Physical mapping of genomes

Physically maps are maps of physical isolated pieces of the genome ie maps of
cloned genomic DNA. A complete physical map of a genome consists of a series of
maps for each chromosome in the haploid chromosome set.

For each chromosome, a complete map consists of a series of continuous

overlapping cloned genomic DNA segments extending from one telomere of the
chromosome to the other.
A physical map of a whole chromosome or a chromosome region provides
direct access to the DNA which in turn facilitates the isolation and characterization of
genes and the systematic sequencing of chromosome DNA.

Physical maps have two main values

1. Previously cloned genes and DNA markers on a genetic map can be localized
to a specific clone on a physical map by hybridization technique like southern
blotting or PCR. This genes us way for marker DNA location and distances
between the markers, which cannot be obtained by genetic map.
2. One of the main strategies for sequencing an entire genome requires a
complete physical map. Therefore, the availability of the physical map
provides an intermediary in moving from genetic maps to complete sequence
maps of the genome.

Construction of physical maps

The vectors that can carry vary large inserts are the most useful because their
use in cloning requires breaking the genome up into fewer pieces than would be
necessary for other vectors.
Cosmids, YAC’s, BAC & PAC are the main types of vectors used.
The maximum size insert size is as follows.
Cosmid  400kb
BAC  300kb they can be amplified in Bacteria isolated & manipulated.
PAC  100kb
YAC  1000kb

The BAC & PAC form fewer hybrid inserts then YAC. Hybrid inserts 
mean cloning genes composed of several fragments of the genome instead of just one.

The goal of physical mapping is to develop a clone overlap that truly reflects
the organization of the intact genome.
The vectors that carry large inserts are useful but the task of creating a
physical map is nothing but a tedious one. Therefore even small genome for tiny
nematode (caenorhabditis elegans) has a 100mb genome therefore 2500 Cosmids are
required to map this genome.
Creating a complete physical map begins by amassing a large number of
randomly cloned inserts. In some manner, sequence identities between end regions of
the clones and then identities are used to infer that the ends of the two clones overlap
and thus the clones come from adjacent regions of the genome. A set of overlapping
clones is called contig.

CONTIG
In the early phases of a genome project, contigs are numerous and represent
separate cloned segments of the genome, but as more and more clones are
characterized, clones are found that overlap two previously separate contigs and these
joining clones then permit the merger of the two contigs into one larger contig (fig 1).
This process of contig merging continues until eventually a set of contigs is
built that is equal to the number of chromosomes. At this point if the contigs in this
set extend out to the telomeres of the chromosome, the physical map is complete.

Let’s now look at techniques for identifying clone overlaps in other words techniques
for turning a set of randomly isolated clones into a contig.
Sequence Tagged Sites STS
Clone finger printing
Radiation Hybrid
End Labeling Method
Hybridization Method
Positional cloning.

Ordering by sequence tagged sites. (YAC, BAC and PAC)

STS mapping has become the preferred method for constructing physical maps
of chromosome regions and whole chromosomes form larger inserts (YAC, BAC and
PAC) genomic libraries.
STS is a short unique sequence of 100 – 300bp that can be specifically
amplified and detected by using defined PCR primers.
The STS derive from sequenced regions of the genome and are used as land
marks for clone classification in establishing a physical map of a genome.
The main logic of the technique is clones that share STS must overlap.
Therefore, the resulting physical maps are called STS content maps.
Large number of STS with an STS of every 50 to 100kb is required to produce
a continuous contig over a long span of a chromosome
For ex :- 1500 – 3000 STS are required for the physical mapping of a chromosome
that consists of about 200MB pairs of DNA and at least 30,000 STS are needed for a
high resolution physical map of the complete human genome.

Preparation / development of an STS content map

a. Large numbers of usable STS sequences are collected by sequencing the ends
of a random set of clones from a genomic library.
b. Two PCR primers per STS sequence are developed in such a way that a given
pair of primer will uniquely amplify a single STS.
c. Now PCR is used to screen individually all the clones containing a particular
STS that is amplified by the primer pair used to yield PCR amplification
product.
 Then this PCR product is detectable because it incorporates labeled nucleotide
triphosphates during its synthesis.
The samples of the DNA in the PCR reaction mixtures for each of the clone
after STS amplification with a specific primer pair are examined for labeled DNA
products.
Then on the basis of STS that are shared between clones a computer program
is used to deduce a likely set of overlapping clones and to a limited extent the relative
positions of some of the STS (fig 2).

Assembling / ordering by clone finger printing method (Cosmid

Libraries)

Small insert contigs are often more useful for the study of genes and large
scale DNA sequencing projects than the large insert contigs.
Here cosmid libraries are used frequently as source for assembling region specific or
whole chromosome contigs.
Here a multiple restriction enzyme digestion is carried out which generate a
set of bands whose number and position are a unique finger print of that clone then
the pattern of bands generated by each separate clone can be aligned and the bands
from different clones can be aligned by computer to determine if there is any overlap
between the inserted DNA’s.
Here what is determined is the proportion of bands that are shared between
two clones but the order of the band is not determined here but only the proportion of
shared bands.
For Ex:- Four clones are digested with multiple restriction enzymes and the resulting
complex mixture of restriction fragments is separated on the basis of size by gel
electrophoresis. The bands containing the fragments are stained to show their
location. The number of identically sized bands for each pair of digests is determined.
A and B digests share > 50% of the bands as do the B and C digests indicating that
they come from overlapping regions of the genome. No other pair of digests shares a
significant number of bands fig 3.

