molecular markers

1. Molecular Markers
Dr. Nawfal Hussein
Email: nawfal_hm@yahoo.com
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2. 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. 2

3. 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,3

4.  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:
GAGGGTGGNGGNTCTGAGGGTGGNGGNTCTGAGGGTGGNGGNT
CT
 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.
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5.  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).
 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 favorable5The activation of the lac operon in E. coli.

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7.  Restriction fragment length polymorphism
(RFLP):
 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.
 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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8.  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.
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9. 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. 9

10. 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.
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11. 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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12. Amplified Fragment Length Polymorphism
(AFLPs)
 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. 12

13. 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. 13

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