Chromosome combing
Encyclopedia
Chromosome combing is a technique used to produce an array of uniformly stretched DNA
that is then highly suitable for nucleic acid hybridization studies such as fluorescent in situ hybridisation
(FISH) which benefit from the uniformity of stretching, the easy access to the hybridisation target sequences , and the resolution
offered by the large distance between two probes, which is due to the stretching of the DNA by a factor of 1.5 times the crystallographic length of DNA.
DNA in solution (i.e. has a random-coil structure) is stretched by retracting the meniscus
of the solution at a constant rate (typically 300 γm/s). The ends of DNA strands, which are thought to be frayed (i.e. open and exposing polar groups) bind to ionisable groups coating a silated glass plate at a pH
below the pKa
of the ionizable groups (ensuring they are charged enough to interact with the ends of DNA). The rest of the DNA, which is mostly dsDNA, cannot form these interactions (aside from a few ‘touch down’ segments along the length of the DNA strand) so is available for hybridisation to probes. As the meniscus retracts, surface retention creates a force that acts on DNA to retain it in the liquid phase; however this force is inferior to the strength of the DNA’s attachment; the result is that the DNA is stretched as it enters the air phase; as the force acts in the locality of the air/liquid phase, it is invariant to different lengths or conformations of the DNA in solution, so DNA of any length will be stretched the same as the meniscus retracts. As this stretching is constant along the length of a DNA, distance along the strand can be related to base content; 1 µm is approximately equivalent to 2 kb.
DNA regions of interest are observed by hybridising them with probes labelled by haptens like biotin; this can then be bound by one or more layers of fluorochrome-associated ligands (such as immunofluorescence antibodies). Multicolour tagging is also possible. This has several potential uses, typically as a high-resolution physical mapping technique (e.g. for positional cloning), an example of which was the correct mapping of 200 kb of the CAPN3 gene region, or the mapping of non-overlapping sequences (since the distance between two probes can be accurately measured). It is therefore useful for finding exons, microdeletions, amplifications, or rearrangements. Before the combing improvement, FISH was too low-res to be of use in this case. With this technique, the resolution of FISH is theoretically limited only by the resolution of the epifluorescence microscope; in practice, resolutions of around 2&Nbsp;µm are obtained, for DNA molecules usually 200–600 kb long (though combing-FISH has been used with some success on molecules in excess of 1 Mb long), and there may be room for improvement through optimisation. Since DNA analyses using this technique are single-molecule, we can compare genomes from different cells and find anomalies; the implications for diagnosis of cancer and other genetic alterations are evident.
However, it is the uses for the study of DNA replication that most interest us. This is a highly regulated process that is reliant on a specific programme of temporal and spatial distribution of activation of origins of replication. Each origin is at distinct genetic locus and must fire only once per cell cycle. This technique allows us to get a genome-wide view of the firing of origins and propagation of the replication. As no assumptions are made about the sequence of the origin of replication, this technique is particularly useful for mapping Origin of replication
in eukaryote
s (who are not thought to have precisely-defined initiation sequences).
Strategies involving combing recently replicated DNA typically involve incorporating modified nucleotides (such as BrdU, bromodeoxyuridine) into the nascent DNA, then fluorescently detecting it. As replication forks spread bidirectionally from the Origin of replication
at (approximately) equal speeds, then ori position can be inferred. Replacing the modified nucleotide with another after a certain amount of time allows us to develop a time-resolved picture of the firing of sites, and the kinetics of replication forks. Pause sites can be identified, merged replication forks resolved, and the frequency of ori firings in different time periods to be studied. It was shown in in vitro studies of Xenopus laevis egg extract to increase as S phase progresses. In another study, this time on Epstein-Barr Virus episomes, hybridised probes were used to visualise the regional distribution of firing events; a particular zone showed preference for firing, whilst a few pause sites were also inferred.
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
that is then highly suitable for nucleic acid hybridization studies such as fluorescent in situ hybridisation
Fluorescent in situ hybridization
FISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
(FISH) which benefit from the uniformity of stretching, the easy access to the hybridisation target sequences , and the resolution
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
offered by the large distance between two probes, which is due to the stretching of the DNA by a factor of 1.5 times the crystallographic length of DNA.
