Mutation detection by Real- Time PCR

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      Written by:SwetaYadav  

Introduction:

 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 ideally suited for analysis of single nucleotide polymorphisms (SNPs) present in genetic disorders and has been increasingly used for this purpose since the advent of real-time PCR and as whole genome sequences have become available. It requires methods that are rapid, sensitive, specific and inexpensive, and several real-time methods have evolved which fulfil these requirements. Several methods exist for detection of somatic mutations by real-time PCR. These methods include use of allele-specific competitive blocker PCR, blocker–PCR, realtime genotyping with locked nucleic acids, restriction enzymes in conjunction with real-time PCR, and allele-specific kinetic PCR in conjunction with modified polymerases. Additional methods include ARMSPCR, TaqMAMA, and FLAGPCR. These methods require either the use of modified bases, special enzymes, or additional proprietary reagents or procedures. We wished to develop a simple, robust, highly sensitive, and selective method that is compatible with standard processes used for gene expression analysis by real-time RT-PCR. The variability of nonspecific amplification typically requires a process of trial and error when designing highly selective mutation assays.

 Principle of Mutation Detection: 

The detection of mutations is a fast growing field of increasing importance for many areas of science including diagnosis of human disease, pharmacogenetics, drug development and microbiology. Mutations are classed as one or more changes in the DNA bases and can include large re-arrangements such as translocations, inversions and gene insertions/deletions or small alterations such as point mutations and base insertions/deletions (SNPs). SNPs are the commonest type of DNA sequence variations and can occur once every 100-300 bases. As the requirement for rapid, reliable, sensitive and inexpensive methods for SNP detection grow the number of techniques available also increases, each with inherent strengths and weaknesses. All of the available platforms are semi-automated and none require additional post-PCR handling for example, agarose gel analysis. Depending on the platform used real-time PCR is suitable for low to medium sample throughput. The greatest advantage of these systems is the quality of the data that is generated, known mutations are easily detected and there are possibilities with some of the systems to detect new mutations. Due to increased demand there is now a number of commercially available tests developed, especially for the Applied Biosystems Sequence Detection Systems and the LightCycler.

Fig 2: Diagram illustrating the assay method

Applications of Real-Time PCR for Mutation Detection: 

As the ability to generate huge amounts of sequence data develops, information on sequence changes increases and this can be exploited for many purposes such as clinical diagnostics and microbiology. Molecular methods for SNP detection have been available for some time, however most of these methods are time consuming and cumbersome to perform. The development of real-time PCR has led to an explosion in cheap and rapid mutation detection methods. The applications in disease diagnostics for Factor V Leiden and Cystic Fibrosis will be explored, as well as the detection of antibiotic resistance mutations in bacteria.

Conclusion:

 As a result of whole genome sequencing projects there has evolved a need to be able to accurately detect mutations in a cost-effective, rapid and simple approach. Real-time PCR is suited for this approach and a number of different systems, both platforms and chemistries, have been evaluated for this purpose. Traditional methods that were used included restriction enzyme digestion, sequencing, single-stranded conformation polymorphism (SSCP) and specific PCR followed by direct hybridization. Most of these methods are time consuming, slow and can often be expensive to perform especially when a large number of mutations are to be analyzed. Other new technologies have been applied to mutation detection and some may offer advantages over real-time PCR. Microarrays have been applied to mutation detection and allow numerous sequences to be analyzed in parallel. Microarrays consist of amplifying PCR products that are either attached to the surface of a slide and are interrogated with specific probes or alternatively the specific probes are attached to the slide and the PCR products hybridized to them. In this way mutations can be detected in a large scale format. The major advantage of this method is the ability to scan for large numbers of mutations in one experiment. However, at present it is difficult to distinguish all polymorphisms and difficult to detect low-level changes (Kirk et al., 2002). Sequencing has always had a role in mutation detection because of its accuracy and robustness although it is a time consuming and expensive method. Real-time PCR offers a method for mutation detection that is extremely flexible, a large number of different approaches can be applied depending on the target of interest. The methods are all semi-automated and offer low to medium throughput depending on the platform used. The methods are very accurate in identifying the mutation and all platforms offer a close-tubed system that minimizes cross-contamination in the laboratory. Additionally, the methods are relatively inexpensive and are easy to perform with simple analysis of results.

Reference: 

1. doi:10.1371/journal.pone.0004584.g001 

2. John Morlan, Joffre Baker, Dominick Sinicropi, Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method. Genomic Health, Inc., Redwood City, California, United States of America. 

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 6. What is Real-Time PCR (qPCR)? | Bio-Rad Laboratories