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MOLECULAR

MARKERS AND
THEIR
APPLICATIONS
Definition

A molecular marker is a DNA sequence that is readily


detected and whose inheritance can easily be monitored .

What are Molecular Markers?


Specific fragments of DNA that can be identified within the
whole genome.

Molecular markers are the general assays that allow


detection of the sequence differences between two or more
individual.
Molecular markers are found at specific locations of the
genome.

They are used to 'flag' the position of a particular gene or


the inheritance of a particular characteristic or desired
characteristics
Marker systems are tools which is used to mark a trait in living organism

MORPHOLOGICAL MOLECULAR MARKERS:


MARKER: Variation in macro-molecules
Classical markers
BIOCHEMICAL GENETIC MARKERS
MARKERS RFLP
ISOENZYME

PROTEIN AFLP
Low polymorphism
Requires expression of trait / RAPD
gene
Dominance effect
Expression sex limited Depend upon sequence of DNA
Expressed late in life
They are protein produced by expression of gene
DNA markers
Non-PCR Based,
RFLP- Restriction fragment length polymorphism.
PCR Based
RAPD- Random amplification of polymorphic DNA.
AFLP-Amplified fragment length polymorphism.
SCAR-Sequence characterize amplified region.
STS- Sequence tagged sites.
EST-Express sequence tags.
SNP-Single nucleotide polymorphism.
SSR-Simple sequence repeats
CAPS-Cleaved amplified polymorphic sequences.
Properties of Ideal Genetic Marker

it must be polymorphic.
Co-dominant inheritance.
A marker should be evenly and frequently
distributed throughout the genome.
It should be easy, fast and cheap to
detect.
It should be reproducible.
High exchange of data between
laboratories.
DEFINITION:- RAPD that is defined by differences
between individuals in terms of DNA
regions either being or not being amplified in a
polymerase chain reaction primed by random
oligonucleotides sequences.

It is a type of PCR reaction, but the


segments of DNA that are amplified are
random.
RAPD creates several arbitrary, short primers (812
nucleotides), then proceeds with the PCR using a large
template of genomic DNA

RAPD- The full form of RAPD is RANDOM AMPLIFIED


POLYMORPHIC DNAs are obtained by using a PCR
equipment or a thermo cycler.

RAPD - is a lab technique used to amplify


unknown(random) DNA segments
1866- Mendel described the
inheritance
pattern of conceptual
hereditary
units now called genes.

1980- Botstein et al suggested


DNA
marker RFLP
RAPD analysis is a PCR based
molecular marker technique.
Single short oligonucleotide
primer is arbitrarily selected
to amplify a set of DNA
segments distributed
randomly throughout the
genome.
APPLICATION
Molecular marker is a gene / DNA
sequence with
a known location on a chromosome that
can be
used to identify cells.

A marker must be polymorphic that it


must exist
in different forms so that chromosome
carrying
the mutant gene can be distinguished
from the
chromosome with the normal gene by
marker.
RAPD involves following steps:-

1.The DNA of a selected species is isolated.

2. An excess of selected
decaoligonucleotide
added.

3. This mixture is kept in a PCR equipment


and
is subjected to repeated cycles of DNA
denaturation-renaturation-
DNAreplication.

4. During this process, the


decaoligonucleotide
RAPD involves following steps:-

5.DNA replication extend the decaoligonucleotide and


copy the sequence continuous with the sequence
with
which the selected oligonucleotide has paired.

6.The repeated cycles of denaturation - renaturation-


DNA
replication will amplify this sequence of DNA.

7. Amplification will takes place only of those regions


of
the genome that has the sequence complementary
to
the decaoligonucleotide at their both ends.

8. After several cycles of amplification the DNA is


subjected to gel electrophoresis.

9. The amplified DNA will form a distinct band. it is


RAPD & ITS APPLICATION

Fig. no. 1 diagrammatic view of


RAPD & ITS APPLICATION
1. Molecular genetic markers have been
developed into powerful tools to analyze
genetic relationships and genetic diversity.

2. As an extension to the variety of existing


techniques using polymorphic DNA markers,
the Random Amplified Polymorphic DNA
(RAPD) technique may be used in molecular
ecology to determine taxonomic identity,
assess kinship relationships, analyze mixed
genome samples, and create specific probes.

