Digital polymerase chain reaction
Therefore, nucleic acids may be quantified by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample.
Several different methods can be used to partition samples, including microwell plates, capillaries, oil emulsion, and arrays of miniaturized chambers with nucleic acid binding surfaces.
[citation needed] The benefits of dPCR include increased precision through massive sample partitioning, which ensures reliable measurements in the desired DNA sequence due to reproducibility.
Also, dPCR is highly quantitative as it does not rely on relative fluorescence of the solution to determine the amount of amplified target DNA.
It provides absolute quantification because dPCR measures the positive fraction of samples, which is the number of droplets that are fluorescing due to proper amplification.
[67] However, it is difficult to measure these allelic variations with high precision using other methods such as qPCR, thus making phenotypic and disease associations with altered CNV status challenging.
[74][6] Digital PCR has been used to uncover both germline and somatic variation in gene copy number between humans[75] and to study the link between amplification of HER2 (ERBB2) and breast cancer progression.
[76][77][78][27] Partitioning in digital PCR increases sensitivity and allows for detection of rare events, especially single nucleotide variants (SNVs), by isolating or greatly diminishing the target biomarker signal from potentially competing background.
Rare mutation detection occurs when a biomarker exists within a background of a highly abundant counterpart that differs by only a single nucleotide variant (SNV).
Digital PCR has been shown to be capable of detecting mutant DNA in the presence of a 200,000-fold excess of wild type background, which is 2,000 times more sensitive than achievable with conventional qPCR.
[17][80] Researchers have used liquid biopsy to monitor tumor load, treatment response and disease progression in cancer patients by measuring rare mutations in circulating tumor DNA (ctDNA) in a variety of biological fluids from patients including blood, urine and cerebrospinal fluid.
[85] The application of liquid biopsy tests have also been studied in patients with breast,[86] colorectal,[87][88] gynecologic,[89] and bladder cancers[81][90] to monitor both the disease load and the tumor’s response to treatment.
[31] One can often achieve greater sensitivity and precision by using dPCR rather than qPCR to quantify RNA molecules in part because it does not require use of a standard curve for quantification.
[95][96] Additionally, the improved specificity of complex PCR amplification reactions in droplets has been shown to greatly reduce the number of iterations required to select for high affinity aptamers in the SELEX method.
[101] Chip-based Digital PCR (dPCR) is also a method of dPCR in which the reaction mix (also when used in qPCR) is divided into ~10,000 to ~45,000 partitions on a chip, then amplified using an endpoint PCR thermocycling machine, and is read using a high-powered camera reader with fluorescence filter (HEX, FAM, Cy5, Cy5.5 and Texas Red) for all partitions on each chip.
[102] dPCR rose out of an approach first published in 1988 by Cetus Corporation when researchers showed that a single copy of the β-globin gene could be detected and amplified by PCR.
[46] In 1999, Bert Vogelstein and Kenneth Kinzler coined the term “digital PCR” and showed that the technique could be used to find rare cancer mutations.
[110][111][112] In recent years, scientists have developed and commercialized dPCR-based diagnostics for several conditions, including non-small cell lung cancer and Down’s Syndrome.
[113][114] The first dPCR system for clinical use was CE-marked in 2017 and cleared by the US Food and Drug Administration in 2019, for diagnosing chronic myeloid leukemia.
