An honest effort to present molecular marker in easiest way both informative and conceptual. Hybridization based (non-PCR) and PCR based markers are discussed to the point with suitable diagram.

1. Molecular Marker
Shovan Das
B.Sc (Agriculture)
Bidhan Chandra krishi viswavidyalaya

2. Molecular Marker: Molecular marker is a DNA or gene sequence within a recognized
location on a chromosome which is used as identification tool. In the pool of unknown DNA
or in a whole chromosome, these molecular markers help in identification of particular
sequence of DNA at particular location.
Molecular Marker’s variations arise due to:
 Base pair changes.
 Rearrangements (translocation or inversion).
 Insertions or deletions.
 Variation in the number of tandem repeats.
Quality for a good molecular marker:
 Genetic markers should be highly polymorphic in nature.
 They should be selectively neutral.
 Assay for detecting markers should be simple and rapid.
 Genetic markers should occur frequently within genome.
 The genetic marker (gene) should show codominant inheritance pattern.
 They should be highly reproducible.
 They should not interact with other markers while using multiple markers at a
same time.
 Single copy and no pleiotropic effect.
 High availability (un-restricted use) and suitability to be duplicated or
multiplexed.
Molecular markers can be classified into:
A. Hybridization based markers
B. PCR based markers
1. RFLP (Restriction Fragment Length Polymorphism):
It was invented by Alec Jeffreys in 1984. It is a
hybridization based technique.
Process: Extraction of high quality DNA
Digestion of DNA with restriction
endonuclease Analysis of restriction
fragments using gel-electrophoresis
Transfer fragments to membrane Hybridize
Hybridization Based Markers

3. with radioactively labelled probe Detection by autoradiography.
Applications Advantages Disadvantages/Limitations
1. Applied for genetic
fingerprinting/
profiling.
2. Identification of
inherited diseases,
carrier of that
diseases, genetic
mapping, and
heterozygous
detection.
3. Biological parentage,
paternity access.
1. Co-dominant marker
allows determination
of homozygosity &
heterozygosity.
2. Stable &
reproducible, gives
constant result over
time & location.
3. No prior information
on DNA sequence is
required.
4. Relatively simple
technique.
1. Need high quality
DNA.
2. Need to develop
polymorphic probes
3. Costly, laborious and
tedious process.
4. Sensitivity and more
precautions for
contamination
required.
1. RAPD (Random Amplified
Polymorphic DNA):
RAPD was developed by Williams et al.
and AP-PCR (Arbitrary primed PCR) was
developed by Welsh and McClelland in
1990 independently. Both are same
technique. This is the technique to amplify
through PCR random DNA segments
primed by random 10-mer oligonucleotide
sequences (arbitrary primer of 8-12 base
pairs).
Process: Isolation of DNA Denaturation
of DNA (at 94⁰c, 1min) DNA strands seperated Annealing of primer (36⁰c, 2min)
Complementary strand synthesis (72⁰c, 1.5 min, 35-45 cycles) Amplified product
seperated by gel electrophoresis Bands detected by Ethidium bromide Staining.
Applications Advantages Disadvantages/Limitations
1. RAPD is applicable
for the mapping of
genome, analysing
linkage, and
individual species
genotyping.
2. Identification of
somatic hybrids.
1. Requires no
information about
DNA probes & DNA
sequence.
2. Requires only small
amount of DNA.
3. Quick, simple,
efficient & requires
1. RAPD markers are
dominant in nature so
it has restrictions for
mapping purpose.
2. It has demerits as
poor reproducibility,
yields faint products,
problems occur in
PCR Based Markers

4. 3. Fingerprinting of
individuals.
low cost
comparatively.
band scoring.
2. AFLP (Amplied Fragment Length Polymorphism):
Zabeau and Vos invented the AFLP
technique in 1993. AFLP is based on a
selectively amplifying a subset of
restriction fragments from a complex
mixture of DNA fragments obtained
after digestion of genomic DNA with
restriction endonucleases.
Process: Restriction of DNA (using a
frequent cutter and a rare cutter
restriction enzyme) Ligation of
oligonucleotide adapters to both ends of fragment Selective amplifications of sets of
restriction fragments Analysis of results in gel electrophoresis or PAGE
Autoradiography
Applications Advantages Disadvantages/Limitations
1. Study of genetic
identity, identification
of clones and cultivars.
2. Used in phylogenetic
studies of closely
related species.
1. Very sensitive.
2. Good reproducibility.
3. No need of prior
knowledge of the
DNA sequences
taken.
1. Markers are
dominant so can’t
tell surely about the
type of a trait.
2. Need purified, high
moecular weight
DNA.
3. SSR (Simple Sequence Repeats) or Microsatellites or STM (Short Tandem Repeats)
or STM (Sequence Tagged Microsatellites) or VNTR (Variable Number of Tandem
Repeats):
Developed by Jeffrey et al. in 1985. SSR or microsatellites are 1-6 bp long repeat sequences
(usually less tham 100 times).
Process: Microsatellite library construction Identification of unique microsatellite loci
Identification a suitable area for primer design Obtaining a PCR product Evaluation
of banding pattern Assessing PCR product for polymorphisms.
Applications Advantages Disadvantages/Limitations
1. Used in
genetic
diversity,
population
genetic study.
2. Parentage
1. Highly
polymorphic, high
reproducibility,
codominant
nature.
2. Locus specificity
1. Assay is costly if sucient primer
sequences for the species of
interest are not available.
2. Errors in genotype scoring
occurs if alterations are seen in
primer annealing sites.

