PHYSICAL MAPPING STRATEGIES IN GENOMICS

Genome mapping The creation of a genetic map assigning DNA

fragments to chromosomes, which provide the first level of genome mapping.

When a genome is first investigated, this map is nonexistent. The map improves with the scientific progress and is perfect when the genomic DNA sequencing of the species has been completed.

During this process, and for the investigation of differences in strain, the fragments are identified by small tags. These may be genetic markers or the unique sequence-dependent pattern of DNA-cutting enzymes.

The term mapping is used in two different context but both of these are inter-related.

Genetic mapping uses classical genetic techniques (e.g. pedigree analysis or breeding experiments) to determine sequence features within a genome.

Using modern molecular biology techniques for the same purpose is usually referred to as physical mapping.

Types of genome mapping There are two types of genome

mapping,

Genetic mappingPhysical mapping

Genetic Mapping

A genetic map is a representation of the distance between two DNA elements based upon the frequency at which recombination occurs between the two.

The first genetic map of the chromosome was constructed by Alfred Sturtevant, using data from Drosophila mating crosses collected by Thomas Morgan .

Physical Mapping The physical map of a genome is a map of

genetic markers made by analyzing a genomic DNA sequence directly.

Along with genetic maps, physical maps for each chromosome within the genome can be constructed.

A variety of different techniques have been used to construct physical maps in the absence of complete DNA sequence.

Techniques Involved In Constructing Physical Maps

Restriction maps Radiation hybrid maps STS maps

Restriction Maps Restriction map is a

map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes.

In molecular biology, restriction maps are used as a reference to engineer plasmids

or other relatively short pieces of DNA, and sometimes for longer genomic DNA.

There are other ways of mapping features on DNA for longer length DNA molecules, such as mapping by transduction.

Method Of Making These Restriction Maps

The experimental procedure first requires an aliquot of purified plasmid DNA for each digest to be run. Digestion is then performed with each enzyme chosen. The resulting samples are subsequently run on an electrophoresis gel, typically on agrose gel.

The first step following the completion of electrophoresis, is to add up the sizes of the fragments in each lane. The sum of the individual fragments should equal the size of the original fragment, and each digest's fragments should also sum up to be the same size as each other.

If fragment sizes do not properly add up, there are two likely problems.

In one case, some of the smaller fragments may have run off the end of the gel. This frequently occurs if the gel is run too long.

In second case the possible source of error is that the gel was not dense enough and therefore was unable to resolve fragments close in size.

Cont… This leads to a lack of separation of fragments

which were close in size. If all of the digests produce fragments that add up one may infer the position of the REN (restriction endonuclease) sites by placing them in spots on the original DNA fragment that would satisfy the fragment sizes produced by all three digests.

EcoRI HindIII EcoRI + HindIII

Radiation Hybrid Mapping Radiation hybrid

mapping (also known as RH mapping) is a technique for mapping mammalian chromosomes.

It carries a small DNA fragment from the genome of other organism, e.g. human.

Irradiating human cells with X-rays causes random breaks with in the DNA.

The size of these fragments decreases as the dose of these rays increases.

These radiation levels are sufficient to kill the human cells but hybridization of these cells with hamster cells in vitro rescue these cells.

Then these human cells are then further analyzed for the genetic markers it carries.

STS Maps STS( sequence tagged

site) is a DNA fragment of 100-200 bp in length generated by PCR using primers based on already known sequence.

The genomic sites are tagged by its ability to hybridize with that sequence.

STS can be generated from previously cloned genes, or from other random non-gene sequences

An STS map of the human genome has now been constructed using a series of radiation hybrids respectively.

ADVANTAGES OF THESE PHYSICAL MAPPING STRATEGIES

Provide approx. distance and order between DNA sequences.

Yields a framework onto which sequence information can be applied.

Restriction mapping provide highly reliable fragment ordering and distance estimation.

RH mapping allows the relative likelihoods of alternative marker orders to be determined.

The RH procedure was used to map 14 DNA probes from a region of human chromosome 21, spanning 20 Mbp.

Cont… STS technique has been used to order

inserts from individual human chromosomes in a YAC library.

But it was later rejected as some YAC’s contained DNA from more than one human genome sequence.

The physical maps proved useful in producing ordered library clones.