MOLECULAR MARKERS (Aid for Genetic Analysis)
MOLECULAR MARKERS (Aid for Genetic Analysis)
1 Ms. Iqra Ahmad 2 Dr. Muhammad Naeem
1 Biochemistry 2 Business Administration
Laureate Folks
International
https://laureatefolks.blogspot.com;
laureatefolks@gmail.com
1
Introduction
Various
genotypes have not been distinguished at the phenotypic level due to the low
genome coverage and marker dominance. Initial work on biological markers to
find genomic variations on animals was based on morphological and biochemical
markers. According to Batley and Edwards (2009) weaknesses have been seen in
both of these techniques over time. Morphological markers are dependent on age,
limited to gender, and can be modified by the environmental influence whereas,
biochemical markers have been reported to show a low degree of polymorphism (Batley & Edwards, 2009). Molecular
biology has become advanced to deal with such problems and has come up with
various significant techniques to develop molecular markers. With the advanced
tools in molecular biology, important evolutionary, taxonomic and ecological
research questions can be answered. Moreover, the applications of molecular
biology in statistics and population genetics have provided insights for the
more efficient development of livestock. According to Beuzen et al (2000), the
efficiency of methods related to population genetics has increased due to their
drawback of measurement in low heritability. Only those characteristics are
selected that can easily be detected in a large animal population (Beuzen et al., 2000).
There
can be several criteria for the selection of animals from a large population.
Traditional selection methods were less efficient as they involved using
various unfavourable genetic correlations such as protein content in milk
production. Also, the characteristics like the survival rate are lately
expressed and can be considered as a productive criterion for the selection.
Teneva
in 2009 reported that various advanced molecular biology techniques can detect
polymorphisms and variations in the selected individuals of a population. These
populations include specific portions of DNA. Such variations can be used to
make the genetic map and to measure differences among possible genetic markers
to find the effects of these variations on traits. Gene mapping by molecular
biology techniques has also been used. In this way, different kinds of
molecular markers have been developed. Different problems arise during this
process and with time some of these problems are solved and simplifications
have been developed in the process of developing molecular markers. Thus new
ways have been established to study breeding in animals and genetics in
livestock (husbandry & 2009, 2009).
As
aforementioned, there are limitations of chromosomal and biochemical markers
(i.e. protein markers). Detection of genetic variations at genetic levels by the
use of molecular markers has eliminated these limitations. Molecular markers
are in huge number within the genome and can provide a huge amount of
information as well. Molecular markers are heritable, this advantage makes them
the reason to be chosen over phenotypic biomarkers.
2
Types
of Molecular Markers and Their Applications:
Analysis
of quantitative traits is done by molecular markers by use of their property to
detect the position and function of genes. The use of different molecular
markers is discussed below.
2.1
Restriction fragment length polymorphism:
Restriction
fragment length polymorphism or RFLP is used for the detection of mutations at
specific small regions of DNA. These small regions are called restriction sites
(Botstein et al., 1980). It is a
technique that can characterize and differentiate different species according
to the length of fragments obtained by restriction enzymes. Restriction enzymes
are endonucleases that cleave or hydrolyze DNA at particular locations. Different
lengths are achieved by cleavage of DNA of different species by the same endonucleases.
RFLP is a very efficient genetic marker that was developed to find
polymorphisms at the DNA level (Marwal & Gaur, 2020).
2.1.1
Steps involved in RFLP analysis:
1. The
first step is the extraction of DNA for any biological sample. The sample can
be blood, semen, saliva, etc.
2. The
extracted DNA is then purified and exposed to restriction endonucleases. For
example, the restriction enzyme cuts the DNA wherever AATT occurs. Every species
has its own set of genetic bases and the repetition of bases differs as well.
The restriction enzyme cuts on every AATT and DNA converts into small fragments
depending upon the position of the AATT pattern in the DNA.
3. DNA
has a negative charge so do the resulting fragments of DNA. These fragments are
passed through the gel electrophoresis. On gel, they move towards the positive
electrode and their speed depends on their size. Small fragments reach the
bottom faster leaving the larger fragments on the upper side of the gel. Thus
they separated on the base of their size, but are not visible yet.