5. and genome
mapping.
3. Individual
genotyping.
random dispersion
throughout most
genomes.
3. Readily analysed
by PCR and easily
detected on PAGE.
3. Chances of homoplasy ( some
characters are present in more
than one species but not present
in their common ancestor
because of convergence
evolution).
4. ISSR (Inter Simple Sequence Repeat):
It was reported by Ztetikiewicz et al. in 1994. ISSR is a single primer designed from SSR
region that act as forward & reverse primer and amplify the region between two closely
spaced and oppositely oriented SSRs.
Process: Primer designed from SSR region Primers are annealed with DNA & PCR
amplification done ISSR amplified product obtained Analyse through Gel-
electrophoresis.
Applications Advantages Disadvantages/Limitations
1. Study of gene
mapping, gene
tagging, distinct strain
identication, and
parental recognition.
1. ISSR doesn’t require
previous knowledge
of genome.
2. Highly polymorphic.
1. ISSR has less
reproducibility and
non-homology of
identical.
2. Dominant marker.
5. SCAR (Sequence Characterized Amplied Region):
SCAR is based on PCR-agarose gel electrophoresis. It uses longer primers of 15-30
nucleotides yielding high reproducibility. It needs prior sequence information for primer
designing. As PCR is used, it needs only low quantities of template DNA.
Applications Advantages Disadvantages/Limitations
1. For studying gene
mapping and
largely used in
plant selection
studies.
1. Simple, reliable and
reproducible.
2. Codominant in nature
and locus-specic
1. Sequence information
needed.
2. Require effort and
expense for designing
specific primers.
6. CAPS (Cleaved Amplied Polymorphic Sequence):
CAPS marker was described by Konieczny and Ausubel in 1993 for genetic mapping. CAPS
is identical to RFLP and referred as PCR-RFLP.
Process: Amplification of DNA by PCR Production of monomorphic PCR products
Digestion by restriction endonuclease to show polymorphism Screening through
electrophoresis by staining with ethidium bromide.

6. Applications Advantages Disadvantages/Limitations
1. Used for gene
mapping.
1. Codominant in nature
and are locusspecic.
1. Sequence information
needed.
2. Low level of
polymorphism.
7. ESTs (Expressed Sequence Tags):
ESTs are molecular markers synthesized by partial sequencing of random cDNA clones.
Once cDNA is synthesized, then a few hundred nucleotides (500 - 800bp) from either end (3’
or 5’ end) is sequenced to create two dierent kinds of ESTs. Sequencing of the 5’ end of
cDNA produces a 5′ EST which usually codes for a protein. These regions tend to be
conserved across species and do not change much within a gene family. Sequencing of 3’ end
of cDNA produces a 3′ EST which is non-coding, or untranslated regions (UTR) and it is less
conserved among species. Therefore, ESTs are sub-sequence of cDNA which represents tag
for the entire cDNA.
Applications:
 EST is used for the whole genome sequencing and studying gene of interest.
 It is used for cloning gene of interest and gene mapping.
8. SCoT (Start Codon Targeted) polymorphism:
This method was developed based on the short conserved region flanking the ATG start
codon in plant genes. SCoT uses single 18-mer primers in polymerase chain reaction (PCR)
and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose
gel electrophoresis.
Applications:
 To assess genetic diversity and structure.
 Identify cultivers and QTL mapping.
 DNA fingerprinting in different species.
9. SNP (Single Nucleotide Polymorphism):
SNP was invented by Lander in 1996. SNP is formed when any alteration/mutation occurs in
single nucleotide (A, T, C, or G). The point mutation as such substitutions, insertions or
deletions in single nucleotide it represents SNP. SNPs are based on hybridization of detected
DNA fragments with SNP chips (DNA probe arrays) and the SNP allele is named with
respect to the hybridization results.
Applications:
 Widely used in biomedical research for comparing the case and control groups of
disease.
 It is also used in studying phylogenetics, genetic variation etc.

7. Applications of molecular marker:
 It plays a crucial role in gene mapping by identifying the position of linked genes in
the chromosome which inherited together.
 It also detects any alteration in a sequence of DNA or any genetic oddity. It ascertains
genes involved in genetic disorders.
 It is used to determine different characters in a DNA sequence which is used to
distinguish between individuals, populations or species.
 Different types of genetic polymorphism can be used as genetic markers.
 Used in forensic for criminal identification, paternity test etc.
 Very useful in diagnosing genetic diseases, treatments.
Disadvantages of molecular markers:
 Costly and time consuming process.
 Lack of detectable polymorphism in certain crops.