Muhammad Irfan

PhD Zoology
(04-arid-1150)

BIOMARKERS
Biomarkers are the characteristics that distinguish one individual or population from the
others. Biomarkers can be phenotypic, biochemical, cytological or molecular markers.
Phenotypic markers include colour, texture and shape etc. these markers may appear at certain
age so they are not easy to use. Biochemical markers include carbohydrates, proteins and lipids
biomarkers. Abnormal chromosome number/shapes can be used as cytological markers.

Biomarkers can be used for phenotyping and genotyping of the living organisms by using
phenotypic or molecular characteristics. Evolutionary relatedness of the living organism can be
studied by using these markers. Biochemical markers can be used to detect contamination in
food and water etc. Biomarkers can be used for disease diagnosis and as forensic evidences.

Molecular markers are sites where differences in DNA sequences occur among members
of the same species. They reveal polymorphisms at the DNA level. These differences can be in
either coding or non-coding regions. The differences on the DNA level are single nucleotide
polymorphisms (SNPs), insertions or deletions (Indels) and variable number of tandem repeats
(VNTRs). These differences are due to errors in replication/proof reading or insertion/deletion of
a segment of chromosome.

Molecular markers can be linked markers or direct markers depending on their position in
genome. The linked markers are those markers which are near to a gene but are not the part of
gene. However, direct markers are part of a gene.

There are three generations of molecular markers depending on history and

characteristics. The first ever used molecular marker is restriction fragment length polymorphism
(RFLP). Oldest DNA marker technology that relies on the difference in DNA sequences between
two samples that creates or removes a restriction site. Restriction sites are palindromic sequences
of 4 or more bases which are recognized and cut by specific endonucleases (restriction enzymes).
DNA samples from contrasting individuals or populations are digested witha restriction enzymes
and fragments are separated by gel electrophoresis. DNA is then subjected to Southern blotting
and probe used to span the restriction sites. This technique is obsolete as it takes a weak to be
completed and moreover, mutations may not always alter restriction sites.

A Variable Number Tandem Repeat (or VNTR) is a location in a genome where a short
nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes,
and often show variations in length between individuals. Each variant acts as an inherited allele,
allowing them to be used for personal or parental identification. Their analysis is useful in
genetics and biology research, forensics, and DNA fingerprinting.
Muhammad Irfan
PhD Zoology
(04-arid-1150)
Individual repeats can be removed from or added to the VNTR via recombination or
replication errors, leading to alleles with different numbers of repeats. Flanking the repeats are
segments of non-repetitive sequence, allowing the VNTR blocks to be extracted with restriction
enzymes and analyzed by RFLP, or amplified by the polymerase chain reaction (PCR) technique
and their size determined by gel electrophoresis Main difference is RE does not cut within the
probe but on flanking regions. This technique relies on short sequences (10 - 100 bp) repeated
within the restricted fragment. Differences in the number of repeats can be detected by difference
in length.

A type of PCR reaction, but the segments of DNA that are amplified are random. Several
arbitrary, short primers (8-12 nucleotides) are created, 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 a RAPD reaction. No knowledge of the
DNA sequence for the targeted gene 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 in a system in which relatively few DNA sequences are compared (it is not suitable for
forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has
some limitations in the use of degraded DNA samples. Its resolving power is much lower than
targeted, species specific DNA comparison methods, such as short tandem repeats. In recent
years, RAPD has been used to characterize, and trace, the phylogeny of diverse plant and animal
species.

Microsatellites, also known as Simple Sequence Repeats (SSRs), or sometimes Short

Tandem Repeats (STRs), are repeating sequences of 1-6 base pairs of DNA. Microsatellites are
typically neutral and co-dominant. They are used as molecular markers in genetics, for kinship,
population and other studies. They can also be used to study gene duplication or deletion.

Inter Simple Sequence Repeat (ISSR) marker system is another newly developed method,
which relies on one primer for PCR that anneals to an SSR region and amplifies region between
inversely oriented adjacent SSRs. ISSR assay can be undertaken for any species that contains a
sufficient number and distribution of SSR motifs and has the advantage that genomic sequence
data is not required. This technique amplifies large numbers of DNA fragments per reaction,
representing multiple loci from across the genome; it is an ideal method for fingerprinting
varieties.

Single nucleotide polymorphisms (SNPs) are bi-allelic markers, which unravels

polymorphism between individuals due to change of a single nucleotide. These single nucleotide
variations arise because of point mutations. The detection of SNPs needs only a plus/minus assay
with automation. However, adequate sequence information is necessary.

