The polymerase chain reaction (PCR) machine, or thermocycler, is a cost-effective and highly efficient tool used to amplify small segments of DNA or RNA. PCR combines principles of nucleic acid replication with complementary nucleic acid hybridization to exponentially produce specific target DNA/RNA sequences by a factor of 107 within a matter of hours.
It works by binding primers to the target sequence and extending this using a Taq polymerase. PCR is in three stages, and there are also a huge variety of modifications of conventional PCR that exist. PCR systems are sought-after systems for many studies, including testing for viral infections such as COVID-19, and the option to lease these machines makes them accessible to start-up companies and researchers as well as big-name companies.
What Does A Thermocycler Do?
A Polymerase Chain Reaction (PCR) machine, or thermocycler, is commonly used to make millions of copies of an initially small segment of DNA. It is also referred to as “molecular photocopying,” and allows scientists to amplify DNA enough to study in detail. It is a valuable piece of equipment in many laboratory and clinical processes. This is because analysis of DNA requires significant amounts of any given sample. A single piece of DNA is nearly impossible to work with for molecular and genetic analyses, so PCR amplification is essential.
The amplified DNA produced by PCR has a variety of uses. These uses can include the investigation of genetic diseases, DNA fingerprinting, and the detection of bacteria or viruses. PCR is an integral part of much biomedical research and criminal forensics.
How PCR Works
The steps highlighted here are considered standard.
1. The initial DNA sample which is to be copied
2. Primers (small sections of DNA which initiate the PCR reaction and bind to the DNA you intend to copy)
3. A heat-resistant taq polymerase enzyme
4. DNA nucleotide bases (also known as dNTPs)
5. A buffering agent and salts required to create the ideal conditions for the reaction
The polymerase chain reaction process can be broken down into three stages:
1. Denaturing – The DNA template is heated to separate it into two pieces of single-strand DNA. (94-95°C)
2. Annealing – The temperature is lowered, enabling the primers to bind to the DNA and initiate polymerisation. (50-56°C)
3. Extending – The temperature is raised again and, using the original strands as templates, a new strand of DNA is made by an enzyme (usually taq polymerase). (72°C)
One cycle of PCR results in two strands of DNA. Each of these strands contains one original strand and one newly-made strand.
The three stages of a PCR make up one full cycle. This cycle is usually repeated around 30 times, and each one doubles the number of DNA copies made. This allows scientists to create the millions or billions of copies usually needed to research a single piece of DNA.
The cycling process of PCR is automatic, and a billion or so copies can be created in just 2-3 hours.
Types of PCR Machines
In addition to the standard methods of PCR, several modifications have been developed. By altering how PCR machines operate, they can now be used for a wider variety of applications. Some of the most common types of PCR are:
1. Conventional PCR machine
The conventional polymerase chain reaction is used to amplify a target DNA sequence to several million in a short amount of time, usually in just 2-3 hours. It allows the replication of cellular genetic material using a polymerase enzyme to construct specific fragments of DNA.
The polymerase enzyme works alongside a primer which is connected to a strand of DNA, allowing for the synthesis of specific parts of the DNA strand. The result of using primers in this way is the amplification of a chosen DNA sequence, up to millions or billions of copies.
Conventional PCR is used in many areas of study, including medical and diagnostic research, forensic studies and research, selective DNA isolation, amplification and quantification of DNA.
Quantitative PCR (qPCR), also called real-time PCR, or RT-PCR, is a variation of the standard polymerase chain reaction which uses just one machine to combine the amplification of a target DNA sequence with the quantification of the concentration of the DNA in any given reaction. This is done using fluorescence-detecting thermocyclers.
Compared to conventional PCR, qPCR provides a faster alternative to facilitate analysis by detecting products in real-time during the exponential phase. Fluorescent dyes signal DNA of interest, and the amount of fluorescence generated is determined by the quantity of DNA present.
There are various models of real-time PCR available, but they all have common features: a standard thermal cycler platform coupled with an excitation source (usually a laser or tungsten lamp), a camera for fluorescence detection, and computer and software for data processing.
qPCR can be used in genotyping and quantification of pathogens, microRNA analysis, cancer detection, microbial load testing and GMOs detection.
Reverse Transcription PCR (RT-PCR) is a variant on conventional polymerase chain reaction which amplifies target RNA. The addition of reverse transcriptase (RT) enzyme before PCR means it is possible to amplify and detect RNA targets. During RT-PCR, RNA molecules are converted into complementary DNA (cDNA). Single-stranded cDNA is converted into double-stranded DNA using DNA polymerase. The resulting DNA molecules can then be used and amplified in a PCR reaction.
RT-PCR is used in gene insertion, research methods, genetic disease diagnosis and cancer detection.
Nested PCR is a modification of PCR where non-specific binding is prevented to increase the sensitivity and specificity of the reaction. Nested PCR works by having the first set of primer bind to the outside of the target DNA and amplifies a larger fragment, while a second set of primer binds specifically at the target site in successive PCR reactions.
Nested PCR is great for use in phylogenetic studies and in the detection of different pathogens. This is due to the higher sensitivity brought by Nested PCR compared to conventional PCR. Even if a sample contains lower DNA, Nested PCR allows this sample to be amplified.
5. Hot Start
Hot start PCR is a new form of the conventional polymerase chain reaction which reduces the occurrence of undesired products and formation of primer-dimers due to non-specific DNA amplification at room temperatures. This works by keeping the different elements of the reaction separate until the mixture reaches the denaturation temperature after heating. Hot start PCR can often increase product yields in comparison to conventional PCR. It also requires less effort than conventional PCR and reduces the risk of contamination.
6. Digital, aka dPCR
Digital PCR devices, or dPCR, are the most accurate devices on the market. They provide absolute counts of target DNA with enhanced increased sensitivity, precision, and reproducibility. Digital polymerase chain reaction is poised to disrupt molecular analysis technology on every level. There are two types of dPCR machines:
- Droplet Digital PCR (ddPCR): ddPCR uses Taq polymerase to amplify targeted DNA in a complex sample. To simplify the processing, ddPCR emulsifies samples in oil and uses fluorescence to process and analyze the results. Before analysis, the sample is divided into droplets and thermocycled, then run through a 96 well PCR plate. This separation process is the primary difference between ddPCR and qPCR
- qdPCR: The qdPCR process is based on integrated fluidic circuits (chips). While chip-based techniques have a narrower dynamic range, they provide extremely precise sample partitioning and vastly lower variance.
On the forefront of detection technology, ddPCR and qdPCR are top-of-the-line processing machines with a high price tag to match. Fortunately, it’s cheaper to lease.
How Much Does a PCR Machine Cost?
The cost of a PCR machine can vary enormously depending on your needs. A basic PCR machine can have as low as a $5,000 list price, while rtPCR systems can range from $15,000 to over $90,000 for a top-of-the-line unit. If you’re looking to purchase a PCR machine, you may have to sacrifice performance and features for price and performance.
The prohibitive cost of one of the defining techniques of modern life science molecular biology makes PCR systems inaccessible for any facility on a limited budget. If you’re in the market for a high-end PCR machine and constrained by your budget, leasing might be the option you’re looking for.
Leasing vs. Buying PCR Machines
There are a number of advantages to leasing a PCR machine through Excedr compared to purchasing. Here are a few highlights:
- Minimize upfront costs. Payments are 100% tax deductible operating expenses. Leasing can be less expensive than purchasing.
- You don’t need to buy or worry about annual service contracts, because all repairs and maintenance are covered in the lease.