The brain is an amazing organ. Even more amazingly, the human genome contains instructions for building, using, maintaining and repairing this extraordinarily complicated tissue. However, with this complexity comes increased chances that things can go wrong. These issues can manifest as changes in a person’s perception, behavior, mobility – almost anything that affects the way we interact with our world. We are only beginning to understand the interplay between genetic changes and brain function, but it is important for us to do so, both for economic reasons but also for the affected individual and their loved ones.
Beyond sequencing
Analysis of fragment length by CE
Fragment analysis is a highly flexible method that involves the separation of different-sized and differentially labeled DNA fragments by CE. One of the most common methods of generating fragments for analysis is polymerase chain reaction (PCR). Because of the flexibility afforded with the choice of PCR primers, a specifically sized fragment corresponding to a PCR target sequence is straightforward to generate. Along with the ability to label fragments with up to four different fluorophores, researchers have a large degree of flexibility in experimental design. Neuroscience researchers rely on fragment analysis on CE instruments as an important tool for understanding genetic anomalies in neuropathologies.
Different fragment analysis applications
Triplet repeat neurodegenerative diseases
For example, a class of neurodegenerative diseases, known triplet repeat diseases, are linked to expansions of microsatellite repeats found within translated or untranslated gene regions of certain genes (1). The pathology often arises when the repeat sequence, which in normal alleles can range from 15 to 40 repeats, expands to exceed a disease specific threshold, usually greater than 45 repeats. The disease severity may worsen from generation to generation due to de novo germline expansion of repeats. It is therefore critical to monitor the repeat length of these microsatellite sequences, especially in individuals who are at-risk.
Polymerase chain reaction followed by fragment length analysis is the most common – indeed, the preferred – method to analyze changes in repeat length in these diseases. Fragment analysis by CE has been recommended by the European Molecular Quality Genetics Network (EQMN) as the default analytical method for analyzing triplet repeat length in spinocerebellar ataxias (SCAs, 2) and Huntington disease (3). Fragment analysis continues to be an important tool as protocols are developed to make use of new information (for example, 4). Johnson et al. used fragment analysis to characterize the CTG repeat length in the DMPK gene in myotonic dystrophy type 1 from dried newborn blood spots (5). Another study (6) used fragment analysis to confirm that repeat expansions in C9orf72, ATX1 or ATX2 are risk factors for amyotropic lateral sclerosis (ALS) in a Hungarian population.
Kim et al. described CAG repeat length expansion in the atrophin 1 (ATN1) gene in a patient with both dentatorubral-pallidoluysian atrophy (DRPLA) and Parkinson disease (7). And triplet repeat expansions were screened in 1620 hereditary ataxia cases and family members in a study of Turkish patients (8).
Analysis of gene length variations in other types of disease
Repeat length variations in microsatellite sequences can also be useful markers for other neuropathologies. For example, a study of Czech subjects found that deletions, detected by fragment analysis, could be part of genetic anomalies found in hereditary neuropathies (9). Finally, in a study published by Moran et al, a genetic addition risk score (GARS) for individuals at-risk for drug and alcohol addiction was developed that made use fragment analysis in five genes as part of their panel (10).
Detecting pathogens in neurological samples
The flexibility of fragment analysis is also illustrated by making use of it as a tool for multiplexed PCR (11). A recent study made use of a multiplex PCR panel capable of detecting 18 different pathogens to analyze cerebrospinal fluid samples for viral encephalitis (12). Panels like these are extremely valuable to researchers, since the number of targets can be maximized using a limited amount of sample. The authors conclude that “…this assay is less laborious and [more] accurate…” and can be a useful tool for detecting viral encephalitis.
Fragment analysis: an invaluable tool for neuroscience research
Whether it is analysis of triplet repeat lengths, indel detection in coding sequences, microsatellite allele length linkage analysis, or multiplexed pathogen detection, these and other studies have shown that the flexibility and ease-of-use of fragment analysis makes it a valuable addition to the genetic analysis toolbox for neuroscience researchers.
We have provided some examples of how fragment analysis can be useful for understanding the function and dysfunction of neuronal cells. However, this is not a complete or comprehensive list. Other methods and examples are described in the Fragment Analysis User’s guide, linked below. The flexibility of fragment analysis assays and their applications to research may be limited only by the investigators’ imagination and experimental creativity.
Download the Fragment Analysis User’s Guide here
Fragment analysis has been applied in a number of fields, including oncology, infectious disease, rare and under-diagnosed genetic diseases, and neuroscience. While techniques such as Sanger sequencing and next-generation sequencing (NGS) are also used, fragment analysis offers the accuracy of capillary electrophoresis combined with faster, simplified workflows and multiplexing capabilities.
In this guide we provide thorough coverage of the principles of fragment analysis technologies, the basics of multi-color capillary electrophoresis, the steps in the various workflows, required materials, experimental design strategies, and pre-developed solutions for some commonly-used applications.
For more information on how a range of Applied Biosystems technologies can help you with your neuroscience research, visit thermofisher.com/abcomplexdisease.
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