genome-scale DNA and RNA sequencing has
revolutionized biology and is having increased
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impact on clinical medicineManipulation of a DNA sequence and construction
of chimeric molecule provides a means of studying
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how a specific segment of DNA controls function
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Understanding molecular genetics technology is
important for several reasons:
1. it offers a rational approach to understand the
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molecular basis of disease. For example, familial
hypercholestrolemia, sickle cell disease, the
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thalassemias, muscular dystrophy as well asmore complex multifactorial diseases like
vascular and heart disease, Alzheimer
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disease,cancer,obesity and diabetes
2. Human proteins can be produced in abundance
for therapy
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3. Proteins for preparation of vaccine and for
diagnostic testing can be easily obtained
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4. This technology is used both to diagnose existing
diseases as well as to predict the risk of developing a
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given disease and individual response topharmacologic therapeutics-so called personalized
medicine
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5. Special techniques have led to remarkable advances
in forensic medicine, which have allowed for the
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molecular diagnostic analysis of DNA from singlecells.
6. Finally, in extremely well understood disease,
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potentially curative gene therapy for disease cause by
single gene deficiency
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Normal gene Variations
There is normal variation of DNA sequence just as it is
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true of more obvious aspect of human structure
Polymorphisms, occur approximately once in every
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500 to 1000 nucleotidesThere are also genomic deletions and insertions of
DNA as well as single base substitutions.
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In healthy people, these alterations in noncoding
regions of DNA or sites that cause no change in
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function of encoded proteinThis heritable polymorphism of DNA structure can be
associated with certain disease within a large kindred
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Gene Variations Causing Disease
Classic genetics taught that genetic diseases were due
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to point mutation that lead to an impaired proteinGenetic disease could result from derangement of any
of the steps leading from replication to transcription
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to RNA processing/transport and protein
synthesis,PTMs etc
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This point is again nicely illustrated by examination ofbeta-globin gene
Defective production of beta globin results in variety
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of diseases and is due to many different lesion in and
around Beta globin gene
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Point Mutations
The classic example is sickle cell disease, which is
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caused by a mutation of a single base i.e. A-to-TDNA substitution
This in turn results in an A-to-U change in mRNA
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corresponding to the sixth codon of the beta-
globin gene.
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The altered codon specifies a different amino acidi.e GLU to VAL
This causes a structural abnormality of the beta
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globin molecule leading to hemoglobin
aggregation and red cell "sickling".
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Other point mutations in and around Beta
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globin gene result in decreased or, in some
instances, no production of beta globin
causing beta thalassemia
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The thalassemias are characterized bydefects in the synthesis of haemoglobin
subunits, and so beta thalassemia results
when there is insufficient production of beta-
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globin.Deletions, Insertions and Rearrangements of DNA
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Studies of bacteria,viruses,yeasts,fruit flies, andnow humans show that pieces of DNA can move,
or transpose from one place to another within a
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genome via a process of DNA transposition.
The deletion of critical piece of DNA, the
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rearrangements of DNA within a gene, or theinsertion or amplification of a piece of DNA within
a coding or regulatory region can all cause
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changes in gene expression resulting in disease
Molecular analysis of thalassemias produces numerous example
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of these processes-particularly deletions-as cause of disease
Deletions in the alpha-globin cluster, located on chromosome 16,
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cause alpha-thalassemia.A similar analysis could be made for a number of other diseases.
If the mutation destroys or creates a restriction enzyme site, the
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technique of RFLP can be used to pinpoint the lesion
Deletions or insertions of DNA larger than 50 bp can often be
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detected by southern blotting while PCR can detect much smallerchange in DNA structure
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Pedigree AnalysisSickle cell disease again provides an excellent example of how
RDT can be applied to the study of human disease
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The substitution of T for A in template strand of DNA in beta
globin gene changes the sequence in the region and destroys a
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recognition site for restriction enzyme MstII.Pedigree analysis has been applied to a number of genetic
diseases and is most useful in those caused by deletions and
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insertions or rare instances in which restriction endonuclease
cleavage site is affected
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Such analyses are now facilitated by the PCR reaction, which canamplify and hence provide sufficient DNA for analysis from just a
few nucleated cells.
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Prenatal Diagnosis
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If the genetic lesion is understood and a specificprobe is available, prenatal diagnosis is possible
DNA from cells collected from small volume of
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amniotic fluid can be analyzed by Southern blot
transfer, and much smaller volume if PCR ?based
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assays are usedFetus with the restriction pattern AA is normal, if
with the SS pattern will develop the disease.
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PCR is an in vitro method for amplifying a selected
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DNA sequencePCR permits the synthesis of millions of copies of a
specific nucleotide sequence in few hours
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It can amplify the sequence, even when the targeted
sequence makes up less than one part in a million of
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total initial sampleThe method can be used to amplify DNA sequences
from any source, including viral,bacterial,plant or
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animal
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Procedure1.
Constructing primer
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It is not necessary to know the entire nucleotide sequence
of the target DNA in the PCR method.
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However, it is necessary to know the nucleotide sequenceof short fragment on each side of target DNA
The nucleotide sequence of the flanking regions are used to
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construct two, single-stranded oligonucleotides,which are
complementary to the respective flanking sequences
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The 3'OH end of each oligonucleotide points towards thetarget sequence
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2. Denaturing DNAThe target DNA to be amplified is heated to 95 degree
Celsius to separate dsDNA in to single strands
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3. Annealing primersThe separated strands are cooled to 50 degree Celsius
and the two primers anneal to a complementary
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sequence on the DNA.4. Extending primers
DNA pol and DNTP are added to the mixture to initiate
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the synthesis of two new strands which arecomplementary to the original DNA strands.
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Applications
1. Comparison of a normal gene to its mutant forms
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Forensic analysis of DNA samples
Detection of low-abundance nucleic acid sequences
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Prenatal diagnosis and carrier detection of cysticfibrosis
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