DNA markers

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Molecular Markers
♠ DNA polymorphisms: genetic markers produce measurably different phenotypes,
either at the visual level or at the biochemical level. With the rapid development of
molecular techniques that permit examination of precise details of DNA sequence, it
has become possible to use other types of changes in DNA sequence, including
naturally occurring differences among individuals, as genetic markers. Differences
among individuals at particular genomic sites that can be used as genetic markers are
commonly referred to as polymorphisms.
♠ Molecular (DNA) marker: piece of DNA that may be different between organisms , or
Locations or addresses along chromosomes .Like addresses on a road or street map
, Genetic markers can be used for a wide variety of purposes, ranging from chromosomal
mapping to forensics and pedigree analysis.
♠ Types of Molecular (DNA) markers:
 Non PCR-based markers : such as RFLPs
 PCR-based markers: such as RAPDs, SSRs, AFLPs, SNPs etc………
Tandem repeat (satellite DNA) A sequence of nucleotides repeated one or more
times, consecutively, in the same molecule.
Minisatellites (Variable number tandem repeats (VNTR):
 Segments of DNA consisting of short tandem repeats (10-80bp) (ex:
GAGGGTGGNGGNTCTGAGGGTGGNGGNTCTGAGGGTGGNGGNTCT
 Typically there may be from five to 50 tandem repeats. In mammals, VNTRs are
common and are scattered over the genome, although they tend to be found close to
the telomeres.
 Due to unequal crossing over, the number of repeats in a given VNTR varies among
individuals. Although VNTRs are non-coding DNA and not true genes, nonetheless
the different versions are referred to as alleles.
 Some hyper-variable VNTRs may have as many as 1,000 different alleles and give
unique patterns for almost every individual. This quantitative variation may be used
for the identification of individuals by DNA fingerprinting.
Microsatellites (Short tandem repeat polymorphism (STRP):
 Regions of DNA that consist of multiple tandem repeats of short sequences of
nucleotides (2–10 bp) (ex CACACA….., notated as (CA)n.
 It is found in various genomes (including e.g. human, fungal, bacterial)
 It is vary in length (i.e. in number of repeated subunits), even in closely related
subjects, and are useful e.g. as genetic markers and in forensic profiling. A given
microsatellite sequence may exhibit expansion (i.e. increase in the number of
tandemly repeated units) or contraction (involving deletion of nucleotides).

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 Variation in the number of repeated units may arise e.g. by slipped-strand
mispairing; microsatellite DNA is found in both coding and non-coding sequences of
certain genes, and such instability may give rise to disorders such as huntington’s
disease, myotonic dystrophy and fragile x disease.
 Make good genetic markers because they each have many different 'alleles' - ie.
there can be many different lengths of the repeat region. An allele is defined by
the number of repeats there are at the same location. With many alleles, most
individuals are heterozygous, giving power to note association between marker
allele and performance in progeny inheriting a favorable linked QTL allele.
♠ Minisatellite and Microsatellite markers can be detected through the use of restriction
endonucleases that cut on either side of the repeat, followed by Southern blotting and
detection with a probe specific for the repeated sequence. Alternatively, Through the
PCR reaction, which uses the unique sequences either side of the repeat sequences
as primer binding sites, microsatellite DNA can be specifically amplified. The
individual alleles carried at particular microsatellite loci can then be determined by
accessing the size of the amplified fragment through agarose gel electrophoresis.
Restriction fragment length polymorphism (RFLP):
o Restriction enzymes: enzymes cut DNA wherever they find the appropriate
nucleotide sequence (eg. Eco R1 cuts at the 'recognition sequence' GAATTC). If
there is a mutation at this sequence, no cut is made and the resulting DNA
fragment is longer. Also mutation to give a new recognition sequence gives a pair
of shorter fragments. Genetic differences (polymorphisms) of this type are known
as Restriction Fragment Length Polymorphisms.
o When a specific cloned DNA probe is used to analyze a Southern blot of human
(or other) DNA, a limited number of restriction fragments of specific and
characteristic lengths will be identified. Because single base mutations can either
create additional restriction sites or destroy pre-existing sites, DNA preparations
from different individuals frequently exhibit different patterns of size distribution of
restriction fragments that hybridize with a particular probe. In many cases, the
genetic polymorphisms that generate RFLPs will have no obvious phenotypic

