PCR based technologies:
PCR and its derivatives are widely used at GenePath Diagnostics as diagnostic tools by themselves or as a preparatory step for more complex diagnostic protocols. Real-time PCR assays (typically real-time SYBR Green + melt analysis based assays or TaqMan / 5’ exonuclease based assays) are the most commonly used approaches at GenePath for PCR based diagnostics. These highly quantitative techniques enables amplification of specific regions of interest in nucleic acid molecules (DNA or RNA) and detection in real-time, in a closed tube system. As there is no post-PCR sample handling involved, the risk of amplicon carry-over contamination (which is one of the biggest drawbacks of conventional PCR) and errors associated with human handling are minimized. Our optimized assays using highly efficient detection chemistries and sensitive instrumentation allow for the detection of extremely low amounts of DNA in clinically relevant biological matrices. Typical uses of real-time PCR include pathogen detection, gene expression analysis (comparing mRNA levels, as in the case of certain cancers), single nucleotide polymorphism (SNP) analysis or analysis of allelic variations in genomic regions, and analysis of chromosomal abnormalities such as deletions, inversions and translocations.
Fragment analysis based technologies:
Capillary electrophoresis is used to separate amplified DNA for a variety of different molecular applications. Some of these applications include:
- Multiplex Ligation Dependent Probe Amplification (MLPA):
This variation of PCR enables relative quantification of copy number variations and detection in genomic DNA of up to 50 target sequences simultaneously, with a single pool of probes. The MLPA method – and its close relative Methylation Sensitive (MS) MLPA – are used for detection of copy number variations such as deletions and duplications and changes in methylation patterns in genetic disorders Duchenne Muscular Dystrophy, Prader Willi Syndrome, etc.
- Insertion Deletion (InDel) detection:
Fragment sizes can also be used to detect the presence of larger sized InDels, which are challenging to detect, in genes like CALR, FLT3 and NPM1 associated with hematological neoplasms.
- Triplet repeat disorder detection (TP-PCR):
Triplet repeat disorders are a class of genetic disorders caused by changes in numbers of repeating 3-nucleotide base motifs within genes. These can result in conditions like myotonic dystrophy, Fragile X syndrome and Huntington Chorea. These are challenging to diagnose by conventional PCR and by sequencing techniques. Triplet primed PCR (TP-PCR), a powerful technique that combines PCR with fragment analysis, is used to diagnose patients and carriers of these diseases.
- Microsatellite Instability (MSI):
Microsatellite instability is an effect that results from changes in the lengths of certain polymorphic markers across the genome. The lengths of these markers are used as surrogates for damage to DNA repair genes that are implicated in certain cancers like colon and endometrial cancer. MSI analysis combines PCR with fragment analysis and is used to assist in patient management. Recently MSI data is also being used to predict response to immunotherapy drugs.
- Maternal Cell Contamination (MCC):
When prenatal samples (e.g. amniotic fluid or chorionic villi) are being tested, it is critical to distinguish DNA sourced from the fetus versus DNA from the mother. Maternal cell contamination analysis is the testing of polymorphic genetic markers that serve as unique fingerprints for the fetus and mother using a combination of PCR and fragment analysis.
Sequencing based technologies:
DNA sequencing is used for a variety of applications including genetic testing, oncology testing (both predictive and therapeutic), infectious disease testing, and microbiome (metagenomic) testing. At GenePath we use the following platforms for DNA sequencing:
- Capillary/Sanger Sequencing:
This gold standard method for reading the bases of DNA up to 1000 nucleotides at a time played an essential role in the sequencing of the human genome. This method is useful for smaller genes or localized hotspots of mutations and has been applied for DNA mutation testing in disorders such as cystic fibrosis and beta thalassaemia.
- Next Generation Sequencing (NGS):
These cutting edge methods extend the sequencing process across millions of reactions in a massively parallel fashion, rather than being restricted to a single or a few DNA fragments. The newly generated fragments of DNA are then reassembled like a giant jigsaw puzzle, thus enabling rapid sequencing of large stretches of DNA – ranging from a group of candidate genes or all coding regions in a genome (exome sequencing) to the entire human genome. This technique enables comprehensive analysis of conditions that involve multiple and/or
GenePath’s proprietary CODE-SEQ platform:
GenePath has developed a novel highly-scalable platform, CODE-SEQ, capable of simultaneously detecting multiple genetic aberrations such as single nucleotide variations (SNVs), copy number variations (CNVs) like deletions and duplications, and changes in methylation patterns at multiple sites across the genome in a single assay. The assay may be used to detect variations in a few sites across a large number of samples (e.g. high throughput testing at the scale of populations for conditions such as Spinal Muscular Atrophy) or a large number of loci in a smaller number of samples (e.g. intellectual disabilities).