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The digital polymerase chain reaction is the evolutionary refinement of the conventional type of PCR procedure. The method is used to quantify, and clone, and amplify the nucleic acids extracted from the organism. Here, the nucleic acids are RNA, DNA, and cDNA. The major difference between the digital pcr and conventional pcr or rt pcr is the method of measuring the nucleic acids. In a digital polymerase chain reaction, the measurement of nucleic acids is more subtle and is less prone to errors. While in the traditional pcr steps, the measurement of nucleic acids is prone to errors and contaminants. If an inexperienced person handles the pcr procedure, she or he can make any kind of mistake that would affect the pcr amplification process drastically. But in a digital polymerase chain reaction, the person handling the steps is not considered important. As the chance of errors is nil and the pcr amplification can also happen in a good way.
It is observed that a digital pcr measurement of the nucleic acids is quantitative and discrete. They measure a certain variable at a time with the help of analog measurements. Here, the measurements are made per the DNA patterns that prevailed in the monitor screen. The traditional polymerase chain reaction carries out one reaction at one single time. But dpcr carries a single PCR reaction into various parts which can be seen on the screen. This whole purpose of separation is to pave the way for studying the variations among the amplifications of Pcr.
A digital polymerase chain reaction is considered a revolutionary step in the scientific world. It precisely quantifies the nucleic acids with precision. Digital pcr is called the third generation of the traditional polymerase chain reaction. DPCR enables the complete quantification of the pcr amplification of PCR. The technique is precise with the measurements. Molecular detection is also done with ease in digital pcr. Rare mutations and copy number variations can also be measured with the digital polymerase chain reaction.
The principle of digital polymerase chain reaction is the same as that of conventional polymerase chain reaction. Here, the quantification of nucleic acid is done with precision. The pcr amplification is done to measure the DNA used. To amplify the genes, the enzyme – DNA polymerase I. With the amplification of DNA, dpcr also works in the comparison of the nucleic acids. The pcr amplification cycles are compared with the digital pcr steps. However, some factors complicate the calculation of comparisons. But they can be outperformed by drawing good techniques of measurement practices.
The factors which affect the DPCR process are exponential amplification cycles and different contaminants. Even the low amounts of DNA amounts can also be a concern of problem. The main principle of digital polymerase chain reaction is the quantitation of the nucleic acids in the pcr reaction cycles. Hence, a good and enough amount of nucleic acids are used in the DPCR process to give out good and high-end results.
In the process of digital pcr, the single reaction of polymerase chain reaction is separated into many sub-steps. Like, one single amplification product is partitioned into ten thousand small products in the dpcr. The nano-liters are used in the process which makes it different from the conventional pcr steps.
The sample is made of TaqMan assay which has the taq polymerase enzyme and fluorescence probes for indicating the DNA and RNA. Some primers and matrix mix are also present in the pcr reaction mixture. The pcr machines have the mixture wells which consists of DNA polymerases, dNTPs, and MgCl2 with some reaction buffers. All these are present in the optimal concentrations in the pcr reaction mixture. The same happens in reverse transcriptase pcr.
For partitioning the amplification products, several methods are used, such as :
After the successful partitions of the DNA samples, the reaction cycles are then made to run with the DNA samples. When the cycles of pcr are completed, the samples are checked for fluorescence with the help of a binary number of “0” or “1”. Now, the fraction is calculated out and data is recorded. The partitioning of samples gives space for assuming the Poisson distribution. Using the mentioned distribution, one can very accurately calculate the number of droplets formed after running the Pcr cycles.
The above model easily predicts the number of samples containing at least one of the target molecules to increase. Here, the probability of getting a target sequence is increased with the digital polymerase chain reaction.
The major advantage of using the digital polymerase chain reaction is the precision of data is given out. Dpcr uses the massive DNA sampling partitioning that backs the reliable measurements of the desired DNA sequence. In the basic pcr reaction, the errors have more chances to prevail, but the digital pcr nullifies the error rate and gives the perfect measurement.
Apart from all this, the digital polymerase chain reaction gives out the extreme quantitation of the DNA samples. This quantitation of the DNA samples is not possible in the traditional or basic PCR steps. Moreover, the dpcr gives a perfect blend of good data and identification of the target DNA samples.
Digital pcr is used for many types of purposes which uses the steps of pcr very extensively. The major areas of applications of DPCR :
Apart from all these, the dpcr is also used for many applications like absolute quantification and copy number analysis, etc.
Digital polymerase chain reaction enables the complete quantification of the target DNA used in the pcr machines. The nucleic acids are present in the single-molecule resolution which are quantified with the dpcr. However, dpcr does not need any curve analysis to represent the target DNA species. In this case, the dpcr has the potential to tolerate the activity of inhibitor substances. qPCR has all the properties of dpcr but lacks the precision at which the target DNA data is represented.
It is used to quantify data in the specific sequences also to get the right and targeted DNA samples. The detection of biomarkers is also done with the digital polymerase chain reaction. The digital pcr combines with reverse transcription pcr to yield better results.
Any alteration in a copy number refers to the single-copy number of the locus is called a copy number variation. If it appears in the germline cells, it appears in the somatic cells too. These copy number variations are the base of variations in the genome of humans. The dpcr works in these data samples with precision to give out the target DNA data.
Conclusion
The digital polymerase chain reaction is the evolutionary step in the history of polymerase chain reaction. Because of its precision in the data sets, it is widely used for many applications. Dpcr is also helpful in retesting next-generation sequencing. Due to the high quality of data generated by the digital polymerase chain reaction, it is useful in all areas of the scientific world.
