HOME

TheInfoList



OR:

BEAMing, which stands for beads, emulsion, amplification, magnetics, is a highly sensitive
digital PCR Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands includi ...
method that combines emulsion PCR and
flow cytometry Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow ...
to identify and quantify specific somatic mutations present in DNA.


Process

BEAMing begins with the isolation of DNA from a patient’s blood or plasma sample. Target regions of the purified DNA undergo a pre-amplification step with conventional PCR utilizing primers of known sequences to amplify the genetic regions of interest. The amplified DNA templates are then introduced to primers that are covalently bound to magnetic beads via
streptavidin Streptavidin is a 66.0 (tetramer) kDa protein purified from the bacterium ''Streptomyces avidinii''. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation c ...
-
biotin Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...
interactions and are compartmentalized into aqueous microdroplets of a water-in-oil
emulsion An emulsion is a mixture of two or more liquids that are normally immiscible (unmixable or unblendable) owing to liquid-liquid phase separation. Emulsions are part of a more general class of two-phase systems of matter called colloids. Although ...
. The aqueous phase is emulsified with the oil, creating millions of individual water droplets having a diameter of 3-10 microns. Within each droplet, a separate PCR reaction is performed. Due to the small size, each water droplet contains on average a single DNA molecule and a magnetic particle. In addition to the pre-amplified DNA, each emulsion droplet contains the necessary reagents and sequence-directed primer-coated magnetic beads to carry out the emulsion PCR reaction. The microemulsion droplets are temperature cycled using conventional PCR methods. Each DNA template (with the magnetic bead present in the aqueous compartment) is extended and amplified, resulting in a bead coated with thousands of identical copies of the template DNA fragment. Usually a high-fidelity
DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
is used in order to limit errors normally introduced during PCR. This precaution limits the risk of false-positive detection and enables the accurate discrimination of target molecules. Following the emulsion PCR step, the water and oil phase are separated so that the microparticles can be collected in the aqueous phase. The microemulsion droplets are then broken to release the magnetic beads, which have the amplified copies of DNA attached. The beads are magnetically purified and base pair-specific fluorescent probes are attached. This helps distinguish between wild-type and mutant DNA fragments, as one fluorescent probe binds specifically to the wild-type DNA and the other to specific mutant DNA. Each fluorescently labeled bead is analyzed in a flow cytometer, resulting in a separation of mutant from wild-type DNA as well as the ratio of mutant to wild-type DNA present in a sample. The microscopic emulsion droplets used in BEAMing allow for the compartmentalization of DNA segments into single droplets. Emulsion PCR is run on the compartmentalized DNA, enabling hundreds of millions of PCR reactions to run in parallel. This
massively parallel Massively parallel is the term for using a large number of computer processors (or separate computers) to simultaneously perform a set of coordinated computations in parallel. GPUs are massively parallel architecture with tens of thousands of th ...
PCR platform delivers high levels of sensitivity (.001%) for the detection of rare tumor DNA molecules among a large background of wild-type DNA.


Applications

BEAMing is often used in cancer research to conduct assessments of circulating tumor DNA (
ctDNA Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA in the bloodstream that is not associated with cells. ctDNA should not be confused with cell-free DNA (cfDNA), a broader term which describes DNA that is freely circulating in the bloo ...
), also known as a
liquid biopsy A liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. Like traditional biopsy, this type of technique is mainly used as a diagnostic and monitoring tool for ...
. It also allows for the quantification of a sample’s mutant fraction, which can be tracked over time using serial plasma measurements. The method has a sensitivity threshold of 0.01%.


History

In the late 1990s, Vogelstein and Kinzler coined the term “ digital polymerase chain reaction” when conducting research into somatic mutations associated with and potentially causative for
colorectal cancer Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is the development of cancer from the colon or rectum (parts of the large intestine). Signs and symptoms may include blood in the stool, a change in bowel m ...
. A fundamental challenge that digital PCR was designed to address was the detection of minor quantities of a pre-determined somatic mutation in larger cell populations. While both digital and classical PCR can be used in quantitative or qualitative analyses, digital PCR analyzes samples one molecule at a time to produce an all-or-nothing signal thereby increasing the signal-to-noise ratio and overall sensitivity to rare targets. The results from these studies indicated that digital PCR was able to reliably quantify the relative proportion of variant sequences in a DNA sample. BEAMing grew out of digital PCR technology and in 2003 was described in a ''
Nature Methods ''Nature Methods'' is a monthly peer-reviewed scientific journal covering new scientific techniques. It was established in 2004 and is published by Springer Nature under the Nature Portfolio. Like other ''Nature'' journals, there is no external ed ...
'' publication from Vogelstein’s team. In 2005, Vogelstein’s team published their first clinical data applying BEAMing technology to analyze plasma samples of patients with cancer. In a 2008 ''
Nature Medicine ''Nature Medicine'' is a monthly peer-reviewed medical journal published by Nature Portfolio covering all aspects of medicine. It was established in 1995. The journal seeks to publish research papers that "demonstrate novel insight into disease pro ...
'' publication, BEAMing ctDNA measurements were sensitive enough to reliably monitor tumor dynamics. In 2008, Inostics GmbH formed to commercialize BEAMing. In 2014, Inostics was acquired by
Sysmex Corporation is a Japanese company headquartered in Kobe that is engaged in the health care business. Originally called TOA Medical Electronics (a branch of the TOA Corporation), the Sysmex brand was established in 1978, and were mainly involved with haemato ...
to form Sysmex Inostics.


References

{{Reflist Polymerase chain reaction