b'Fragment analysis methods and tools by DNA MMR; however, when deficiencies in the DNA MMR Fragment analysis is a highly flexible method that can be appliedsystem are present, microsatellite replication errors accumulate to a wide variety of research fields. The flexibility afforded within the genome [3]. In addition to its prognostic value for certain the choice of PCR primers (PCR being a necessary first steptypes of cancer, including colorectal [4] and endometrial, MSI in any fragment analysis experiment) means that a specificallycan also serve as a predictive biomarker for immune checkpoint sized fragment corresponding to a PCR target sequence istherapy response [5]. straightforward to generate (Figure 3). Coupling this with theThe Applied Biosystems TrueMark MSI Assay interrogates ability to label fragments with up to four different fluorophores,13 mononucleotide MSI markers. Eight of these markers are researchers have great flexibility in their fragment analysisderived from literature and guidelines from the National Cancer experimental design. Below, we describe some examples ofInstitute [6], and the other five markers were internally identified how fragment analysis can be used in common cancer researchfor monomorphism and high sensitivity in multiple cancer types. problems.The assay also contains two sample identification markers to Mutation detection determine sample mix-up or contamination [7]. To facilitate Oncology researchers frequently need to investigate or verifyanalysis of MSI data, we developed TrueMark MSI Analysis a limited number of variations in one or more genes of interest.Software. This is a desktop-based software that takes the The Applied Biosystems SNaPshot Multiplex System is afragment analysis files of the TrueMark MSI Assay and provides versatile and economical method to perform SNP genotyping.an easy-to-interpret analysis of the results. A groundbreaking Up to ten SNP markers (on different genes) can be investigatedfeature of the software is a proprietary algorithm that can analyze simultaneously by using PCR amplification, followed by dideoxysamples without the need for running normal sample controls singlebase extension (SBE) using an unlabeled primer, andconcurrently, saving time and expense.then capillary electrophoresis of the resulting fragments. AfterLoss of heterozygosity (LOH)electrophoresis and fluorescence detection, the alleles of aMLPA (multiplex ligation-dependent probe amplification) single marker appear as different colored peaks at roughly thetechnology is a flexible technique that is commonly used same size in the electropherogram plot [1]. This technologyto detect aberrations in gene copy number such as LOH. It has been used for simultaneous screening of four hotspotis based on the ligation and PCR amplification of up to 50 mutations in PIK3CA, a gene involved in the phosphatidylinositolmultiplexed pairs of probe oligonucleotides, which hybridize 3-kinase pathway, which plays an important role into the loci of interest. Each oligonucleotide pair is designed cancer pathogenesis [2]. to give an amplification product of a specific length; by using Microsatellite instability assay sequence-tagged ends, all ligated probes can be amplified Microsatellite instability (MSI) is a hallmark of several cancers andwith a single primer pair in a PCR reaction. The forward PCR is characterized by changes to the length of microsatellites dueprimer carries a fluorescent label, allowing for the detection and to defect in mismatch repair mechanisms (MMR). Microsatellitesquantification of size-separated probes on an automated capillary are genetic motifs consisting of 16 base pair repeats. Theseelectrophoresis system [8]. sequences are susceptible to replication errors that can result in deletions and insertions. Normally, these errors are corrected DNA extraction PCR amplication Capillary electrophoresis Data analysisLaserGene A Gene B Gene CGene D Gene EGene F MigratesMigratesLargeSmall slowly quickly fragments fragmentsFigure 3. Basics of a fragment analysis assay. PCR primers are designed such that different target sequences are amplified with primers with different fluorophore labels, and that will generate different sized amplicons. Following PCR, the amplicons are electrophoretically separated by size in a capillary. A laser excites the fluorophores as the fragments migrate through the capillary. The size and color of the resulting fragments reflects the abundance of the target sequence in the sample.Contents 16'