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GENE (Genetic )Markers.docx RAPD AFLP RFLP SSSR SNP ESR
1. RANJEET MAURYA
Genetic Markers
“Genetic markers are known DNA sequences which can be used for studying any kind of
polymorphism”. OR
A genetic marker is a known location on a chromosome used for the identification of
individuals among and between species.
Thus from the definition, we can say that broadly, the Genetic markers are used,
For studying disease gene
For studying any kind of alterations (polymorphism)
Identification of individuals
Identification of species or organisms
In the present article, we will understand some common markers used in the molecular genetic
technique along with their applications.
What is a genetic marker?
RFLP(Restriction fragment length polymorphism)
AFLP (Amplified fragment length polymorphism)
RAPD(Rapid amplified polymorphic DNA)
STR (Short tandem repeats
ISSR(Inter simple sequence repeat)
SCAR (sequence characterized amplified region)
EST (Expressed sequence tag)
SNP(single nucleotide polymorphism
SSR(simple sequence repeat)
2. What is a genetic marker?
“The genetic markers are often known as DNA marker is a known
DNA sequence located in the genome, used in the molecular genetics
for identification of alteration or DNA sequence from the unknown
DNA sample.”
OR
The genetic marker is a known DNA sequence or gene located on
the chromosome which can be applied in the identification of
individual species or organisms or we can use it in the identification
of other genes or DNA sequences.
1) Before that let me give you a brief idea about how different genetic markers are
developed.
2) If we mark some sentences or paragraphs, we can use them every time in exams,
assignments and other activities.
3) Similarly, by marking a specific sequence of DNA, we can use it in different types of
genetic studies.
Let’s take an example:
3. 1) Can you identify something? 11 basepair sequence is repeatedly observed at several
intervals in a sequence and it is present in all three species.
2) Suppose this sequence is abundantly present in all organisms on earth (assume it). Let me
give some name to this marker by using its characteristics.
3) It is observed one after another, tandemly.
4) It is repeatedly observed after a regular interval of sequences hence it is a repeat
sequence.
5) Though the sequence is the same, sequence numbers are different in all three species i.e,
4 repeats in species A, 5 repeats in species B and 7 repeats in species C. So the number
of repeats is variable.
4. 6) Now collect all three characters in a single set: it is a sequence, arranged tandemly,
repeated one after another and variable so we can name it as variable numbers of tandem
repeats.
RFLP: Restriction fragment length polymorphism
With the help of the Restriction digestion by REase, alterations in the same/
homologous DNA sequences can be detected by analyzing fragments of
different lengths, digested with a restriction enzyme.
A single restriction endonuclease gives more specific results by cutting at one specific locus and
producing fragments of different lengths. See the figure on how different DNA fragments are
created by Restriction digestion.
5. 1) Firstly, let us understand the terminology RFLP, alteration (polymorphism) in the length
of different fragments (of DNA) can be analyzed using restriction digestion.
2) Restriction digestion is performed by endonuclease enzymes. It cuts DNA at its specific
restriction site. Millions of restriction sites are present for individual RE in the human
genome.
3) The length of different fragments is identified using blotting, which is now replaced by
sequencing. RFLP is applicable in disease identification, genetic mapping, heterozygous
detection and carrier identification.
4) The RFLP markers are highly locus-specific and co-dominant.
5) Why RFLP markers are locus-specific?
6) Because of the nature of the REase used in the rflp.
7) As we discussed in the previous article, the REases can cut at a specific location where
their restriction site is located. One restriction digestion site generates two different
fragments.
8) Further, it is co-dominant because both alleles in the heterozygous condition (mutant as
well as normal) can be detected by rflp.
9) There are two variations in the RFLP that are most common.
10) The traditional method of the RFLP is the blotting-based probe hybridization method.
11) The PCR-based RFLP method is a newer version, easy and rapid.
6. 12) In the traditional method, the labeled DNA probes called an RFLP probe, used to
hybridize with the digested fragments by the Southern blotting analysis.
13) The polymorphism can be detected by analyzing the hybridization pattern of the
labeled probe using autoradiography
14) In the PCR-based RFLP method, the gene of our interest is amplified using the
polymerase chain reaction and proceed for the restriction digestion using the specific
REase.
