µLAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5 minutes

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.

The Need for Establishing a Universal CTG Sizing Method in Myotonic Dystrophy Type 1

“…It would be interesting to determine how novel, recently described technologies for CTG sizing [11,12] compare to the methods we assessed here. The sizing kit used by Leferink et al, was based on tripled repeat primed PCR, which is a robust and accurate technique to determine the presence of a CTG expanded allele [11].…”

“…Thus, although the kit reported in the Leferink et al study would seem very useful for accurate DM1 diagnosis, it would not be suitable to size CTG expansion. In the study by Malbec et al, repeat sizing was performed with a lab-on-chip system that concentrates, separates, and detects DNA fragments in a very short time (actually, less than 5 min) from femtomolar concentrations of PCR-amplified DNAs [12]. Although this system appears as a good alternative to the sizing methods that we assessed, its accuracy would depend on the design of the primers used and the PCR amplification cycle.…”

“…Indeed, the number of CTG repeats can be inversely and directly related with age of disease onset and clinical severity, respectively [9,10]. Although different approaches have been described to assess CTG expansion size in patients with DM1 [5,[11][12][13][14][15], some methodological issues remain to be solved, mainly related to the inherent repeat instability and technical difficulties when amplifying long CTG fragments.…”

DM1 Phenotype Variability and Triplet Repeat Instability: Challenges in the Development of New Therapies

“…Polymerase chain reaction (PCR) amplifications are difficult for a high number of CTG repeats, and Southern blotting and triplet-primed (TP)-PCR do not allow accurate CTG number measurements. Although optimized protocols have been developed more recently, precise determination of repeat numbers is limited to~1000 CTG repeats [16,17]. In addition, age at the time of diagnosis and somatic instability in the blood are confounding factors that can bias correlation studies [18].…”

Advances in Continuous Microfluidics-Based Technologies for the Study of HIV Infection

“…Here, we focus on continuous microfluidics, which has been less described than the well-known droplet techniques [ 2 , 3 ]. Due to rapidity, low volumes, precise control, multiplexing, and automation, continuous microfluidics is attractive in a vast spectrum of research works [ 4 , 5 , 6 , 7 ]. Microfluidics encompasses the Micro-Total Analysis System known as µ-TAS or “Lab-on-a-Chip” that consists of the integration of multiple functions in a single microfluidic chip.…”