Apollo Laboratory

Molecular Testing using PCR

Apollo-Laboratory offers molecular testing utilizing polymerase chain reaction (PCR) to identify a broad spectrum of pathogens which cause human infections.

Molecular testing yields numerous advantages over traditional infectious disease testing techniques, such as cultures, by identifying pathogens with unparalleled accuracy, speed, specificity and sensitivity. Utilization of this designed methodology and advancements in instrumentation allows our scientists to accomplish what could have taken days for an identification to a matter of hours.

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PCR DNA Strands

PCR Testing (Polymerase Chain Reaction) History

The timeline of intial Polymerase Chain Reaction testing dates back to 1976 involving the isolation of Taq polymerase from the thermophilic bacterium Thermus aquaticus. Finally, molecular biologists possessed an isolated thermostable enzyme with the capacity to duplicate PCR cycling thus eliminating the requirement of injecting new DNA polymerase following each cycle.

Cetus Corporation employed a chemist in its Emeryville, CA facility who was responsible for the progression of the PCR process in 1983. Kary B. Mullis, an American biochemist, additionally was awarded the prestigious Nobel Prize for Chemistry in 1993 for his work in this field. Prior to the evolution of Polymerase Chain Reaction testing, the procedures used to magnify or create duplicates of new combinations of DNA fragments were lengthy and laborious. In comparison, a instrument blueprinted to facilitate PCR reactions, permitted scientists to created millions of reproductions of limited specimens of DNA in just a few hours.

The process Mullis created involved injecting a modest supply of the DNA incorporating the targeted gene into a test tube. Additionally injected into the tube are a batch of loose nucleotides that help tie into the exact copies of the original gene.

The next step in the process is to insert a couple of small DNA segments whose elements have been combined duplicating the segments on either side of the targeted gene. Primers, as they are referred to, locate the right side of the DNA, acting as origination points for DNA copying. Following the addition of the enzyme Thermus aquaticus (Taq), the loose nucleotides attach themselves to a DNA sequence governed progression of the targeted gene positioned among the two primers.

Two strands are created as the DNA double helix splits as a result of the heating of the test tube. This uncovers the DNA sequence of individual strands of the helix. Reducing the temperature permits the primers to naturally join to their corresponding parts of the DNA sample.

Simultaneously, the enzyme joins the floating nucleotides to both the primer and the individual detached DNA strands using the applicable sequence. The entire reaction is completed in about five minutes. The end result is the creation of two double helices consisting of the appropriate portion of the original. The process is replicated via heating and cooling resulting in doubling the number of DNA copies. Following the 30 to 40 cycles, an individual DNA strand is reproduced to hundreds of millions.

 

DNA Sequencing image

The number one drawback to this process was that it was slow and time consuming. Thus, Cetus scientists researched processes to automate everything. This led to the creation of the initial thermocycling machine.

“Mr. Cycle” was invented by Cetus engineers to fulfill the requirement of injecting a fresh enzyme to individual test tubes following the heating and cooling course. The Taq polymerase purifying process culminated in the ability for a vehicle to cycle quicker between contrasting temperatures.

The DNA Thermal Cycler was created in 1985 following a partnership between Cetus the Perkin-Elmer Corporation. Because of both the popularity and quantifiable results of the process, in 1989 Cetus agreed to partner with Hoffman-LaRoche in the creation of PCR testing technology for human diagnostic products.

Polymerase Chain Reaction testing has changed the face of current research, specifically the analysis of genetic defects including the AIDS virus in human cells. This process is extensively utilized by criminologists utilizing blood and hair samples though and solving crimes through DNA comparison. PCR testing is heavily relied upon in the study of evolution resulting from vast quantities of DNA that can be manufactured from fossils.

What is PCR testing?

Polymerase Chain Reaction is otherwise referred to as PCR or molecular testing. This is due to the speed of the process to magnify and replicate minute segments of DNA strands making it an extremely affordable testing process.

By utilizing the enzyme DNA polymerase together with the raw material and individual molecule of DNA,  multiplication to thousands in a few short hours is possible. PCR amplification is the most crucial aspect of the process as it enables the research of large amounts of DNA in both research and genetic studies.

For purposes of Apollo-Laboratory, we utilize the PCR process in the the detection of pathogens for the diagnosis of infectious diseases.