15) The results are analyzed using agarose gel electrophoresis.
16) No tedious labeling and hybridization techniques are required in this method.
17) The PCR-RFLP method is easy, simple, time-efficient and reliable.
18) Polymorphism in the alleles of a gene such as MTHFR and HBS can be detected using
PCR- RFLP.
Application of RFLP:
It is used in polymorphism study, forensics, paternity verification, hereditary disease diagnosis
and DNA fingerprinting and genome mapping.
AFLP: Amplified fragment length polymorphism
The AFLP technique was developed by Zabeau and Vos in the year 1993. The method is based
on the amplification of the selected fragments followed by the digestion of the total genomic
DNA of the selected organism.
7. The process of the AFLP is restriction digestion of the genomic DNA, adapter ligation of the
digested DNA fragments and amplification of DNA fragments in PCR using the adapter specific
primers.
The method was originally developed by the keygen in 1990.
1) The genomic DNA is extracted using one of the DNA extraction methods.
2) The DNA is digested with the help of the restriction endonuclease
3) The digested DNA is ligated with the short adapter sequences. The sequence of the
adapters is known.
4) The fragments are amplified using a set of primers.
5) The results are analyzed on agarose gel electrophoresis followed by autoradiography or
we can use the PAGE technique to differentiate multiple fragments of DNA.
6) The AFLP technique is highly applicable in Crop improvement programs and genomic
analysis of different types of crop varieties.
7) As the primers are based on the adapter data, no previous data on the DNA sequences are
required to construct a marker.
8) Due to the high priming capacity of the primers, it is also used in phylogenetic studies for
different plant varieties.
8. Advantages:
No prior sequence information is required in the AFLP.
Also, the sensitivity, reproducibility and resolution of the AFLP marker are higher as
compared with other markers.
Disadvantages:
The AFLP marker is not co-dominant hence it can not distinguish homozygous from
heterozygous.
RAPD: Random amplification of polymorphic DNA
By using the short, arbitrary DNA primers, the part of the genomic DNAs is randomly
amplified.
9. Pronounced as a “rapid”, it does not require any previous sequence information, the primer of 8
to 12mer randomly amplifies the region of the genomic DNA.
If any mutation or alteration occurred in the region of the primer binding site, the primer can not
bind to that location or the amplified fragment length decreases or increases.
A different pattern of amplification is observed in different sample types. Hence the RAPD can
help in the polymorphism studies.
Detection of RAPD:
The RAPD fragments are between 0.2 to 5.0kb. It can be observed using the ethidium bromide-
stained agarose gel or it can also be analyzed on polyacrylamide gel electrophoresis.
Advantages:
The RAPD markers are abundantly present in the genome and are distributed throughout
the genome.
10. No previous sequence information is needed.
It can amplify a low quantity of DNA.
Disadvantages:
The marker is not locus-specific.
The sensitivity of the RAPD is also lower.
The reproducibility is also very low.
Homozygous and heterozygous are not easily distinguished.
The RAPD results are difficult to interpret because the short primer can amplify any of
the random sequences presented into the genome.
ISSR: Inter simple sequence repeat
The ISSR marker is the inter simple sequence repeat, the method is based on the PCR agarose
gel electrophoresis, detected using the single primer amplification reaction.
Between the oppositely oriented microsatellites, the sequence of 100 to 3000bp fragments of a
sequence is called the ISSR.
Here the advantage of using the ISSR is that no prior sequence information is required for
constructing the primers.
Multiple loci are amplified because of the random distribution of ISSR within the genome.
Also, the low quantity of templates is sufficient to do PCR.
However, it has lower reproducibility and non-homology of similar-sized fragments.
Because of the simple setup of the ISSR, it is employed in gene mapping studies, clone
identification, different strain identification and parental verification.
11. It is a highly polymorphic marker and is used in genetic diversity studies, gene tagging,
evolutionary study and phylogenetic studies.
In this technique, the flanking non-repeated regions of the inversely oriented microsatellites are
amplified using a single primer or pair of primers.