4. Nylon
membrane is put on the porous support and it is covered by the plastic mask.
The gel is slid carefully on the mask. The apparatus is closed and the pump is
started.
5. Prehybridization
takes 4 to 5 hours generally, but if the gel is being used for the first time
prehybridization may require a night at the incubation of 65C. After that
hybridization is performed by adding labelled and boiled probes in the
prehybridization.
6. Incubate
the gel overnight. Add luminescent dyes to visualize the bands of RFLP.
An
overview of this process is shown in flowchart 1.1
2.1.2
Applications of RFLP:
RFLP has a role in the detection of
heritable diseases and genetic mapping. If a patient is diagnosed with a
disease and the route of the disease is found to be genetic the researcher
would analyze the DNA of the patient by RFLP and use this pattern as a
reference to find if someone else in his/her family has the same pattern at the
particular location. The disease can be detected before the symptoms and can be
treated before getting worse. The persons who are at risk of getting the
disease as well as those who are carrying the mutant gene but are the carriers
can be identified.
Early methods of genetic fingerprinting
used the RFLP technique. Genetic fingerprinting is a tool famous for forensic
sciences like for the determination of the samples from the criminals or for
the detection of paternity. It is also used for the identification of diversity
in animals and their breeding patterns.
2.2
Allele-specific
oligonucleotide:
Allele-specific oligonucleotide (ASO) is
a small piece of DNA, 15-20 bases long. ASO is single-stranded and is specific
for a particular portion of DNA or allele. In other words, they are
complementary to the DNA and bind to their sites when added with that particular
DNA target. In Southern blotting, they act as the prob (Marwal & Gaur, 2020).
2.2.1
Applications of allele-specific
oligonucleotides:
They are used in Human Genome Project.
ASO is an effective tool for forensic research and molecular biology. They are
useful for genotype analysis.
The recessively inherited trait of
yellow colour coat in a dog breed called Labrador retriever is due to mutation.
This mutation was first detected by ASO testing. Later on, Golden retrievers
were found to be homozygous for that mutation (Everts et al., 2000).
2.3
Allele-specific
polymerase chain reaction:
Polymerase chain reaction work on the
principle of using a primer from the unmutated or invariant part of the genome
to amplify the portions of DNA near it. Whereas in allele-specific polymerase
chain reaction the strategy is opposite. In this reaction, the primers are
synthesized from the mutated or polymorphic part of the genome located at the 3’
end and amplify sequences neat to it. The primer should be completely matched
to the sequence near the targeted sequences to initiate the replication.
Otherwise, the transcription will not start. The appearance of the amplified
product is the sign of the replication of desired genotype (Wangkumhang et al., 2007).
2.3.1
Applications of an allele-specific
polymerase chain reaction:
The allele-specific polymerase chain
reaction is a relatively easy and cheap method for genotyping SNPs (Single
nucleotide polymorphism) and mutations in the DNA. Population and molecular
genetics as well as pharmacogenomics have applied this tool in recent studies.
b-casomorphin
is an abnormal bioactive peptide that is released from the b-casein gene upon
digestion. The bovine b-casein gene has two variants A1 and B. Both of these
variants may or may not encode b-casomorphin if they are mutated or normal
respectively. An allele-specific PCR (AS-PCR) was designed to differentiate
between them. AS-PCR has successfully distinguished between these two variants
in 41 out of 42 animals in a study (Aileen F Keating, 2008).
2.4
Random
amplified polymorphic DNA:
Random amplified polymorphic DNA (RAPD)
is based on PCR for the generation of primers and molecular markers. As a
result of RAPD of distinct species distinct bands appear which describes these
distinct species as Mendelian genetic markers. Only one primer is used and it
anneals at different sites on DNA. Only those sequences are amplified where
annealing occurs. Thus the number of amplification fragments on the gel depends
on the presence of annealing sites on DNA. Various fragments of DNA are in the
end detected on agarose gel and are called DNA fingerprints (Marwal & Gaur, 2020).
