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PCR Primer Synthesis Tips

1. How are primers synthesized?

Currently, primer synthesis is mainly carried out using the solid-phase phosphoramidite triester method. This technique is widely used in the industry and is the standard for most DNA synthesizers. The main manufacturers of such equipment include ABI/PE. While different machines may vary in terms of yield, reagent consumption, and cycle time, the fundamental principle remains the same.

The Shenneng Gaming Company primarily uses the ABI3900 high-throughput synthesizer for standard primer production. For long-chain synthesis, they rely on Beckman 1000M and PE8909 DNA synthesizers. For modified primers and high OD number synthesis, the ABI394 model is employed.

The phosphoramidite triester method involves immobilizing DNA on a solid support (CPG) and building the sequence from the 3' to the 5' end. Each nucleotide is added through a 3'→5' phosphodiester bond. The process includes several key steps:

  • Step 1: The protected nucleotide attached to the CPG is treated with trichloroacetic acid to remove the DMT protecting group, exposing the 5'-OH group.
  • Step 2: A phosphoramidite monomer, activated by tetrazole, reacts with the free 5'-OH group to form a phosphite linkage.
  • Step 3: A capping step is performed to block any unreacted 5'-OH groups, preventing the formation of short fragments.
  • Step 4: The phosphite is oxidized to a more stable phosphotriester using iodine.

This cycle repeats until the full sequence is built. After synthesis, the primers are cleaved from the CPG using ammonia and purified using methods like OPC, PAGE, or HPLC. They are then concentrated using C18, desalted, and precipitated before being suspended in water and quantified based on OD260 readings.

2. What are the methods of purifying the primers and how to choose them?

There are several purification techniques available, each suited for different applications:

  • C18 Column Desalting: Also known as reversed-phase column purification, it effectively removes salts but not small fragments. Suitable for basic PCR applications, but not recommended for sequencing or cloning.
  • OPC Purification: Uses affinity between the DMTr group and resin. Offers >95% purity and is ideal for primers under 40mers.
  • PAGE Purification: Involves gel electrophoresis and is highly effective for long oligos (>50mers), offering >95% purity.
  • HPLC Purification: Provides >99% purity, ideal for modified or short primers, but is expensive and less efficient for mass production.

3. How is the OD number of the primer quantified?

The OD value is determined using a UV spectrophotometer at 260 nm. A quartz cuvette with a 1 cm path length is used. The solution should be diluted to an OD between 0.2 and 1.0 for accurate measurement. After dissolving the dried primer in a specific volume of water, a 1 ml aliquot is measured. The OD of the stock solution must be adjusted according to the dilution factor.

Proper quantification ensures that the right amount of primer is used in downstream experiments, helping maintain the accuracy and efficiency of PCR reactions.

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