DNA Combing, a technique used in the analysis of single molecules of DNA, offers researchers an opportunity to better understand the dynamics of replication, transcription and individual molecule interactions. While advances in DNA sequencing technology have rapidly enhanced our ability to decode the genome, DNA sequence alone fails to fully explain a cell’s specific proteome. Similarly, ensemble assays, that average DNA characteristics over large populations, fail to address essential differences between individual molecules of DNA. To enable single molecule analysis, a highly dense layer of DNA is immobilized and stretched uniformly. Using DNA Combing, DNA fragments up to 12Mb can be analyzed using various fluorescence imaging techniques.
Plasma Treatment for DNA Combing
DNA combing involves three critical steps, immobilization, alignment and stretching, each of which are enhanced by plasma treatment. Plasma treatment removes nanoscale organic contamination and introduces polar functional groups to the material surface. The hydroxyl groups introduced with plasma treatment react with alkoxy groups on silane, forming strong covalent bonds. In turn, DNA molecules in solution bind vinyl groups (-CH=CH2) of the silane. As a result, the availability of hydroxyl groups on the surface directly impacts the density of DNA molecules immobilized on the material surface. Additionally, the strength of the bonds enables DNA stretching.
Plasma Vs Piranha
The most common alternative to plasma treatment for DNA combing is piranha cleaning, a process that add complexity and safety hazards. Piranha is a mixture of sulfuric acid and hydrogen peroxide that is also used to clean organic residues off substrates and provide a hydroxylated surface. Due to the inherent danger of using Piranha, its use is often limited to clean rooms and trained professionals. In addition, benchtop plasma cleaners are more versatile than chemical treatments, enabling researchers to quickly silanize their substrates following treatment. This may result in more dense silane layers suitable for DNA combing.
Relevant Articles from Harrick Plasma Users
Kaykov A, Taillefumier T, Bensimon A, and Nurse P. “Molecular combing of single DNA molecules on the 10 megabase scale”. Sci. Rep. 2016 6: 19636 10.1038/srep19636
Crut A, Geron-Landre B, Bonnet I, Bonneau S, Desbiolles P, and Escude C. “Detection of single DNA molecules by multicolor quantum-dot end-labeling”. Nucleic Acids Res. 2005 33: e98 10.1093/nar/gni097
Gallo D, Wang G, Yip CM, and Brown GW. “Analysis of Replicating Yeast Chromosomes by DNA Combing”. Cold Spring Harbor Protocols 2016 2016: 90–101 10.1101/pdb.prot085118
Labit H, Goldar A, Guilbaud G, Douarche C, Hyrien O, and Marheineke K. “Silanization of coverslips for DNA combinga”. Biotechniques 2010 11: 50 10.2144/000113002
Park C-Y, Jacobson DR, Nguyen DT, Willardson S, and Saleh OA. “A thin permeable-membrane device for single-molecule manipulation”. Rev. Sci. Instrum. 2016 87: 14301 10.1063/1.4939197