Cleavage Fragment Length Polymorphism (CFLP) CFLP assay relies on

denaturation (melting) of DNA. The single stranded DNA will assume folded hairpin-like
Muhammad Irfan
PhD Zoology
(04-arid-1150)
structures, which are unique to the nucleotide sequence. The endonuclease Cleavase-I
specifically cleaves the junction between single strand and double strand regions. Fragments are
5' labeled, electrophoretically separated and visualized. Polymorphic fragments indicate a
mutation; their length is indicative for the position of the mutated site.

Retrotransposon Based Insertional Polymorphism (RBIP) is a co-dominant marker

system that uses PCR primers designed from the retro transposon and its flanking DNA to
examine insertional polymorphisms for individual retro transposons. Presence or absence of
insertion is investigated by two PCRs, the first using one primer from the retro transposon and
one from the flanking DNA, the second using primers designed from both flanking regions.
Polymorphisms are detected by simple agarose gel electrophoresis or by dot hybridization
assays. Drawback of the method is that sequence data of the flanking regions are required for
primer designing.

Inter retrotransposon Amplified Polymorphism (IRAP) is also a dominant, multiplex marker

system that examines variation in retro transposon insertion sites. IRAP fragments between two retro
transposons are generated by PCR, using outward-facing primers annealing to long terminal
repeats (LTR) target sequences. Fragments are separated by high-resolution agarose gel-
electrophoresis.

Retrotransposon Microsatellite Amplified Polymorphsim (REMAP) is yet another

dominant, multiplex marker system that examines variation in retro transposon insertion sites.
REMAP fragments between retro transposons and microsatellites are generated by PCR, using
one primer based on a LTR target sequence and one based on a simple sequence repeat motif.
Fragments are separated by high resolution agarose gel-electrophoresis.

There are various factors that influence the purposes of molecular markers in biological
explorations. One of them is abundance that molecular markers should be in abundance covering
the entire genome for the development of high-density linkage maps or genome wide DNA
fingerprinting. The appropriate genetic marker technique having high level of polymorphism
should be employed in genome mapping/DNA fingerprinting. There are two possible types of
markers: markers with a single alternative allele (bi allelic) and markers several alternative
alleles (poly allelic). Markers are classified two groups as single locus markers (unique location
on the genome) and multi locus (several locations on the genome) markers. Single locus markers
are preferred for genome mapping while the markers of multi locus nature are employed for
DNA profiling. Among the single locus markers, markers of poly allelic nature are very useful
for DNA profiling.

Markers of bi allelic nature are considered as co dominant when both the alleles are
observed in the hybrid. If one of the two alleles is observed then the marker is considered as
dominant. Co dominant markers are more informative than the dominant markers since
codominant markers can distinguish heterozygotes from homozygotes. This allows the
Muhammad Irfan
PhD Zoology
(04-arid-1150)
determination of genotypes and allele frequencies at loci more precisely. Therefore, co-
dominant markers are preferred over the dominant for genome mapping. RFLP, mini satellites
and PCR-sequencing require technical skills and facilities to carry out radioactive labeling. In
addition, Southern blot hybridizations are part of the RFLP and mini satellite analysis. These
techniques are therefore among the more technically demanding markers. Technical demands for
RAPD and ISSR markers comparatively less. These days many of the markers resolved with
ease either using silver staining or fluorescently labeled primers.

Because only small quantities of template DNA (5-100 ng per reaction) are required,
techniques which are based on the Polymerase Chain Reaction (PCR) currently are preferred.
RFLPs and minisatellites require the largest amount of DNA (5-10 μg per reaction) but Southern
blots may be re probed several times. Intermediate quantities of DNA are needed for AFLP-
analysis (0.3-1 μg per reaction) because endonuclease restriction of the DNA template precedes
the PCR-reaction. Application of PCR-based markers may be relevant when only small amounts
of DNA can be extracted, e.g. when working with tiny organisms. Currently, techniques, which
can be automated, are preferred because they enable increased sample throughput. Although
considerable financial investment still is required, automation may be cost effective when
techniques are applied on a routine basis. Nearly all techniques, which are based on the
Polymerase Chain Reaction (PCR), are amenable to automation. Marker development may be
very time consuming and costly when necessary probes or sequence data for primer construction
are unavailable. In addition, sufficient technical skills and facilities need to be present.
Development of suitable probes for Southern blot hybridizations (e.g. for RFLP-analysis)
requires the construction of a genomic library, the isolation of DNA fragments and the
examination of various probe/restriction enzyme combinations for the ability to detect
polymorphisms.