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effect because they are located in introns or involve "silent" mutations that convert
a codon to different codon specifying the same amino acid.
o Annonymous probes: One major advantage of RFLPs as genetic markers is that
they do not need to have any special properties other than the availability of a
probe that can be used to visualize the alternative patterns of restriction fragments
that are obtained depending on whether a particular cut site is present or absent.
Probes that do not correspond to any known genes are referred to as
annonymous probes. Many useful human RFLPs are identified with annonymous
probes.
o Detection of sickle cell anemia heterozygotes by RFLP: In special cases, the
DNA sequence associated with a genetic disease may generate an RFLP. The
example is sickle cell anemia, which is caused by a one nucleotide substitution in
the coding sequence for beta-globin that converts a glutamic acid to a valine. The
normal allele for beta-globin contains a cut site for the restriction endonuclease
DdeI, which is missing from the sickle cell mutant form. When a Southern blot is
probed with a partial beta-globin sequence that spans the polymorphic cut site,
normal hemoglobin allele yields 2 restriction fragments 175 and 201 nucleotides
long, whereas the sickle cell allele that lacks the cut site yields a single fragment
that is 376 nucleotides long. DNA from an individual who is heterozygous exhibits
both patterns codominantly. Thus the probe identifies bands corresponding to
fragment lengths of 175, 201, and 376 nucleotides. This makes it possible to use
RFLP analysis to determine whether or not healthy siblings (and other relatives) of
a sickle cell anemia patient are heterozygous carriers of the sickle cell allele.
Single Nucleotide Polymorphisms (SNPs): Single Nucleotide Polymorphisms are
based on single base pair polymorphisms. A SNP is a position at which two alternate
bases occur at appreciable frequency. In humans they may number greater than one
in a thousand base pairs. SNPs can be detected by a number of methods; however
a relatively new technology, using DNA chips, can be used for large scale screening
of numerous samples in a minimal amount of time.
Random-amplified polymorphic DNA (RAPD): RAPD begins with a single primer
for PCR that is only about 10 nucleotides in length. Such a primer has about a one in
a million chance of binding at any particular site in a human genome, which means
there are about 3000 such sites. The chance of a second binding on the
complementary strand close enough to support PCR is quite small, such that about 4-
8 amplification products are typically obtained, even with reduced stringency during
the annealing phase. The patterns that are obtained have substantial individuality,
since a one base mutation is likely to be enough to create or destroy a site under the
conditions that are used. In this case, the pattern of inheritance is dominant, since the
assay only sees the presence or absence of a band, making it impossible to
distinguish homozygous positive from heterozygous. However, because of its speed,
RAPD is often used in preliminary testing.

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Amplified Fragment Length Polymorphism (AFLPs)
o AFLP is based on PCR amplification of selected restriction fragments. Like
RAPDs, AFLPs require no prior knowledge of DNA sequences (unlike
microsatellites). The advantage of AFLPs over RAPDs is that they are more
reliable and reproducable (depend less on DNA quality and lab conditions). Also,
the number of polymorhpic loci (molucular markers) that can be detected is 10-100
times greater with AFLPs than with microsatellites or RAPDs.
♠ DNA profiling (DNA fingerprinting) :
 DNA fingerprinting: is distinguishing individuals with DNA analysis. With
appropriate combinations of the procedures described above, it is possible to
identify sets of DNA markers (RFLP, VNTR, STRP, RAPD, and others) that are
highly individualistic. Techniques such as these have made possible a procedure
that has come to be known as DNA fingerprinting, which is now widely used in
criminal investigations to match blood, hair or semen left at a crime scene to that of
suspects. When done properly, such techniques can identify a specific individual
with virtual certainty. Another area where DNA fingerprinting is extremely useful is
in determining paternity. Non-human applications are also possible.
Figure Southern blot of a forensic DNA sample.
The DNA samples from the victim, the defendant's
shirt, and the defendant were treated with the
same restriction enzyme. Here, the banding
pattern of the DNA extracted from the blood on the
defendant's shirt is identical to the victim's DNA
banding pattern and different from the defendant's
pattern. The sizes of the DNA molecules in these

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Principle of multi-locus DNA fingerprinting. Independent mini-satellite loci A, B,
and C are members of one VNTR family related to each other by a small variation in the
common core sequence of 1,000 bp. Each locus has a different number of repeats on
each homologous chromosome, designated as A1 and A2, B1 and B2, and C1 and C2.
In order to generate a DNA fingerprint, DNA is cut with restriction endonuclease that
does not have a recognition site on any repeat. This generates a set of DNA fragments of
different sizes, which is a consequence of the different number of repeats present at a
particular locus. Thus, locus A1 will be represented by 9 kb fragments, locus A2 by 8 kb
fragments, locus B1 by 5 kb fragments, etc. These fragments are separated by agarose
gel electrophoresis, transferred to a membrane, and hybridized to a probe
complementary to the repeated element. The autogram shows a set of hybridization
bands that represent hybridization to each member of the VNTR family. This is called a
DNA fingerprint.