In the next sections, we’re going to get into more detail regarding the PCR process. But for the time being, we need to know that it consists of three steps:

  1. Denaturation
  2. Annealing
  3. Extension

At the completion of each of these stages, the number of DNA molecules doubles and continues through many cycles resulting in large numbers of copies.

The steps of PCR testing

The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with co-factors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.

The basic steps are:

 

PCR-testing-of-DNA-Samples

Denaturation (96°C):

A mixture is created, with optimized concentrations of the DNA template, polymerase enzyme, primers, and dNTPs. The ability to heat the mixture without denaturing the enzyme allows for denaturing of the double helix of DNA sample at temperatures in the range of 94 degrees Celsius.

When DNA is copied, the strands have to separate. Because this looks like a zipper unzipping, we call the process ”unzipping”. More scientifically, the process of DNA strands separating is called denaturation, because it’s no longer in its natural state. Heat can disrupt the DNA’s hydrogen bonds and lead to denaturation. Because heat is involved, this process of turning double-stranded DNA into single strands is sometimes referred to as ”DNA melting.”

Heat the reaction strongly to separate, or denature, the DNA strands. To amplify a segment of DNA using polymerase chain reaction, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called “Taq polymerase” synthesizes – builds – two new strands of DNA, using the original strands as templates.

This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on.

This provides single-stranded template for the next step.

Annealing (55-65°C):

Following denaturation, the sample is cooled to a more moderate range, around 54 degrees, which facilitates the annealing (binding) of the primers to the single-stranded DNA templates.

The process of two strands of DNA rejoining is referred to as annealing. Annealing happens when temperatures drop or return to a level where DNA can be in its natural state.

Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.

Extension (72°C):

In the third step of the cycle, the sample is reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for elongation. During elongation, DNA polymerase uses the original single strand of DNA as a template to add complementary dNTPs to the 3’ ends of each primer and generate a section of double-stranded DNA in the region of the gene of interest.

PCR is only going to amplify a certain region of DNA, not the entire DNA template. The primers represent the starting point for the next step, called the extension step. During the extension, or elongation, step, Taq polymerase binds to each PCR primer and begins adding nucleotides. Note that Taq, like human DNA polymerase, can only add DNA nucleotides in one direction.

This is the reason why you need two primers that are located on either side of the region you wish to amplify. Each primer is oriented to amplify one of the complementary strands. The extension step is so named because the product of PCR continues to extend during this step.

Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

 

 

PCR Cycling:

The cycle of denaturing and synthesizing new DNA is repeated as many as 25 to 40 times, leading to more than one billion exact copies of the original DNA segment. The entire cycling process of polymerase chain reaction is automated and can be completed in 2-4 hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.

That’s because it’s not just the original DNA that’s used as a template each time. Instead, the new DNA that’s made in one round can serve as a template in the next round of DNA synthesis. There are many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can roughly double in each round of cycling.

Gene copies are made using a sample of DNA, and the technology is good enough to make multiple copies from one single copy of the gene found in the sample. PCR amplification of a gene to make millions of copies, allows for detection and identification of gene sequences using visual techniques based on size and charge (+ or -) of the piece of DNA.

Applications of PCR testing

Environmental microbiology:

PCR contributes to our understanding of many environmental issues, particularly where the detection of microorganisms in the environment is required. PCR allows specific target species to be identified and quantified, even when very low numbers exist. One common example is searching for pathogens or indicator species such as coliforms in water supplies.

The PCR technique has been successfully used to explore many issues in environmental microbiology. Some of its environmental applications are listed below:

Sensitive detection of degrading microorganisms in toxic waste and pollutants can be achieved using PCR, which helps efficient biodegradation and bioremediation at the polluted sites.

A gene probe-based PCR testing method has been developed by researchers for the detection of indicator bacteria such as coliforms in water supplies, thus supporting measures that enhance water safety.

Polymerase chain reaction is also used to detect and monitor water-borne microbial pathogens, which pose a major public health hazard.

Consumer genomics:

PCR has enabled personalized genome testing. An industry has sprung up offering consumers tailor-made products and services based on information in their genome. For example, nutrigenomics is a specific form of consumer genomics linking genetic information to information about foods that might be better or worse for particular conditions, like inflammatory bowel disease.

