NAME: MASHAL FATIMA

ROLL NO: 2018-DVME-03

SUBJECT: ANIMAL BREEDING AND GENETICS -1
TOPIC: GENETIC MARKER
SUBMITTED TO: Dr. MUHAMMAD DAWOOD
GENETIC MARKER

Genetic marker is a specific location on a chromosome that is defined by a naked eye

polymorphism as difference in electrophoretic mobility of specific proteins, or a difference in
specific DNA sequence
A genetic marker is a DNA sequence with a known physical location on a chromosome. Genetic
markers can help link an inherited disease with the responsible gene. DNA segments close to
each other on a chromosome tend to be inherited together. Genetic markers are used to track the
inheritance of a nearby gene that has not yet been identified, but whose approximate location is
known. The genetic marker itself may be a part of a gene or may have no known function.
Genetic markers consist primarily of polymorphisms, which are discontinuous genetic variations
that divide individuals of a population into distinct forms. Genetic markers play a key role in
genetic mapping.
Classification of Markers

A) Classical Marker
1-Morphological Marker
Markers that are related to variation in visible traits i.e., shape, size, color are called
morphological markers.
Such markers refer to available gene loci that have obvious impact on morphology of individual.
Genes that affect form, coloration, male sterility, or resistance among others have been analyzed
in many plant species.
Demerits
a. Limited in number
b. Exhibit dominance.
c. Influenced by environmental factors
d. Exhibit pleiotropy.
e. Generally express late into the development of an organism. Hence their detection is
dependent on the development stage of the organism.
2-Biochemical Marker
Markers that are related to variation in proteins and amino acid banding pattern are known as
biochemical markers. A gene encodes a protein that can be extracted and observed
Examples: isozymes and storage proteins.
3-Cytogenetic Marker
Markers that are related to variation in chromosome number, shape, size and banding pattern are
referred to as cytological markers.
It refers to the chromosomal banding produced by different stains; for example, G banding, C
banding, Q banding
B) DNA Marker
A gene or other fragment of DNA whose location in the genome is known is called DNA marker.
It is a unique (DNA sequence), occurring in proximity to the gene or locus of interest. It refers to
any unique DNA sequence which can be used in DNA hybridization, PCR or restriction mapping
experiments to identify that sequence.
Classifications of DNA Marker
1-Based on Gene function
a-Type 1
Markers are associated with gene of known function
e.g. RFLP, EST
b- Type 2
Marker are associated with anonymous genomic function
e.g., RADP, AFLP, SNP, Microsatellite
2- Based on Protocol
a-Hybridization
RFLP
b-PCR
RADP, AFLP, SSR, EST
3-Based on Inheritance
a-Dominant
RADP, AFLP
B-Co-Dominant
SSR, STR, RFLP
Types of genetic markers are:
 RFLP ( Restriction fragment length polymorphism)
  AFLP (or Amplified fragment length polymorphism)
 RAPD (or Random amplification of polymorphic DNA)
 VNTR (or Variable number tandem repeat)
 Micro satellite polymorphism, ( Simple sequence repeat /short tandem repeat)
 SNP ( Single nucleotide polymorphism)
 EST(Expressed sequence Tag)
Restriction Fragment Length Polymorphism (RFLP)
The variation in the restriction DNA fragment lengths between individuals a species is called
restriction fragment length polymorphism (RFLP).
It is a laboratory technique to analyze and compare DNAs of two or more individuals of a
species or of different species. Genetic structure of all the individuals of a species is same, but at
DNA level there are so many single base variations between the individuals due to point
mutation.
Using restriction endonuclease enzymes fragments of DNA is obtained and the desired fragment
is detected by using restriction probes. Southern hybridization using restriction endonuclease
enzymes for isolation of desired length of DNA fragments is an example of RFLP.
Protocol
 Sample collection: Tissues or cells of individuals are collected to extract their DNA. The
samples are collected separately.
 Extraction of desired fragments of DNA using restriction endonuclease (RE) generating
different size of fragments
 The digested fragment are run in polyacrylamide gel electrophoresis or Agarose gel
electrophoresis to separate the fragments on the basis of length or size or molecular
weight.
 The gel is placed in sodium hydroxide (NaOH) solution for denaturation so that single
stranded DNA are formed.
 The single stranded DNA are transferred into charge membrane ie. Nitrocellulose paper
 The labelled RFLP probe is hybridized with DNA on the nitrocellulose paper.
 Figure:1.1 Flow chart of RFLP protocol

