Johan Hofkens*, Volker Leen, Doortje Borrenberghs, Kris Janssen

 

Celestijnenlaan 200 F
KU.Leuven
Heverlee, Belgium

 

DNA sequencing methodologies rely on massively parallel DNA sequencing approaches, which sequence short regions of the genome, from 30 up to 1500 bases in length, followed by a computationally-intensive assembly of these fragments into a genomic DNA sequence. This method requires large amounts of DNA and is labour intensive, relative to optical mapping technologies. For example, sample preparation for so-called next-generation sequencing experiments requires a full day to complete. For some experiments, this is a price that is well worth paying. However, single-base resolution of the DNA sequence is often unnecessary, as genomic differences between species (e.g., microorganisms) or structural variations between individuals within a given species (e.g., humans) can be distinguished using lower-resolution mapping approaches. While optical (restriction) mapping is easier than sequencing, it suffers from two important limitations, namely the scale on which information can be obtained and the speed. The scale is limited by the use of enzymes that break the DNA, into fragments of around 10kb in length. Since sequence reads typically run to around a few hundred bases, there is a void in the scale of information that can be derived from these techniques. Genes are typically of the order of 1 kilobase in length but can run up to several tens of kilobases, placing genetic elements exactly within this gap.

An alternative to restriction mapping was developed in our lab1; the so-called DNA FLUOROCODE. In this technique a DNA methyltransferase is used to direct the labelling of the DNA at sequences reading 5’-GCGC-3’ with a fluorescent probe. The DNA is then analyzed using a wide-field fluorescence microscope with sub-diffraction limit localization of the emitters. This technique allows for a much higher labelling density compared to restriction enzymes, and an unparalleled resolution. In this contribution, I will describe the progress that was made with this concept in terms of labelling, surface deposition of DNA, superresolution imaging2, 3 …I also will discuss alternatives that we develop for stretching the DNA on a surface and applications envisioned.

 

1 Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry, Nucleic Acids Res. (2014), 1-10.

2 Methyltransferase -directed labeling of biomolecules and it’s applications, Angew. Chem., 56 (2017), 5182-5200

3 Combing of genomic DNA from droplets containing pictograms of material, ACSNano. (2015) 809-816

 

Biographic Details

Name Johan Hofkens

Title: Professor

Affiliation, Departmetn of Chemistry, KULeuven Country:Belgium

Phone: +32474437708  E-mail: johan.hofkens@kuleuven.be

Research interests: single molecule microscopy, spectroscopy, biotechnology