Forensic science:

DNA Fingerprinting

PCR is very important for the identification of criminals and the collection of organic crime scene evidence such as blood, hair, pollen, semen and soil. DNA fingerprints (also called DNA profiles), identification of familial relationships, genomic DNA isolation and other molecular diagnostics and biochemical analyses can be undertaken forensically through the use of PCR. PCR allows DNA to be identified from tiny samples – a single molecule of DNA can be enough for PCR amplification.

With the advent of PCR-based DNA fingerprinting, PCR became an invaluable tool in forensic investigations.

Using DNA fingerprinting, tiny fragments of DNA can be isolated from a crime scene and compared to a huge database of DNA of convicts or criminals. It is also useful in ruling out suspects as part of an investigation. DNA fingerprinting is also used in paternity testing, where the DNA from an individual is matched with that of his possible children, siblings, or parents.

Medicine:

PCR has enabled valuable developments in several medical disciplines. For example, it is now used to diagnose and therefore aid in the treatment of many diseases, and it is widely used in research into the diagnosis, treatment and potential cure for a range of many others.

PCR triggered many valuable developments in several medical disciplines.

Microbiology:
PCR is a highly valuable technique in microbiology as it allows crucial observations for organism detection. Organisms such as Mycobacterium tuberculosis can be studied effectively with the help of genotyping. This allows early identification and treatment and greatly impacts public health monitoring.

In virology, PCR helped detect and characterize the nucleic acids of viruses, which enabled comprehensive viral characterization and a greater understanding of the virus behavior during infection. This understanding immensely helped clinical treatment and enhanced further research on the viruses. For instance, PCR is used to detect HIV infection at an early phase even before the antibodies are formed. This is also useful for screening blood samples collected for donation.

Mycology and Parasitology:
PCR technology has also found applications in mycology and parasitology, by enabling early identification of the microorganisms, thus aiding efficient diagnosis and treatment of fungal and parasitic infections.

Dentistry:
The PCR technique has become a standard diagnostic and research tool in the field of dentistry. PCR and other molecular biology techniques enable the diagnosis of infectious microbes that cause maxillofacial infections. This helps in the effective management of conditions such as periodontal disease, caries, oral cancer, and endodontic infections.

Minute quantities of DNA, including ancient DNA, from sources such as hair, bones and other tissues can be amplified using PCR. The DNA can then be identified and analysed, and genomes can be sequenced. These processes allow scientists to further their knowledge and understanding of evolution and paleontology. Genome sequencing Identifying the sequence of bases in the genome of an organism.

It can also aid in phylogenetic studies, leading to greater understanding of organisms’ evolutionary relationships to each other. This information can be useful to scientists in supporting conservation efforts, studying evolution and understanding unique adaptations.

Scientists-Putting-DNA-into-centrifuge image

Food and Agriculture:
Genetic technologies include a range of techniques that enable the modification of existing organisms for the purpose of improving foods and food production. Of course, selective breeding has been around for centuries, but now the genetic code can be altered deliberately.

Genetic Research:
There are many fields of genetic research that use PCR as an essential tool. The majority of these technologies have multidisciplinary applications. These include the creation, detection and monitoring of genetically modified organisms (GMOs), genetic engineering, gene modification, transgenics, cloning, hybridisation, synthetic biology and directed evolution.

PCR has revolutionized scientific research, ever since it was first presented to the outside world in the 1980s. Some of its specialized applications in genetic research are:

  • Rapid amplification of tiny fragments of DNA using PCR enabled several techniques such as southern or northern blot hybridization even when the amount of sample material available was very small.
  • Study of gene expression patterns is another common application of PCR, where in cells or tissues are analyzed in different stages to check for expression of a specific gene. qPCR can be used here to quantitate the level of gene expression.
  • PCR also assists techniques like DNA sequencing using which segments of DNA from an area of interest can be easily amplified to study genetic mutations and their consequences.
  • The Human Genome Project used PCR to indicate the presence of a specific genome segment in a particular clone. This enabled mapping of the clones and collating results from several laboratories.
  • Advanced variants of the PCR technique have been found to be useful in chromosomal analysis techniques that can help in early detection of genetic birth defects in children.
  • PCR augments the traditional method of DNA cloning by amplifying tiny DNA segments for introduction into a vector. By altering the PCR protocol, site-directed or general mutations can be achieved in the DNA fragment of interest.
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