Lecture Notes (M.Sc.

Biotechnology, IInd Semester)

Molecular Marker in Genome Analysis

1. Introduction:
A molecular marker is a molecule contained within a sample taken from
an organism (biological markers) or other matter.

It can be used to reveal certain characteristics about the respective source. DNA, for
example, is a molecular marker containing information about genetic
disorders, genealogy and the evolutionary history of life.

Specific regions of the DNA (genetic markers) are used for diagnosing the autosomal
recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics)
and identity (DNA barcoding).

Further, life forms are known to shed unique chemicals, including DNA, into
the environment as evidence of their presence in a particular location.

Other biological markers, like proteins, are used in diagnostic tests for
complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological
molecular markers are also used, for example, in environmental studies.

2. Types of Genetic Markers:

There are many types of genetic markers, each with particular limitations and strengths.
Within genetic markers there are three different categories: "First Generation Markers",
"Second Generation Markers", and "New Generation Markers".
These types of markers may also identify dominance and co-dominance within the
genome. Identifying dominance and co-dominance with a marker may help identify
heterozygotes from homozygotes within the organism.
Co-dominant markers are more beneficial because they identify more than one allele thus
enabling someone to follow a particular trait through mapping techniques.
These markers allow for the amplification of particular sequence within the genome for
comparison and analysis.
Molecular markers are effective because they identify an abundance of genetic linkage
between identifiable locations within a chromosome and are able to be repeated for
verification.
They can identify small changes within the mapping population enabling distinction
between a mapping species, allowing for segregation of traits and identity.
They identify particular locations on a chromosome, allowing for physical maps to be
created. Lastly they can identify how many alleles an organism has for a particular trait (bi
allelic or poly allelic).

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3. Mapping of Genetic Markers:

Molecular mapping aids in identifying the location of particular markers within the genome.
There are two types of maps that may be created for analysis of genetic material.

First, is a physical map, which helps identify the location of where you are on a
chromosome as well as which chromosome you are on.

Secondly there is a linkage map that identifies how particular genes are linked to other
genes on a chromosome.

This linkage map may identify distances from other genes using (cM) centiMorgans as a
unit of measurement. Co-dominant markers can be used in mapping, to identify particular
locations within a genome and can represent differences in phenotype.

Linkage of markers can help identify particular polymorphisms within the genome. These
polymorphisms indicate slight changes within the genome that may present nucleotide
substitutions or rearrangement of sequence.

When developing a map it is beneficial to identify several polymorphic distinctions

between two species as well as identify similar sequence between two species.

4. Applications:

Assessing variability of genetic differences and characteristics within a species.

Identification and fingerprinting of genotypes.
Estimating distances between species and offspring.
Identifying location of QTL's.
Identification of DNA sequence from useful candidate genes[13]
Species Identification
Genetic variation and population structure study in natural populations
Comparison between wild and hatchery populations
Assessment of demographic bottleneck in natural population
markers assisted breeding

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5. Example of Molecular Marker (RFLP Analysis):

The basic technique for the detection of RFLPs involves fragmenting a sample of DNA
with the application of a restriction enzyme, which can selectively cleave a DNA
molecule wherever a short, specific sequence is recognized in a process known as
a restriction digest.
The DNA fragments produced by the digest are then separated by length through a
process known as agarose gel electrophoresis and transferred to a membrane via
the Southern blot procedure.
Hybridization of the membrane to a labeled DNA probe then determines the length of the
fragments which are complementary to the probe.

A restriction fragment length polymorphism is said to occur when the length of a detected
fragment varies between individuals, indicating non-identical sequence homologies.

Each fragment length is considered an allele, whether it actually contains a coding

region or not, and can be used in subsequent genetic analysis.

There are two common mechanisms by which the size of a particular restriction fragment
can vary. In the first schematic, a small segment of the genome is being detected by a
DNA probe (thicker line).

In allele A, the genome is cleaved by a restriction enzyme at three nearby sites (triangles),
but only the rightmost fragment will be detected by the probe.
In allele a, restriction site 2 has been lost by a mutation, so the probe now detects the
larger fused fragment running from sites 1 to 3.

The second diagram shows how this fragment size variation would look on a Southern
blot, and how each allele (two per individual) might be inherited in members of a family.

In the third schematic, the probe and restriction enzyme are chosen to detect a region of
the genome that includes a variable number tandem repeat (VNTR) segment (boxes in
schematic diagram).
In allele c, there are five repeats in the VNTR, and the probe detects a longer fragment
between the two restriction sites.
In allele d, there are only two repeats in the VNTR, so the probe detects a shorter fragment
between the same two restriction sites.
Other genetic processes, such as insertions, deletions, translocations, and inversions, can
also lead to polymorphisms. RFLP tests require much larger samples of DNA than do
short tandem repeat (STR) tests.

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Fig. 01: Fragment size variation on a Southern blot

6. References:
Gerald Karp - Cell and molecular biology. 5th Edition (2007).
Lewis J. Klein smith and Valerie M. Kish - Principles of cell and molecular biology -
Third Edition (2002)
Richard M. Twyman-Advanced Molecular Biology, First South Asian 'Edition." (1998),
Viva Books Pvt. Ltd.
Benjamin Lewin, Gene IX, 9th EditionJones and Barlett Publishers, 2007.
J.D. Watson, N.H. Hopkins, J.W Roberts, J. A. Seitz.& A.M. Weiner; Molecular Biology
of the Gene, 6th Edition, Benjamin Cummings Publishing Company Inc, 2007.
TA Brown - Genomes 2nd Edition; Bios Scientific Publishers 2002 Harvey Lodish,
Arnold Berk, Chris A. Kaiser, Monty Krieger, Matthew P. Scott, Anthony Bretscher,
Hidde Ploegh and Paul Matsudaira - Molecular Cell Biology, 6th Edition; WH Freeman
2008.
Saiki, R.; Scharf, S; Faloona, F; Mullis, K.; Horn, G.; Erlich, H.; Arnheim, N (1985).
"Enzymatic amplification of beta-globin genomic sequences and restriction site analysis
for diagnosis of sickle cell anemia". Science. 230 (4732): 1350-1354.
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