A single sequence of the oligomer is
used as a primer and provides DNA fingerprints. If the sequence of the oligomer
is changed different lengths of DNA fragments are obtained and a different DNA
fingerprint is viewed. Thus an enormous number of loci can be detected if we
keep on changing the sequence of the primer. This makes RAPD different from
other molecular markers. In contrast, its limitation can be the dominant
expression of alleles, which makes it hard to interpret multi-locus patterns.
Thus, RAPD markers can be defined as
amplification products of unknown DNA base sequences by use of a short, single
and random oligonucleotide sequence (as a primer) and they do not need any
previous knowledge of a DNA base sequence (BARDAKCI, 2001).
2.4.1
Steps involved in random
amplified polymorphic DNA analysis:
1.
2.
|
Flowchart
1.2 showing the analysis of RAPD (Marwal & Gaur, 2020) |
3. The next step is performed in a DNA
thermocycler. Oligonucleotide primers are added for amplification in standard
PCR settings.
4. DNA
has a negative charge so do the resulting fragments of DNA. These fragments are
passed through the gel electrophoresis. On gel, they move towards the positive
electrode and their speed depends on their size. Small fragments reach the
bottom faster leaving the larger fragments on the upper side of the gel. Thus
they separated on the base of their size, but are not visible yet.
5. For
visualization of the separated bands UV light is used. In the end, the genetic
marker is used to find the size of fragments. This genetic marker is a DNA
ruler. An overview of this process is shown in flowchart 1.2
2.5
Amplified
fragment length polymorphism:
Amplified fragment length polymorphism
or AFLP is another very efficient technique to detect polymorphism. This method
has applications in population genetics and kinship analysis. Also, it is used
in systematic analysis. With the use of gel scanners homozygotes can be
distinguished from heterozygotes. As the word amplified describes,
amplification of DNA fragments occurs during AFLP analysis. The principle of
this method is PCR (Polymerase Chain Reaction) based amplification with the use
of restriction endonucleases. Specific oligonucleotides adaptors are added at
the end of each fragment and primers complementary for adopters are used for
PCR (Marwal & Gaur, 2020).
2.5.1
Steps involved in the analysis of
amplified fragment length polymorphism:
1. Preparation
of AFLPs starts with the isolation of DNA and digestion from the two
restriction enzymes (endonucleases) simultaneously. Complete digestion from restriction
endonucleases is required for the generation of AFLP. The more the DNA is digested, high the
quality of AFLP is obtained. Also, the digested pieces are the substrates for
the next step. Fragments should be free from any inhibitors or nucleases.
2. Generation
of DNA template for amplification by PCR is the next step. It is done by
inactivating restriction endonucleases. Heat is used for inactivation. After
that adopters are ligated with the small pieces of DNA. The adapters may be
EcoR and Mse . This ligated DNA is ready for amplification. These adoptors
provide the attachment site for the primers. As the sequence of DNA is unknown
and DNA is digested randomly with restriction endonucleases, the primers can
not be added directly to the fragments of DNA. But, with the addition of adapters,
the primers can bind at the end of each fragment and a maximum number of
fragments can be identified.
3. PCR
is then used in two different reactions. These reactions are called
preamplification and selective amplification respectively. At first, the DNA
fragments are attached to the AFLP primers and amplified. Each AFLP primer has
at least one selective nucleotide. The products from the first PCR cycle are
used as the reactants or templates for the second step, selective
amplification. This amplification is selective because two AFLP primers are
used in it and these primers have at least three selective nucleotides.
4. 5% or 6% denaturing polyacrylamide gel is then
used for the separation of the products from selective amplification. The
banding pattern is obtained and it can be used for the detection of
polymorphism as the molecular markers. An overview of this process is shown in flowchart 1.3.
|
Flowchart 1.3 shows the analysis of AFLP (Marwal & Gaur, 2020) |
2.5.2
Applications of AFLP:
Amplified Fragment Length Polymorphism
(AFLP) analysis can identify more than 50 loci in one reaction.
It
takes PCR to next level. The ease of PCR combines with the preciseness of RFLP,
to a new typing technique in AFLP analysis. It can identify DNA of any origin.
