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It's about disease detection and
Typology: Schemes and Mind Maps
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A disease, in molecular sense, can be defined as any
abnormality in the living system.
The abnormality can be caused due to infection by virus,
bacteria, fungi, parasites, etc.
The abnormality can also arise due to changes in the
molecular structure within the cells.
There are two classes of molecular diagnostic
techniques:
(1)DNA detection methods —which uses nucleic acid
hybridization or the polymerase chain reaction to detect a
specific nucleic acid sequence.
(2) Immunological methods —are based upon the
specificity of an antibody for a particular antigen.
Antibody , also called immunoglobulin, a protective protein produced by
the immune system of an organism in response to the presence of a
foreign substance, called an antigen.
Nucleic acid hybridization:
Hybridization is the process of establishing a non-
covalent , sequence-specific interaction between two or
more complementary strands of nucleic acids into a
single hybrid. There are two types of DNA
hybridization techniques:
a)Radioactive detection system
b) Non-radioactive detection system
Single stranded DNA molecule recognize and specifically bind
to a complementary DNA strand in a mixture of other DNA
strand. This is comparable to a specific key and lock
relationship.
-DNA probe labeled with detector substance is added
-DNA probe pairs with the complementary target DNA
wash unbound DNA probes
Sequence of nucleotide in the target DNA can be identified
Non-Radioactive detecting system:
Principle:
Detection is based on enzymatic conversion of a
chromogenic (colour producing) or chemiluminescent
(light emitting) substrates. Mainly Biotin-labeled
(Biotinylated) nucleotides are incorporated into DNA
probe.
Advantages : Biotin-labeled DNA is quite stable for
about 1 year.
Chemiluminescence detection is very sensitive than
chromogenic detection system
hybridization:
The disease causing organism can be detected very specifically in
biological samples by nucleic acid hybridization i.e. if the nucleic
acid sequence of a disease causing organism is present in sample,
then it can be hybridized with a nucleic acid probe complementary
to the sequence of this target nucleic acid.
For exmple, The parasite Plasmodium falciparum causes malaria in
man. A specific gene (thereby its product) is the causative agent in
this parasite. A complementary DNA probe to this gene is
synthesized chemically with radiolabelled
32
P. This probe is bonded
to a membrane support. Then the biological sample to be analysed
is added, under appropriate conditions of temperature and ionic
strength to promote base pairing between the probe and the target
avidin-biotin system as a powerful tool in biotechnological sciences. Avidin's affinity
for biotin is exploited in wide-ranging biochemical assays, including the replacement
of radioiodine labeled antibodies in radioimmunoassay systems, to give an assay
system which is not radioactive.
b) Diagnosis of genetic disease using restriction
endonuclease:
Sickle-cell anemia is a genetic disease due to the change in a
single nucleotide in the codon for the sixth amino acid of the
beta-chain of the hemoglobin molecule. Individuals containing
the sickle gene can be screened before the expression of the
symptoms.
The principle for the detection is that, within the beta-globin
gene of a normal individual, there are three sites for the
restriction endonuclease Cvn -1, but in sickle-cell gene one of
these sites is lost due to replacement of the single nucleotide.
In the normal gene, the DNA sequence is CCTG A GG whereas
in the sickle-cell anemia gene, the sequence is CCTG T GG.
d) Detection of mutations at different sites within one gene:
Beta-thalassemia is a genetic disease that is caused due to mutation in
beta-globulin at eight or more sites, thus results in low rate of its synthesis.
Hence instead of detecting each mutation separately all the eight sites are
scanned at the same time.
The amplified target DNA is hybridized to the membrane bound probes
under conditions that allow only perfect matches to hybridize. Then
streptavidin with attached alkaline phosphatase is added, the membrane
washed and a colourless substrate is added.
A coloured spot on the membrane appears wherever there is a perfect
nucleotide match between the amplified target DNA segment and one of
the specific oligoneucleotide probes. Where there is no hybridization
(mutant DNA segments) no colour appears.
Importance of DNA Diagnosis of Genetic Diseases
Traditional laboratory tests for the diagnosis of genetic
diseases are mostly based on the estimation of metabolites
&/or enzymes.
Usually done after the onset of symptoms.
DNA analysis can specifically diagnose the inherited disease at
the genetic level.
DNA based tests are useful to discover, well in advance
whether the individuals or their offsprings are at risk for any
genetic diseases.
Example of some Important genetic diseases for which DNA
analysis is used
Common fetal hereditary disease Produce thick and sticky mucus
that clogs lungs and RT.
Defect in CFTR gene that encodes Cystic Fibrosis Transmembrane
Regulator protein located on chromosome 7.
DNA probe has been developed to identify this gene.
Disease developed when 2 recessive genes are present.
Fetal cells obtain from samples of amniotic fluid. It is possible to
know whether the offspring will be victim of CF
Sickle cell anemia :
Occur due to single amino acid change in the β-chain of
hemoglobin. Glutamate at the 6th position of β-chain is
replaced by Valine. This single base mutation can be
detected using restriction enzyme MstII to cut DNA
fragment (RFLP technique) followed by electrophoresis of
DNA fragments. Sickle cell anemia characterized by the
irregular, sickle shape of the erythrocytes. Results in to
anemia
Fragile X syndrome:
Fragile X syndrome due to a genetic defect in X chromosome (a sex
chromosome) affects both males & females. Victims are characterized
by mental retardation.
Have three nucleotide bases ( CGG ) repeated again & again. These
trinucleotide repeats block the transcription process resulting in a
protein deficiency.
This protein is involved in the normal function of the nerve cells, & its
deficiency results in mental retardation. A DNA probe has been
developed for the detection of fragile X syndrome in the laboratory.
P 53 GENE
The gene p53 encodes for a protein with a molecular weight 53
kilodaltons. Thus, p53 is a cancer-suppressor gene and acts as a
guardian of cellular DNA.
BRCA I and BRCA II function in a manner comparable to gene p
protein. E.g., Gene for melanoma susceptibility, in humans are
located on chromosomes 1 and 9