Advantages of RFLP
 Highly Polymorphic
 Co-dominantly inherited
 High reproducibility
 Identified through absence or presence of fragment
Application of RFLP
 Genome mapping: helps in analysis of unique pattern in genome for organism
identification and differentiation. It also helps in determining recombination rate.
 Genetic disease analysis: After identification of gene for particular genetic or hereditary
disease, that gene can be analyzed among other family members.
 To detect mutated gene
 DNA finger printing (forensic test): It is the basis of DNA finger printing for paternity
test, criminal identification etc.
Randomly amplified polymorphic DNA (RAPD)
RADP is defined as difference between individual in term of DNA region ether being or not
being amplified in a PCR reaction primed by random oligonucleotide sequence.
It is a type of PCR to amplify random segments of DNA using single short arbitrarily selected
oligonucleotide primer.
The scientist performing RAPD creates several arbitrary, short primers (10- 12 nucleotides), then
proceeds with the PCR using a large template of genomic DNA, hoping that fragments will
amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from an RAPD
reaction.No knowledge of the DNA sequence of the targeted genome is required, as the primers
will bind somewhere in the sequence, but it is not certain exactly where. This makes the method
popular for comparing the DNA of biological systems that have not had the attention of the
scientific community
Protocol
 Collect sample and isolate DNA
 Denature DNA at 94 c for 1 min: DNA strands are separated
 Decanucleotide enzyme, primer, Taq polymerase are added to reaction
 Anneal primer at 36 c for 2 min to DNA strands
 Complimentary DNA strands synthesis at 72 c for 1.5 min, complete 35 -45 cycles
 Amplified products separated by gel electrophoresis
 Bands are detected by ethidium bromide
Advantages of RADP
 No need of specie specific probe in RADP
 Probes prepared for one specie may also be used for other specie
 Requires only small amount of sample
 Detect dominant variation in DNA
Application of RADP
 Used as genetic marker for constructing genetic maps of organisms
 Determines specific genes in chromosomes
 Widely used in study of genetic polymorphism, genetic structure of population
 Distinguish one individual from others of a specie as fingerprinting analysis.

Assisted fragment length polymorphism (AFLP)

AFLP is based on principle of selectively amplifying the subset of restriction fragment from a
complex mixture of DNA obtained from the digestion of genomic DNA with restriction
endonucleases.
It involves use of RFLP and PCR techniques.
AFLP uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the
sticky ends of the restriction fragments. A subset of the restriction fragments is then selected to
be amplified. This selection is achieved by using primers complementary to the adaptor
sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments
(as described in detail below). The amplified fragments are separated and visualized on
denaturing on agarose gel electrophoresis , either through autoradiography or fluorescence
methodologies, or via automated capillary sequencing instruments.
Protocol
 Collect sample, isolate DNA
 Digestion of DNA using different restriction endonucleases
 Ligate two different adaptor(short ds DNA with sticky ends ) with digested fragment.
 Selected fragments are amplified by pre selective and selective amplification using
adapter and primer respectively.
 Amplified DNA fragments are separated by polyacrylamide gel electrophoresis and
visualized by ethylene bromide.
Advantages of AFLP
 No need of known genomic sequence
 High reproducibility
 By changing selective nucleotide different parts of genome can be visualized
Uses and Applications of AFLP
 Used to detect various polymorphism in different genomic region simultaneously.
 Used for identification of genetic variation in closely related polymorphism.
 Linkage studied to generate maps for QTL analysis.
Single Nucleotide Polymorphism (SNP)
Single nucleotide polymorphism (SNP) describes polymorphisms caused by point Mutations that
give rise to different alleles containing alternative bases at a given Nucleotide position within a
locus.
The genomic distribution of SNPs is not homogenous; SNPs occur in non-coding regions more
frequently than in coding regions or, in general, where natural selection is acting and "fixing" the
allele (eliminating other variants) of the SNP that constitutes the most favorable genetic
adaptation.Other factors, like genetic recombination and mutation rate, can also determine SNP
density.
Protocol
 Collect sample and Isolate DNA
 Digest DNA with restrictions enzymes
 Ligate adaptor and amplify digested DNA fragments and sequence DNA.
 Analysis of DNA by software and identify SNP.
Advantages of SNP
SNPs have become a focal point in molecular marker Development as they are
 most abundant polymorphism in any organism,
  adaptable to automation,
 reveal hidden polymorphism, not detected with other Markers and methods
Applications of SNP
 Gene discovery and mapping
 Diagnostic and risk profiling
 Gene function identification
 Response prediction