Figure 3.a. DNA finger print.

Figure 3.b. The physical map derived from the data in part (a). Clones A and C both
overlap clone B but do not overlap each other. Clone D is from some where else in the
genome since it doesn’t overlap any of the other three clones.

End labeling method

The more efficient clone finger printing methods have involved restriction
nuclease treatment of clones and separation of radio isotopes labeled restriction
fragments on gels.
Modern methods of clone finger printing involve restriction nucleases to
achieve a high degree of automation using end labeling the clones or by using
fluorescence labeling and detection systems.
Ordering by hybridization method

If two clones contain sequences in common they will hybridize to each other.
In principle one clone can be used as a probe to identify overlapping clones by using
the DNA from one clone as a probe to hybridize. Hybridization is more sensitive than
finger printing or STS content and is often used to detect overlap between adjacent
contigs.
The only problem encounter with hybridization is a high rate of false positive
results. One of the reason is the presence of repetitive elements in the probe hybridize
to a large number of unrelated clones.
This problem is solved by using probe that carries only single sequence DNA
and use PCR to amplify the sequences between adjacent Alu elements.
Here PCR uses primers that are designed to anneal the repetitive DNA
sequences and direct amplification of the DNA between adjacent repeats.
This repeats of a particular type are distributed fairly randomly in Eukaryotic
genomes with varying distances between them. So, a variety of product sizes are
obtained when these primers are used with clones of Eukaryotic DNA.
If a pair of clones gives PCR products of the same size they must contain
repeats that are identically spaced because the cloned DNA fragments overlap.

Identical repeats repeats

Primer

PCR region spans the region

between adjacent repeats
Interpretation

• The shared bonds suggest that clones overlap.

• And also the amplified products are used as hybridization probe to identify
other clones in the library that contain the same sequence and therefore
overlap with the first clone.

Positional cloning

The creation of a contig has many similarities to a gene cloning strategy

known as positional cloning. This form refers a strategy in which a gene is cloned
based on its mapped position along a chromosome.
This approach has been successful in cloning of many human genes, like that
of genetic diseases when mutated.
The common method used in positional cloning is known as chromosome
walking. To initiate this type of experiment, a genes position relative to a marker must
be known from mapping studies. i.e., a gene may be known to be fairly close to a
previously mapped gene or marker. This will provide a starting point to molecularly
‘walk’ toward the gene of interest.
Ex: - The figure below considers a chromosome walk in which the goal is to locate a
gene that will call gene A.
Previous genetic mapping studies have revealed that gene A is ultimately
cloned to another gene called gene B that has been previously cloned.
Gene A and gene B have been deduced from genetic crosses and said to be
approximately apart. To start this chromosome walk, a cloned DNA fragment of gene
B is used as a starting point to walk to gene A.
Here to walk from gene B to gene A a series of library running methods are
followed.
For Ex: - The starting materials are a cosmid library and a clone containing gene B.
A small piece of DNA from gene B clone is inserted into another vector. This
sub clone is radio labeled and used as a probe to screen a cosmid library.
This will enable researchers to identify a second clone that is cloned to gene
A.
A sub clone from this second clone is used to screen the library a second time. This
will allow the researchers to identify clone that is even closer to gene A. This repeated
pattern of sub cloning and library screening is used to walk toward gene A. here the
number of steps required to reach the gene of intrest depends on the distance between
the starting and ending points.

Radiation hybrid maps.

This is based on the frequently with which marker are found on the same
fragment of DNA often the genome has been fragmented by irradiation with x-rays.
(In RH mapping a human cell line is irradiated with x-rays and then nit is fused to
hamster cells. Here the radiation fragments are such that, they cannot be
independently maintained in the hybrid unless they become incorporated into hamster
chromosomes)
(Here each hybrid line retains about 20% of the donor human genome in fragments)
of about 10MB (Then these panel of lines are beamined for co-retention of different
STS markers). (If two markers are co retained more frequently than would be -------
by chance). (Then this genes evidence that they are linked. The distance between
merkers is estimated from the frequently of convention). The map units are centro
rays.
The importance of RH maps is they provide an independent method of
mapping STS sites.
This RH maps can detect linkage between STS over a much longer distance
than can be detected by the overlap of clones. They therefore provide a frame work to
link clone contigs together.
Each chromosome represents a complete set of chromosomes (genes) in a cell
line. A human cell line is irradiated with x-rays which fragments the chromosome.
These irradiated cells are fused to a hamster cell line. The fragments of human
chromosome become incorporated into the hamster chromosomes.

Markers adjacent to each other in the human genome (AB) show the same
pattern of retention in the hybrid lines, i.e., they are co-retained marker C is distant
from A and B and shows a different co-retention pattern compared with either A or B.