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In August 2020, an updated version of the guidelines for the digital PCR method was published to account for improvement in machinery, technologies, and techniques since the original 2013 release. Additional guideline steps were added for data analysis, while also providing a more simplified checklist table for researchers to use. [ 15 ]
The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.
Quantitative PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples. [3] This measurement is made after each amplification cycle, and this is the reason why this method is called real time PCR (that is, immediate or simultaneous PCR).
The design of appropriate short or long primer pairs is only one goal of PCR product prediction. Other information provided by in silico PCR tools may include determining primer location, orientation, length of each amplicon, simulation of electrophoretic mobility, identification of open reading frames, and links to other web resources. [7] [8] [9]
The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR. Multiplex-PCR was first described in 1988 as a method to detect deletions in the dystrophin gene. [1] It has also been used with the steroid sulfatase gene. [2]
MLST databases contain the reference allele sequences and sequence types for each organism, and also isolate epidemiological data. The websites contain interrogation and analysis software which allow users to query their allele sequences and sequence types. MLST is widely used as a tool for researchers and public healthcare workers.
OLIGO Primer Analysis Software is a software for DNA primer design. [1] [2] The first paper describing this software was published in 1989. [3]The program is a real time PCR primer and probe search and analysis tool.
Currently, a lot of PCR and hybridization assays have been approved by FDA as in vitro diagnostics. [47] NGS assays, however, are still at an early stage in clinical diagnostics. [48] To do the molecular diagnostic test for cancer, one of the significant issue is the DNA sequence variation detection.