The extra-pair parentage frequency was
analyzed in the population of bluethroat (Luscinia svecica momentum). There
were 162 nestlings from thirty-six families as a sample. Three pairs of primers
were used and the probability reached 93%. Although, when the families were
considered independently the probability was reached 99%. According to the
results, there was at least one extra-pair young in 63.8% of all broods. The
results show that extra-pair fertilizations are common. Although, with this
technique, parentage exclusions cannot be attributed to maternity, extra-pair
paternity, or both (Questiau et al., 1999).
The reason is that brood parasitism has
never been described in Luscinia svecica momentum, so it means that the
exclusions are because of the extra-pair males. This research demonstrated that
the dominant markers of AFLP are useful to study the mating system of taxa for
which microsatellite primers are not available (Questiau et al., 1999).
3
Conclusion:
Molecular
genetic analysis and its various techniques have added great understanding about
genetics in animals. Significant knowledge has been discovered about the genetic
basis of behaviour and structure of different animals. One of the major
applications of molecular genetic analysis is its role in DNA
scrutinization. By this scientists can find out unique and similar DNA
sequences among species.
4
References:
Aileen F Keating.
(2008). (PDF) A note on the evaluation of a beta-casein variant in bovine
breeds by allele-specific PCR and relevance to β-casomorphin.
https://www.researchgate.net/publication/237798209_A_note_on_the_evaluation_of_a_beta-casein_variant_in_bovine_breeds_by_allele-specific_PCR_and_relevance_to_b-casomorphin
BARDAKCI, F. (2001).
Random Amplified Polymorphic DNA (RAPD) Markers. Turkish Journal of Biology,
25(2), 185–196. https://dergipark.org.tr/en/pub/tbtkbiology/140160
Batley, J., &
Edwards, D. (2009). Genome sequence data: Management, storage, and
visualization. BioTechniques, 46(5 SPEC. ISSUE), 333–335.
https://doi.org/10.2144/000113134
Beuzen, N. D., Stear, M.
J., & Chang, K. C. (2000). Molecular markers and their use in animal
breeding. The Veterinary Journal, 160(1), 42–52.
https://doi.org/10.1053/TVJL.2000.0468
Botstein, D., White, R.
L., Skolnick, M., & Davis, R. W. (1980). Construction of a genetic linkage
map in man using restriction fragment length polymorphisms. American Journal
of Human Genetics, 32(3), 314.
https://doi.org/10.17348/era.9.0.151-162
Everts, R. E.,
Rothuizen, J., & Van Oost, B. A. (2000). Identification of a premature stop
codon in the melanocyte-stimulating hormone receptor gene (MC1R) in Labrador
and Golden retrievers with yellow coat colour. Animal Genetics, 31(3),
194–199. https://doi.org/10.1046/J.1365-2052.2000.00639.X
husbandry, A. T.-B. in
animal, & 2009, undefined. (2009).
Molecular markers in animal genome analysis. Scindeks.Ceon.Rs, 25(6),
1267–1284. http://scindeks.ceon.rs/article.aspx?artid=1450-91560906267T
Marwal, A., & Gaur,
R. K. (2020). Molecular markers: tool for genetic analysis. Animal
Biotechnology, 353–372. https://doi.org/10.1016/B978-0-12-811710-1.00016-1
Questiau, S., Eybert, M.
C., & Taberlet, P. (1999). Amplified fragment length polymorphism (AFLP)
markers reveal extra-pair parentage in a bird species: The bluethroat (Luscinia
svecica). Molecular Ecology, 8(8), 1331–1339.
https://doi.org/10.1046/J.1365-294X.1999.00703.X
Wangkumhang, P.,
Chaichoompu, K., Ngamphiw, C., Ruangrit, U., Chanprasert, J., Assawamakin, A.,
& Tongsima, S. (2007). WASP: A Web-based Allele-Specific PCR assay
designing tool for detecting SNPs and mutations. BMC Genomics, 8.
https://doi.org/10.1186/1471-2164-8-275
This comment has been removed by a blog administrator.
ReplyDelete