DNA in solution (i.e. has a random-coil structure) is stretched by retracting the meniscus
Meniscus
The meniscus is the curve in the upper surface of a liquid close to the surface of the container or another object, caused by surface tension. It can be either convex or concave. A convex meniscus occurs when the molecules have a stronger attraction to each other than to the material of the...
of the solution at a constant rate (typically 300 γm/s). The ends of DNA strands, which are thought to be frayed (i.e. open and exposing polar groups) bind to ionisable groups coating a silated glass plate at a pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
below the pKa
PKA
PKA, pKa, or other similar variations may stand for:* pKa, the symbol for the acid dissociation constant at logarithmic scale* Protein kinase A, a class of cAMP-dependent enzymes* Pi Kappa Alpha, the North-American social fraternity...
of the ionizable groups (ensuring they are charged enough to interact with the ends of DNA). The rest of the DNA, which is mostly dsDNA, cannot form these interactions (aside from a few ‘touch down’ segments along the length of the DNA strand) so is available for hybridisation to probes. As the meniscus retracts, surface retention creates a force that acts on DNA to retain it in the liquid phase; however this force is inferior to the strength of the DNA’s attachment; the result is that the DNA is stretched as it enters the air phase; as the force acts in the locality of the air/liquid phase, it is invariant to different lengths or conformations of the DNA in solution, so DNA of any length will be stretched the same as the meniscus retracts. As this stretching is constant along the length of a DNA, distance along the strand can be related to base content; 1 µm is approximately equivalent to 2 kb.
DNA regions of interest are observed by hybridising them with probes labelled by haptens like biotin; this can then be bound by one or more layers of fluorochrome-associated ligands (such as immunofluorescence antibodies). Multicolour tagging is also possible. This has several potential uses, typically as a high-resolution physical mapping technique (e.g. for positional cloning), an example of which was the correct mapping of 200 kb of the CAPN3 gene region, or the mapping of non-overlapping sequences (since the distance between two probes can be accurately measured). It is therefore useful for finding exons, microdeletions, amplifications, or rearrangements. Before the combing improvement, FISH was too low-res to be of use in this case. With this technique, the resolution of FISH is theoretically limited only by the resolution of the epifluorescence microscope; in practice, resolutions of around 2&Nbsp;µm are obtained, for DNA molecules usually 200–600 kb long (though combing-FISH has been used with some success on molecules in excess of 1 Mb long), and there may be room for improvement through optimisation. Since DNA analyses using this technique are single-molecule, we can compare genomes from different cells and find anomalies; the implications for diagnosis of cancer and other genetic alterations are evident.
However, it is the uses for the study of DNA replication that most interest us. This is a highly regulated process that is reliant on a specific programme of temporal and spatial distribution of activation of origins of replication. Each origin is at distinct genetic locus and must fire only once per cell cycle. This technique allows us to get a genome-wide view of the firing of origins and propagation of the replication. As no assumptions are made about the sequence of the origin of replication, this technique is particularly useful for mapping Origin of replication
Origin of replication
The origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
in eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s (who are not thought to have precisely-defined initiation sequences).
Strategies involving combing recently replicated DNA typically involve incorporating modified nucleotides (such as BrdU, bromodeoxyuridine) into the nascent DNA, then fluorescently detecting it. As replication forks spread bidirectionally from the Origin of replication
Origin of replication
The origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
at (approximately) equal speeds, then ori position can be inferred. Replacing the modified nucleotide with another after a certain amount of time allows us to develop a time-resolved picture of the firing of sites, and the kinetics of replication forks. Pause sites can be identified, merged replication forks resolved, and the frequency of ori firings in different time periods to be studied. It was shown in in vitro studies of Xenopus laevis egg extract to increase as S phase progresses. In another study, this time on Epstein-Barr Virus episomes, hybridised probes were used to visualise the regional distribution of firing events; a particular zone showed preference for firing, whilst a few pause sites were also inferred.