“Physical maps”
A map generated by genetic techniques is rarely sufficient for directing the
sequencing phase of a genome project.
A plethora of physical mapping techniques has been developed to address
this problem, the most important being:
● Restriction mapping, which locates the relative positions on a DNA
molecule of the recognition sequences for restriction endonucleases;
● Fluorescent in situ hybridization (FISH), in which marker locations
are mapped by hybridizing a probe containing the marker to intact
chromosomes;
● Sequence tagged site (STS) mapping, in which the positions of
short sequences are mapped by PCR and/or hybridization analysis of
genome fragments.
1. Restriction mapping:
Genetic mapping using RFLPs as DNA markers can locate the
positions of polymorphic restriction sites within a genome, but very
few of the restriction sites in a genome are polymorphic, so many
sites are not mapped by this technique. This is what restriction
mapping achieves, although in practice the technique has limitations
which mean that it is applicable only to relatively small DNA
molecules.
The basic methodology for restriction mapping:
● The simplest way to construct a restriction map is to compare the
fragment sizes produced when a DNA molecule is digested with two
different restriction enzymes that recognize different target
sequences. An example using the restriction enzymes EcoRI and
BamHI.
, ● First, the DNA molecule is digested with just one of the enzymes and
the sizes of the resulting fragments are measured by agarose gel
electrophoresis.
● Next, the molecule is digested with the second enzyme and the
resulting fragments again sized in an agarose gel.
● The results so far enable the number of restriction sites for each
enzyme to be worked out, but do not allow their relative positions to
be determined.
● Additional information is therefore obtained by cutting the DNA
molecule with both enzymes together. The double restriction enables
three of the sites to be mapped.
● However, a problem arises with the larger EcoRI fragment because
this contains two BamHI sites and there are two alternative
possibilities for the map location of the outer one of these. The
problem is solved by going back to the original DNA molecule and
treating it again with BamHI on its own, but this time preventing the
digestion from going to completion by, for example, incubating the
reaction for only a short time or using a suboptimal incubation
temperature. This is called a partial restriction and leads to a more
complex set of products, the complete restriction products now being
supplemented with partially restricted fragments that still contain one
or more uncut BamHI sites. The size of one of the partial restriction
fragments is diagnostic and the correct map can be identified.
A map generated by genetic techniques is rarely sufficient for directing the
sequencing phase of a genome project.
A plethora of physical mapping techniques has been developed to address
this problem, the most important being:
● Restriction mapping, which locates the relative positions on a DNA
molecule of the recognition sequences for restriction endonucleases;
● Fluorescent in situ hybridization (FISH), in which marker locations
are mapped by hybridizing a probe containing the marker to intact
chromosomes;
● Sequence tagged site (STS) mapping, in which the positions of
short sequences are mapped by PCR and/or hybridization analysis of
genome fragments.
1. Restriction mapping:
Genetic mapping using RFLPs as DNA markers can locate the
positions of polymorphic restriction sites within a genome, but very
few of the restriction sites in a genome are polymorphic, so many
sites are not mapped by this technique. This is what restriction
mapping achieves, although in practice the technique has limitations
which mean that it is applicable only to relatively small DNA
molecules.
The basic methodology for restriction mapping:
● The simplest way to construct a restriction map is to compare the
fragment sizes produced when a DNA molecule is digested with two
different restriction enzymes that recognize different target
sequences. An example using the restriction enzymes EcoRI and
BamHI.
, ● First, the DNA molecule is digested with just one of the enzymes and
the sizes of the resulting fragments are measured by agarose gel
electrophoresis.
● Next, the molecule is digested with the second enzyme and the
resulting fragments again sized in an agarose gel.
● The results so far enable the number of restriction sites for each
enzyme to be worked out, but do not allow their relative positions to
be determined.
● Additional information is therefore obtained by cutting the DNA
molecule with both enzymes together. The double restriction enables
three of the sites to be mapped.
● However, a problem arises with the larger EcoRI fragment because
this contains two BamHI sites and there are two alternative
possibilities for the map location of the outer one of these. The
problem is solved by going back to the original DNA molecule and
treating it again with BamHI on its own, but this time preventing the
digestion from going to completion by, for example, incubating the
reaction for only a short time or using a suboptimal incubation
temperature. This is called a partial restriction and leads to a more
complex set of products, the complete restriction products now being
supplemented with partially restricted fragments that still contain one
or more uncut BamHI sites. The size of one of the partial restriction
fragments is diagnostic and the correct map can be identified.
