Polymerase Chain Reaction (PCR) Process

Time is of the essence. You need to amplify your DNA sample fast as you face daily PCR challenges. You can be as particular as possible at every stage of your PCR process, but the reality is—your results are only as good as the products and equipment you use. PCR is challenging enough without worrying about if your equipment is going to produce exactly what you need. This is where you can rely on us. We’ve been helping customers like you get their PCR and qPCR protocols done rapidly and resourcefully for decades. Our comprehensive offering of products can help you get the results you demand and perhaps faster than you ever imagined. From sample collection and prep to PCR/qPCR protocols and sample analysis, find a comprehensive offering of proprietary and popular products including centrifuges, PPE, sample prep reagents, thermal cyclers, qPCR units, reagent test kits with and without Mastermix, microplates, plate sealers, electrophoresis systems, and more.

Regardless of your industry, we can help you with your PCR process. We’ve defined this sensitive and very challenging PCR process into the 4 stages below to help you select the products you need quickly and get back to what matters the most.

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Step One — Collection

Preservation & Stabilization

DNA RNA Preservation & Stabilization

A DNA or RNA sample to be tested is collected using swabs, needles, single-use tweezers or other tools. The sample can be from saliva, blood, hair, ancient bone, skin scraping, plant, soil, etc. It is placed into a tube or container. A stabilizing reagent is added to preserve the sample. If the sample is not already at a research facility, it will be transported safely so it can be prepared for the PCR process and tested.

 

Step Two — Preparation

Nucleic Acid Extraction & Purification

vortext mixer

The sample is extracted from its container and the nucleic acid is purified via a lysis method, organic extraction, and/or column extraction/purification. Once the sample is purified and extracted, a Mastermix is added. This contains DNA primers, DNA polymerase (enzyme), and a nucleotide solution mix. This solution is now ready for the PCR process.

 

Step Three — Amplification

Conventional PCR or Real-Time PCR

Conventional PCR or Real-Time PCR machines

The solution is placed into a thermal cycler (either PCR or qPCR) to begin the replication/amplification process. This initial DNA/RNA sample is replicated into millions of duplicate DNA/RNA segments. The PCR process involves three steps:



• Denaturation: The solution contained in the tube is heated to at least 94°C (201.2°F) using a thermal cycler. The heat breaks the hydrogen bonds of the original DNA sample and separates the DNA into single strands.

• Annealing: The sample mixture is then cooled to between 50-60°C (122-140°F) allowing the DNA primers and the DNA polymerase enzyme to bind to the individual strands of DNA that were separated by the heat.

• Extension: The nucleotides (A, T, C, G) from the added mixture solution will pair with the individual separated strands of DNA that resulted from the heating process. Once joined together, they form a new complementary strand of DNA.


This process provides a new duplicate double-stranded DNA molecule formed from each of the single strands of the original sample molecule. This three-step process is done 35 to 40 times in order to amplify the DNA/RNA into millions of duplicate segments.


Step Four — Analysis

Post PCR Clean-Up

electrophoresis system

The amplified DNA/RNA segments from the PCR process are ready for analysis. The most popular method used to accomplish this is called gel electrophoresis. In electrophoresis, basically the DNA/RNA samples are loaded into one end of a gel and placed into an electrophoresis system where an electrical current is passed through the gel. The DNA/RNA segments are separated into bands resembling a laddering pattern, according to their electrical charge and size. Once the gel is stained and viewed under UV light, the PCR processed DNA/RNA segments are compared against a known control source.

 

The amplified DNA segments from the PCR process are ready for analysis. The most popular method used to accomplish this is called gel electrophoresis. In gel electrophoresis, the DNA segments are loaded into wells within a gel placed in an electrophoresis system. For comparison, other DNA segments of known size are included. An electrical current is passed through the gel, separating the DNA segments according to their size. This results in a banding pattern resembling a ladder. The positioning of the amplified DNA segments relative to the known markers reveals the identity of the sample from the PCR process.