Abstract
We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.
Highlights
Microfluidic technologies have a demonstrated potential for nucleic acids analysis, genetic testing, and sample preparation[1,2,3,4]
When operating μLAS in a single channel system[8], determining molecular weight (MW) and concentration was time-consuming because the comparison between a fragment of interest and a MW marker had to be performed sequentially
We designed a new microfluidic system that enables the separation, concentration, and sizing of DNA fragments within minutes using miniscule amounts of material. We expect this to be advantageous for routine molecular biology work, as it would save time usually spent during agarose gel electrophoresis
Summary
Microfluidic technologies have a demonstrated potential for nucleic acids analysis, genetic testing, and sample preparation[1,2,3,4]. We applied μLAS to the sizing of expanded trinucleotide repeats using minute amounts of amplified DNA, including samples derived from individuals with Huntington’s disease (HD) and myotonic dystrophy type 1 (DM1). In some instances, when the repeat tract is large, a triplet-primed PCR is required in which one of the primers used anneals within the repeat tract[22] This method has the advantage of being simple and most molecular biology laboratories have the necessary equipment. Care is taken during these procedures, including the use of Southern blotting, to confirm any negative molecular diagnostics This increases the time, labour, and cost of the tests. SP-PCR has the advantage of detecting single alleles and can determine both allele size and the length heterogeneity within a sample It is unaffected (and is blind to) the presence of interruptions. There is a need for novel methods to determine repeat sizes that are sensitive, that provide information about repeat size variation, and that reduce the time and effort required to obtain the results
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