Linearization and Labeling of Single-Stranded DNA for Optical Sequence Analysis.
Rajib BasakFan LiuSarfraz QureshiNeelima GuptaCe ZhangRenko de VriesJeroen A van KanS Thameem DheenJohan R C van der MaarelPublished in: The journal of physical chemistry letters (2019)
Genetic profiling would benefit from linearization of ssDNA through the exposure of the unpaired bases to gene-targeting probes. This is compromised by ssDNA's high flexibility and tendency to form self-annealed structures. Here, we demonstrate that self-annealing can be avoided through controlled coating with a cationic-neutral diblock polypeptide copolymer. Coating does not preclude site-specific binding of fluorescence labeled oligonucleotides. Bottlebrush-coated ssDNA can be linearized by confinement inside a nanochannel or molecular combing. A stretch of 0.32 nm per nucleotide is achieved inside a channel with a cross-section of 100 nm and a 2-fold excess of polypeptide with respect to DNA charge. With combing, the complexes are stretched to a similar extent. Atomic force microscopy of dried complexes on silica revealed that the contour and persistence lengths are close to those of dsDNA in the B-form. Labeling is based on hybridization and not limited by restriction enzymes. Enzyme-free labeling offers new opportunities for the detection of specific sequences.
Keyphrases
- single molecule
- atomic force microscopy
- nucleic acid
- living cells
- photodynamic therapy
- high resolution
- genome wide
- high speed
- copy number
- single cell
- binding protein
- circulating tumor
- dna methylation
- loop mediated isothermal amplification
- small molecule
- computed tomography
- cell free
- dna binding
- fluorescence imaging
- pet ct
- circulating tumor cells
- genome wide analysis