A novel and rapid method for molecular diagnostics uses.
The worldwide molecular diagnostic market was worth $9 billion in 2009. It is estimated that the market will expand to $15.5 billion in 2014 and $42.5 billion in 2019. DNA sequencing is emerging as one of the most promising contributors to growth in this market. The identification of disease-associated mutations/variations has greatly increased owing to advancements in genomic sequencing. Therefore, sequencing of multiple discontiguous DNA segments has become necessary for the diagnosis of a particular disorder. However, current methods for the simultaneous analysis of multiple DNA fragments suffer from low specifity and lengthy processing times. The present technology offers a quick and simple means to obtain a polynucleotide sequence that is composed from multiple genomic loci.
· Assessment of the presence of a disease.
· Determining if the subject is a carrier for a particular disease (e.g. in prenatal genetic testing).
· Determining a predisposition to a disease.
· Classifying a disease (e.g. companion diagnostic to determine which tumors contain genetic alterations of the target)
· Determining a severity of disease (grade or stage).
· Monitoring disease progression.
· Forecasting an outcome of the disease and/or prospects of recovery.
· The method can be useful in the analysis of genetic variations of an individual that have an effect on drug metabolism, drug interactions, and the responsiveness to a drug or to multiple drugs (i.e. pharmacogenomics).
· Monitoring food, liquids or air samples for the presence of an undesired biological contamination.
· The technology can also be applied be used to confirm or identify the relationship of a DNA of unknown sequence to a DNA of known origin or sequence (e.g. in the field of forensic science).
· In contrast to a classical multiplex PCR reaction, the current invention produces a single PCR product, which can then be sequenced with a single primer.
· In contrast to a classical multiplex PCR reaction, no undesired intermediate sequences are generated using the present technology.
· The method is flexible and can be modified to accommodate various diagnostic applications
The present invention – namely the Linker-Induced Overlapping Recombination PCR method - enables the promotion of an endonuclease-independent, single-step, PCR procedure to aid in disease diagnosis. The method allows for amplification of a DNA product that comprises a multitude of linked fragments, each fragment being indicative of a disease. By combining just diagnostically relevant fragments into one polynucleotide fragment, the amount of sequencing may be significantly reduced. The method may be particularly applicable to prenatal genetic testing whereby a multitude of diseases can be detected and diagnosed by a single reaction. The underlying rational is based on a hierarchical PCR reaction in which by using an additional primer in a limited concentration an intermediate product is generated to a limited amount. The intermediate product then serves both as template and primer for the consecutive reaction that produces the desired end-product which is then amplified by flanking primers. Although all the ingredients are present in the PCR tube at onset of the reaction, the process proceeds through predefined steps guided by the concentration and complimentarity of the different primers included.