3. Main advantages of the RAPD technology


include:-
. Suitability for work on anonymous
genomes
. Applicability to problems where only
limited quantitie of
DNA are available
. Efficiency and low expense.
ADVANTAGE
S
1. Morphological character represents the
actual phenotype importance while protein

& DNA markers are important only as


arbitrary loci used for linkage mapping &
often do not corresponds directly to
specific phenotypes.

2. It provides a quick and efficient screening for


DNA sequence based polymorphism at many
loci.

3. It involve no radioactive assays.


LIMITATION
S
1. Nearly all RAPD markers are dominant, i.e. it is not
possible to distinguish whether a DNA segment is
amplified from a locus that is heterozygous (1 copy)
or homozygous (2 copies).

2. Co-dominant RAPD markers, observed as


different-sized DNA segments amplified from the
same locus, are detected only rarely.

3. PCR is an enzymatic reaction, therefore the


quality and concentration of template DNA,
concentrations of PCR components, and the PCR
cycling conditions may greatly influence the
outcome.

4. Mismatches between the primer and the template


may result in the total absence of PCR product as
well as in a merely decreased amount of the
CHARACTERI RAPD RFLP AFLP
STICS
PRINCIPLE DNA Restriction DNA
amplification digestion amplification
DETECTION DNA staining Southern DNA staining
blotting
PRIMER Yes (random None Yes (selective
REQUIREMENT primer) primer)
PROBE None set of specific None
REQUIREMENT probes
DOMINANT/ Dominant Co dominant Dominant (co
CODOMINANT dominant )
APPLICATIONS
genetic diversity/polymorphism,
germplasm characterization,
genetic structure of populations,
hybrid purity,
genome mapping,
developing genetic markers linked to a trait in
question,
population and evolutionary genetics,
plant and animal breeding,
animal-plant-microbe interactions,
pesticide/herbicide resistance.
RFLP

Definition

The variation(s) in the length of DNA fragments produced


by a specific restriction endonuclease from genomic DNA
s of two or more individuals of a species
Principle

Restriction fragment length polymorphism (RFLP) technology was


first developed in the 1980s for use in human genetic
applications and was later applied to plants.

By digesting total DNA with specific restriction enzymes, an


unlimited number of RFLPs can be generated.

RFLPs are relatively small in size and are co-dominant in nature.

If two individuals differ by as little as a single nucleotide in the


restriction site, the restriction enzyme will cut the DNA of one but
not the other. Restriction fragments of different lengths are thus
generated.

All RFLP markers are analyzed using a common technique.


However, the analysis requires a relatively complex technique
that is time consuming and expensive.
Principle
The hybridization results can be visualized by
1. Autoradiography (if the probes are radioactively labeled), or
2. Chemiluminesence (if non-radioactive, enzyme-link methods are
used for probe labeling and detection).

Any of the visualization techniques will give the same results. The
visualization techniques used will depend on the laboratory condition
Principle
Advantages

-simple method as no sequence specific information is required


-codomonant markers
-it is not depend on PCR

Disadvantages

-it required large amount of highly pure DNA


-it require constant supply of probes
-it is laborious to identify suitable markers
-it is time consuming
-it requires expertise in autoradiography
Applications of molecular markers
MEASURE OF GENETIC DIVERSITY
FINGER PRINTING
GENOTYPIC SELECTION
GENOTYPIC PYRAMIDYING AND
INTROGRESSION
INDIRECT SELECTION USING QUANTITATIVE
TRAITS LOCI (QTLS)
MARKER-ASSISTED SELECTION
IDENTIFICATION OF GENOTYPE
Advantages
-need small amount of DNA
-it involves non-radioactive assay
-it does not required specific probe libraries
-it provide quick and efficient screening for DNA
sequence based on polymorphism at many loci

Disadvantages
-it is inherited as dominant traits
-there is a bands due to relatively short primer
-the production of non-parental bands in the offspring of
known pedigree warrants its use with extreme care
-it is sensitive to change in PCR conditions
YO U
AN K
T H