Download MBBS (Bachelor of Medicine and Bachelor of Surgery) Latest Recombinant DNA Technology Lecture PPT
The development of recombinant DNA
techniques, high-throughput screening ,low cost
genome-scale DNA and RNA sequencing has
revolutionized biology and is having increased
impact on clinical medicine
Manipulation of a DNA sequence and construction
of chimeric molecule provides a means of studying
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
molecular basis of disease. For example, familial
hypercholestrolemia, sickle cell disease, the
thalassemias, muscular dystrophy as well as
more complex multifactorial diseases like
vascular and heart disease, Alzheimer
disease,cancer,obesity and diabetes
2. Human proteins can be produced in abundance
for therapy
3. Proteins for preparation of vaccine and for
diagnostic testing can be easily obtained
4. This technology is used both to diagnose existing
diseases as well as to predict the risk of developing a
given disease and individual response to
pharmacologic therapeutics-so called personalized
medicine
5. Special techniques have led to remarkable advances
in forensic medicine, which have allowed for the
molecular diagnostic analysis of DNA from single
cells.
6. Finally, in extremely well understood disease,
potentially curative gene therapy for disease cause by
single gene deficiency
Normal gene Variations
There is normal variation of DNA sequence just as it is
true of more obvious aspect of human structure
Polymorphisms, occur approximately once in every
500 to 1000 nucleotides
There are also genomic deletions and insertions of
DNA as well as single base substitutions.
In healthy people, these alterations in noncoding
regions of DNA or sites that cause no change in
function of encoded protein
This heritable polymorphism of DNA structure can be
associated with certain disease within a large kindred
Gene Variations Causing Disease
Classic genetics taught that genetic diseases were due
to point mutation that lead to an impaired protein
Genetic disease could result from derangement of any
of the steps leading from replication to transcription
to RNA processing/transport and protein
synthesis,PTMs etc
This point is again nicely illustrated by examination of
beta-globin gene
Defective production of beta globin results in variety
of diseases and is due to many different lesion in and
around Beta globin gene
Point Mutations
The classic example is sickle cell disease, which is
caused by a mutation of a single base i.e. A-to-T
DNA substitution
This in turn results in an A-to-U change in mRNA
corresponding to the sixth codon of the beta-
globin gene.
The altered codon specifies a different amino acid
i.e GLU to VAL
This causes a structural abnormality of the beta
globin molecule leading to hemoglobin
aggregation and red cell "sickling".
Other point mutations in and around Beta
globin gene result in decreased or, in some
instances, no production of beta globin
causing beta thalassemia
The thalassemias are characterized by
defects in the synthesis of haemoglobin
subunits, and so beta thalassemia results
when there is insufficient production of beta-
globin.
Deletions, Insertions and Rearrangements of DNA
Studies of bacteria,viruses,yeasts,fruit flies, and
now humans show that pieces of DNA can move,
or transpose from one place to another within a
genome via a process of DNA transposition.
The deletion of critical piece of DNA, the
rearrangements of DNA within a gene, or the
insertion or amplification of a piece of DNA within
a coding or regulatory region can all cause
changes in gene expression resulting in disease
Molecular analysis of thalassemias produces numerous example
of these processes-particularly deletions-as cause of disease
Deletions in the alpha-globin cluster, located on chromosome 16,
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
technique of RFLP can be used to pinpoint the lesion
Deletions or insertions of DNA larger than 50 bp can often be
detected by southern blotting while PCR can detect much smaller
change in DNA structure
Pedigree Analysis
Sickle cell disease again provides an excellent example of how
RDT can be applied to the study of human disease
The substitution of T for A in template strand of DNA in beta
globin gene changes the sequence in the region and destroys a
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
insertions or rare instances in which restriction endonuclease
cleavage site is affected
Such analyses are now facilitated by the PCR reaction, which can
amplify and hence provide sufficient DNA for analysis from just a
few nucleated cells.
Prenatal Diagnosis
If the genetic lesion is understood and a specific
probe is available, prenatal diagnosis is possible
DNA from cells collected from small volume of
amniotic fluid can be analyzed by Southern blot
transfer, and much smaller volume if PCR ?based
assays are used
Fetus with the restriction pattern AA is normal, if
with the SS pattern will develop the disease.
PCR is an in vitro method for amplifying a selected
DNA sequence
PCR permits the synthesis of millions of copies of a
specific nucleotide sequence in few hours
It can amplify the sequence, even when the targeted
sequence makes up less than one part in a million of
total initial sample
The method can be used to amplify DNA sequences
from any source, including viral,bacterial,plant or
animal
Procedure
1.
Constructing primer
It is not necessary to know the entire nucleotide sequence
of the target DNA in the PCR method.
However, it is necessary to know the nucleotide sequence
of short fragment on each side of target DNA
The nucleotide sequence of the flanking regions are used to
construct two, single-stranded oligonucleotides,which are
complementary to the respective flanking sequences
The 3'OH end of each oligonucleotide points towards the
target sequence
2. Denaturing DNA
The target DNA to be amplified is heated to 95 degree
Celsius to separate dsDNA in to single strands
3. Annealing primers
The separated strands are cooled to 50 degree Celsius
and the two primers anneal to a complementary
sequence on the DNA.
4. Extending primers
DNA pol and DNTP are added to the mixture to initiate
the synthesis of two new strands which are
complementary to the original DNA strands.
Applications
1. Comparison of a normal gene to its mutant forms
Forensic analysis of DNA samples
Detection of low-abundance nucleic acid sequences
Prenatal diagnosis and carrier detection of cystic
fibrosis
This post was last modified on 30 November 2021