Making DNA the PCR Machine Way

A PCR machine (also known as a thermal cycler or a thermocyler) is an instrument that amplifies a single copy of DNA to generate millions more copies in about 2 hours.

PCR machines are used for applications such as cloning, sequencing, genotyping and expression analysis.

There are 2 types of Thermal Cyclers available:

1) Standard Thermal Cycler

2) Real-Time PCR Thermal Cycler / qPCR Thermal Cycler

Traditional or standard PCR detects amplification product at the end point of the reaction using agarose gel. It does not provide exact quantification of product. It provides an estimation of quantity by comparing the intensity of the band on a gel to a known standard.

Real-time PCR (rtPCR)detects PCR amplification during the early exponential (log) phase of the PCR reaction where exact doubling is occurring. Detection of product is being measured while reaction is occurring in real time. Quantification of product is exact and expressed in numbers.

In a nutshell the PCR process looks like this:

1) Purify your DNA sample.

2) Denature the DNA. This requires separating the double strands into single strands by way of heating to 95 degrees C.

3) Anneal the primers. 2 printers bind to specific regions of the DNA that’s targeted for amplification.

4) Extend the primers. An enzyme called Taq polymerase binds to the primers and synthesizes a new DNA strand at a temperature of 72 degrees C.

Instrument Considerations

There are many design specifications to consider in choosing a thermal cycler, such as block format, sample throughput, reaction speeds, and high-tech smart lids.

Many instruments have specific block designs to accommodate needs of the laboratory. These blocks can accommodate multi-well plates or single tubes or tube strips of various sizes. Sample throughput for the machines can also range from an individual reaction up to 480,000 samples.

Reaction speeds also can vary from standard reaction speeds to fast speeds where PCR protocols can be done in less than 15 minutes. Temperature range of instruments can vary from 0-100 degrees C to 4-99 degrees C. There are many options to consider in choosing the right thermal cycler for your laboratory.

Real-Time PCR Thermal Cyclers

Real-Time PCR (rt-PCR) thermal cycler or Quantitative PCR (qPCR) thermal cyclers are instruments that amplify and quantify targeted DNA as it occurs in real time. This procedure detects and quantifies DNA sequences simultaneously during the exponential phase of the PCR reaction.

The main benefit of rtPCR is the ability to provide quantification of end product expressed in numbers. Standard PCR can provide only an estimation of quantity based on comparison with known standards on an agarose gel.

Limitations of Standard PCR:

• Low Sensitivity
• Not quantified, can only estimate amount of product when compared to a known standard on a gel
• Need to purify product post-PCR
• Non-automated

Advantages of Real-Time PCR:

• PCR reaction measured during exponential phase of reaction and can provide quantification of product expressed in numbers
• Sensitive – can detect 2 fold change in product
• Can be automated
• No post-PCR processing

In addition, rtPCR reactions differ slightly from standard PCR reactions. The rtPCR reaction is prepared the same but with the addition of either a fluorescent dye that binds to ds DNA that detects PCR product as it accumulates during reaction or a fluoregenic hybridization probe specific to the target gene. When the reaction is run on a rtPCR thermal cycler, the levels of fluorescence are measured by a detector.

rtPCR machines can be used on standard PCR applications such as cloning, sequencing as well as new applications that would have been less effective with standard PCR. Gene expression analysis, protein expression analysis, microRNA and noncoding RNA analysis, genotyping, genetic variation analysis, and mutation detection are examples of applications used for rtPCR. Some instruments can also be used for validated applications such as food and water testing, human identification, pharmaceutical analytis and animal health testing.

There are also other instrument specifications to think about when choosing a rtPCR thermal cycler. Formats (microtube, chip, arrays, cards) vary as well as the well size. Some machines can handle low throughput and some are more suited for high throughput. Some machines can provide < 5,000 data point readings per day while some (like the next generation sequencers that use rt PCR technology) can provide greater than 1,000,000 data point readings per day.

The table below summarizes the options to consider when choosing the right instrument that would fit your laboratory needs.