Expressed Sequence Tag(EST)

Expressed sequence tags (ESTs) are fragments of mRNA sequences derived through single
sequencing reactions performed on randomly selected clones from cDNA libraries.
Protocol
 Isolate the messenger RNA (mRNA) from a particular tissue (e.g., liver)
 Treat it with reverse transcriptase. Reverse transcriptase is a DNA polymerase that uses
RNA as its template. Thus it is able to make genetic information flow in the reverse
(RNA ->DNA) of its normal direction (DNA -> RNA).
 This produces complementary DNA (cDNA). Note that cDNA differs from the normal
gene in lacking the intron sequences.
 Sequence 200–500 nucleotides at both the 5′ and 3′ ends of each cDNA.
 Examine the database of the organism's genome to find a matching sequence.

Advantages of EST
 sequences can be generated rapidly and inexpensively
 they do not have to be checked for sequencing errors as mistakes do not prevent
identification of the gene from which the EST was derived.
Applications of EST
 Identify unknown genes and to map their position within a genome
 Construction of genome map
 To obtain data on gene expression and regulation
Variable Number Tandem Repeat Sequence (VNTRS)
VNTR are the repeated DNA sequences at a defined locus. The repeats are clustered together and
oriented in the same direction. Individual repeats can be added or removed through replication
and recombination errors. This forms alleles with different numbers of repeats.
The DNA segments vary in different individuals and are hence beneficial in identifying
individuals in case of a crime scene or a paternity dispute. This is known as DNA fingerprinting.
The tandem repeat sequences of DNA are also termed “satellite DNA”. These are of main
following types:
a-Minisatellites
In a Minisatellites, each repeat ranges from 9 to 100 base pairs. It is an array of tandem repeats
500 to 300,000 base pairs long.

Minisatellites have been found in association with important features of the human genome such
as gene regulation, imprinting, and chromosomal fragile sites. They provided the first highly
polymorphic, multiallelic markers for linkage studies.
b-Microsatellite(STR/ SSR)
The repeats are very short, with 2-6 base pairs each. The whole array ranges from 10,000 to
100,000 base pairs long. They are therefore called short tandem repeats or simple sequence
repeats.
Microsatellite markers are inherited from both parents. Therefore, they are useful for paternity
tests. Highly polymorphic loci increase our ability for parental analysis.
Unlike point mutations, which affect only a single nucleotide, micro-satellite mutations lead to
the gain or loss of an entire repeat unit, and sometimes two or more repeats simultaneously.
Thus, the mutation rate at micro-satellite loci is expected to differ from other mutation rates,
such as base substitution rates. One proposed cause of such length changes is replication
slippage, caused by mismatches between DNA strands while being replicated during
meiosis.DNA polymerase, the enzyme responsible for reading DNA during replication, can slip
while moving along the template strand and continue at the wrong nucleotide.
Protocol
 Collect sample, isolate DNA
 Design primer from genomic library
 Amplify by PCR
 Visualize bands of microsatellite using polyacrylamide gel having ethidium bromide in it
 Analyze data and interpret results.
Uses of Microsatellite
 markers are co-dominant transmission (the heterozygotes can be distinguished from
homozygotes)
 locus-specific in nature
 highly polymorphic and
Applications of microsatellite
 DNA profiling, also known as "genetic fingerprinting
 They are also widely used in kinship analysis (most commonly in paternity testing)
 used for mapping locations within the genome, specifically in genetic linkage
 analysis to locate a gene or a mutation responsible for a given trait or diseases
 used for studies of gene duplication or deletion