Download MBBS Recombinant DNA Technology Lecture PPT

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