User:OganM/Denaturation mapping

$I(s)=I_{ds}p_{ds}(s)+I_{ss}*[1-p_{ds}(s)]$

{{I(s)=I_{ds}p_{ds}(s)+I_{ss}*[1-p_{ds}(s)]}}

Modern application of denaturation mapping: DNA is stained with a dye(green) that dissociates from single stranded DNA, changes in the environment like temperature increases that are not enough to completely dissociate the molecule causes parts of it to dissociate in a predictable manner from AT rich regions (red). Phosphorescence emissions from this partially denatured molecule is measured with a lens. The graph shown is a simplified output of such an analysis.

Denaturation Mapping is a form of optical mapping, first described in 1966 that is used to characterize DNA molecules without the need for amplification or sequencing. It is based on the differences between the [[Nucleic acid thermodynamics |melting temperatures]] of AT-rich and GC-rich regions. Even though modern sequencing methods reduced the need for denaturation mapping, it is still being used for specific purposes, such as detection of large scale structural variants.

Methodology

When subjected to denaturing factors like increased heat or chemicals like formamide in low levels, DNA is partially denatured in a predictable pattern based on its nucleotide content in different regions. This allows unique fingerprints or ‘barcodes' to be generated for molecules with different sequences not unlike restriction mapping. In the earliest forms of denaturation mapping, denatured molecules are fixed with formaldehyde or glyoxal and visualized using electron microscopy. Ethidium Bromide, a fluorescent molecule that intercalates into duplex DNA, is used to monitor the extend of denaturation. More recently microfluidics are also used for denaturation mapping of single molecules . Nanofluidic channels can be used to stretch DNA linearly, which is then denatured with heating and formamide. If the DNA is stained with dye that unbinds in the case of melting, then fluorescence microscopy can be used to detect denatured regions

Uses

Main advantage of denaturation mapping is that large-scale organization of the genome is left intact during process.That means long-range structural variants can be detected easily. For detection of large structural variants, it is possible to use this technique to make alignments on these barcodes that will enable one to determine the origins of fragments. For instance it was possible to demonstrate this on T4GT7 DNA that circularly permutates in individual viruses .

Furthermore, fingerprints generated by denaturation sequencing can be used as a reference for de novo assembly of shotgun sequencing data. First round of de novo assembly produces hundreds to thousands of contigs, which can be computationally profiled in terms of their melting temperature. These profiles can be compared to results of an actual denaturation experiment to map the contigs.

Notes

Might be useful in assembly (scaffold for shotgun) genetic heterogeneity (not likely though requires too much change on a region) Detect large variations described in 1966? melting calculations from 2010 paper formamide denaturant AT rich melts easier

References

Innman, Ross B (1966), ‘A denaturation map of the λ phage DNA molecule determined by electron microscopy

External links

• example.com