Molecular Combing

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Molecular Combing (engl., Synonym chromosome combing , to about German, molecular combing ') is a biochemical and biophysical methods for alignment and stretching of polymers such as chromosomal DNA .

principle

The chromosomal DNA is attached to a surface, e.g. B. by hybridization to attached oligonucleotides or by covalent attachment to a silanized surface, e.g. B. a cover slip . By lowering a meniscus at a speed of about 300 μm / s, the DNA is aligned. The DNA preferably remains in the liquid phase. The tensile forces of the sinking meniscus are less than the forces of hybridization, which is why the DNA remains bound to the surface and is aligned and stretched. The DNA is stretched to about 2  kb / μm (150% of the contour length of a random coil , due to a force of 65 pN), which results in improved resolution in the following microscopic observations.

The molecular combing is, inter alia, in fluorescence in situ hybridization used (FISH). Molecular combing results in a resolution of 300 bp in the FISH .

Applications

Due to the improved resolution, FISH z. For example, the speed of the replication fork during the incorporation of BrdU can be measured, the length of DNA can be determined more precisely or unstable chromosomes (e.g. in cancer ) can be examined.

Individual evidence

  1. ↑ a b A. Bensimon, A. Simon, A. Chiffaudel, V. Croquette, F. Heslot, D. Bensimon: Alignment and sensitive detection of DNA by a moving interface. In: Science. Volume 265, Number 5181, September 1994, ISSN  0036-8075 , pp. 2096-2098, PMID 7522347 .
  2. ^ A b R. Lebofsky, A. Bensimon: Single DNA molecule analysis: applications of molecular combing. In: Briefings in functional genomics & proteomics. Volume 1, Number 4, January 2003, ISSN  1473-9550 , pp. 385-396, PMID 15239885 .
  3. ↑ X. Michalet, R. Ekong, F. Fougerousse, S. Rousseaux, C. Schurra, N. Hornigold, M. van Slegtenhorst, J. Wolfe, S. Povey, JS Beckmann, A. Bensimon: Dynamic molecular combing: stretching the whole human genome for high-resolution studies. In: Science. Volume 277, Number 5331, September 1997, ISSN  0036-8075 , pp. 1518-1523, PMID 9278517 .
  4. ↑ a b N. Ohmido, K. Fukui, T. Kinoshita: Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH). In: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences. Volume 86, Number 2, 2010, ISSN  1349-2896 , pp. 103-116, PMID 20154468 , PMC 3417561 (free full text).
  5. ↑ C. Conti, B. Saccà, J. Herrick, C. Lalou, Y. Pommier, A. Bensimon: Replication fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells. In: Molecular biology of the cell. Volume 18, number 8, August 2007, ISSN  1059-1524 , pp. 3059-3067, doi : 10.1091 / mbc.E06-08-0689 , PMID 17522385 , PMC 1949372 (free full text).
  6. ^ J. Herrick, A. Bensimon: Introduction to molecular combing: genomics, DNA replication, and cancer. In: Methods in molecular biology (Clifton, NJ). Volume 521, 2009, ISSN  1064-3745 , pp. 71-101, doi : 10.1007 / 978-1-60327-815-7_5 , PMID 19563102 .