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Mutation Detection Methods using PCR

PCR based methods can be used for the detection of single point mutations (SNPs) present in genetic disorders


The detection of mutations in the genome or transcriptome is of great importance for the diagnosis of genetic disorders, as well as pre-symptomatic testing, conformational diagnosis as well as forensic identity testing. To detect genetic syndromes, two groups of tests are available, molecular and cytogenetic tests.


After the identification and definition of mutations, diagnostic methods or tests can be used to find them using techniques such as allele-specific oligonucleotide hybridization, allele-specific amplification, ligation, primer extension and the artificial introduction of restriction sites.


PCR allows mutation detection, however, PCR itself does not detect the actual mutation. PCR generates an amplicon that is then analyzed by some other method to find possible variations within the amplicon. PCR based methods only detect mutations that have been previously identified by some other techniques if now sequencing step is added.

Real-time PCR is well suited for analysis of single nucleotide polymorphisms (SNPs). Real-time PCR detects SNPs unique to human diseases and is a valuable technique in pharmacogenetics, clinical microbiology and drug development in comparison to methods that use sequencing, single-strand conformation polymorphism, and restriction digestion.

Modern real-time PCR based methods have now become more rapid, sensitive, specific and inexpensive. Real-time PCR monitors the exponential phase of PCR using fluorescently labeled molecules. The PCR amplicon amount present in a reaction tube is directly proportional to the amount of the starting material specific to the PCR primer pair during the exponential phase. Therefore, the amount of emitted fluorescence is directly proportional to the amount of amplicon. This amount is also proportional to the starting amount of the target sequence allowing measurement of the target copy number.

PCR based methods

Real-Time PCR

Real-time PCR using nonspecific DNA-binding dyes

Real-time PCR using labeled probes