLIFICATION GUIDE

PCR is the acronym for polymerase chain reaction. PCR is a molecular biology technique that is used to make many copies of a specific piece of DNA molecule or fragment. PCR reaction occurs in a small reaction tube known as Eppendorf tube, and this reaction is made possible with the help of the equipment known as thermocycler or PCR machine. The thermocycler is a piece of equipment that has both a heating and cooling mechanism. And the three major stages of the PCR reaction step occurring in the thermocycler include:

  • Denaturation
  • Annealing
  • Extension

Denaturation step is the first stage of the PCR reaction. Denaturation stage helps to uncoil or unwind the double strands of the template DNA into single strands. It is generally a 2 minute initial denaturation step at 95oC. Usually, subsequent denaturation step is normally done between 30 seconds and 1 minute. The denaturation temperature is usually high (e.g. 95oC as aforesaid).

Annealing step is the next stage after denaturation. The annealing stage helps the unwound single strands of the template DNA to complementarily anneal or join to a specific primer. Annealing step is usually done for 30 seconds to 1 minute; and the annealing temperature is normally lower than the denaturation temperature. Annealing temperature is usually in the range of 50-55oC.

Extension step is the next stage after annealing. It is usually the last stage of the PCR reaction process. The temperature range for the extension stage is usually between 72-74oC. This stage is usually performed for about 1 minute first, and then a final extension can be undertaken for another 2-5 minutes at the same temperature.

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MicroDok

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