STR: Short tandem repeats
The short tandem repeats/ simple sequence repeats are also called a microsatellite. The
microsatellite is the shorter repeats sequences present into the genome thousands or millions of
times.
In other words, we can say that the STRs are tandem repeats but are much shorter than the
VNTR. The repeats in STR are 2 to 6 base pairs per repeat.
The STR is highly polymorphic, the number of repeats differs between individuals even though
the loci are homologous. Because of this reason, the STR marker is the first choice in forensic
analysis.
12. Due to the difference in the repeat number each individual has a different STR profile.
SCAR: Sequence characterized amplified region
The SCAR stands for sequence characterized amplified region.
It is easy to use, reliable and reproducible.
The present marker is based on the PCR-agarose gel electrophoresis, uses longer primers of 15-
30 nucleotides hence the reproducibility increases anonymously.
It is a locus-specific and co-dominant marker.
13. Unlike the RAPD, it required sequence information for designing the primer for SCAR.
It is widely used in plant marker assistance selection studies.
EST: Expressed sequence tag
Expressed sequence tag is a 500 to 800bp sub-sequence of a short stretch nucleotides of the
expressed gene or the cDNA.
A small segment of the entire cDNA library is sequenced for the generation of the EST.
The EST marker is actually a short stretch of the individual cDNA, which represents a tag for the
entire cDNA.
This tag can be used in the study and identification of the entire gene, thus is no need to sequence
an entire gene.
We will discuss more on the EST in some other articles.
SNP: Single nucleotide polymorphism
SNP is a single nucleotide polymorphism, a type of unique marker in comparison with other
markers enlisted here.
Due to the addition or deletion of a single nucleotide in a genome, the SNP originated.
Millions of SNPs are present in the genome, however, they may or may not be pathogenic.
The SNP marker is used in the Genome-wide association studies.
14. By comparing the case and control groups, the association of a particular SNP associated with a
particular disease can be determined.
For more detail read our article on SNP: An Introduction To Single Nucleotide Polymorphism
(SNP).
SSR: Simple sequence repeat
A simple sequence repeat is a microsatellite repetitive DNA marker, typically ranging from 5 to
50 times in a genome.
The marker is highly variable. It is more similar to the short tandem repeats. We had discussed
STR in the above segment.
15. The SSR marker or the microsatellite marker is co-dominant and restriction digestion is not
involved in this process thus, advantageous over the RFLP.
It is a marker based on the polymerase chain reaction.
Applications of genetic markers:
The genetic markers are employed in the identification of traits related to disease or inherited
abnormal conditions.
The genetic markers are also utilized in the identification of species, individuals or organisms.
Thus the DNA markers like the VNTR and STRs are one of the first choices in DNA
fingerprinting, parental verification and maternal cell contamination.
It is also applicable in population studies and genetic variations studies.
The markers like RAPD and SSCP have great value in plant genetic studies such as plant
hybridizations studies, DNA barcoding, plant breeding, creating stress-resistant species, etc.
Furthermore, genetic markers can also be used in medicinal plant research and phylogenetic
analysis.
It is frequently used in marker-assisted breeding programs.
RFLP, SNP and AFLP like genetic markers are now routinely used in genetic disease analysis
and diagnosis.
Furthermore, gene mapping and genetic screening can be done using genetic markers.
The techniques like AFLP have great value in the characterization and identification of genetic
variations in closely related species.
16. Besides these several other applications are:
1) Quantitative analysis of bio-sample.
2) Species and speciation studies.
3) Sex determination can be done using the Y-specific markers.
4) Quantitative trait loci identification and characterization.
5) Seed testing.
6) Marker-assisted selection.
7) Cloning and gene transfer experiments.
8) The SNP-like genetic markers have great value in genome-wide association studies.
PGR Use Management
Sources:
Young, N.D. Plant Growth Regul (1993) 12: 229. https://doi.org/10.1007/BF00027203
Raza. S., Shoib. W. and Mubeen H. “Genetic markers: importance, uses and
applications.” International Journal of Scientific and Research Publications; 2016: 6(3):
221-223. Link.