Download MBBS Recombinant DNA Technology Lecture PPT

The development of recombinant DNA

techniques, high-throughput screening ,low cost

--- Content provided by FirstRanker.com ---

genome-scale DNA and RNA sequencing has

revolutionized biology and is having increased

--- Content provided by FirstRanker.com ---

impact on clinical medicine

Manipulation of a DNA sequence and construction

of chimeric molecule provides a means of studying

--- Content provided by FirstRanker.com ---

how a specific segment of DNA controls function

?Continue.........

--- Content provided by FirstRanker.com ---

Understanding molecular genetics technology is

important for several reasons:
1. it offers a rational approach to understand the

--- Content provided by FirstRanker.com ---

molecular basis of disease. For example, familial

hypercholestrolemia, sickle cell disease, the

--- Content provided by FirstRanker.com ---

thalassemias, muscular dystrophy as well as

more complex multifactorial diseases like

vascular and heart disease, Alzheimer

--- Content provided by FirstRanker.com ---

disease,cancer,obesity and diabetes
2. Human proteins can be produced in abundance

for therapy

--- Content provided by FirstRanker.com ---

3. Proteins for preparation of vaccine and for

diagnostic testing can be easily obtained

--- Content provided by FirstRanker.com ---

4. This technology is used both to diagnose existing

diseases as well as to predict the risk of developing a

--- Content provided by FirstRanker.com ---

given disease and individual response to

pharmacologic therapeutics-so called personalized

medicine

--- Content provided by FirstRanker.com ---

5. Special techniques have led to remarkable advances

in forensic medicine, which have allowed for the

--- Content provided by FirstRanker.com ---

molecular diagnostic analysis of DNA from single

cells.

6. Finally, in extremely well understood disease,

--- Content provided by FirstRanker.com ---

potentially curative gene therapy for disease cause by

single gene deficiency

--- Content provided by FirstRanker.com ---

Normal gene Variations
There is normal variation of DNA sequence just as it is

--- Content provided by FirstRanker.com ---

true of more obvious aspect of human structure

Polymorphisms, occur approximately once in every

--- Content provided by FirstRanker.com ---

500 to 1000 nucleotides

There are also genomic deletions and insertions of

DNA as well as single base substitutions.

--- Content provided by FirstRanker.com ---

In healthy people, these alterations in noncoding

regions of DNA or sites that cause no change in

--- Content provided by FirstRanker.com ---

function of encoded protein

This heritable polymorphism of DNA structure can be

associated with certain disease within a large kindred

--- Content provided by FirstRanker.com ---

Gene Variations Causing Disease
Classic genetics taught that genetic diseases were due

--- Content provided by FirstRanker.com ---

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

--- Content provided by FirstRanker.com ---

to RNA processing/transport and protein

synthesis,PTMs etc

--- Content provided by FirstRanker.com ---

This point is again nicely illustrated by examination of

beta-globin gene

Defective production of beta globin results in variety

--- Content provided by FirstRanker.com ---

of diseases and is due to many different lesion in and

around Beta globin gene

--- Content provided by FirstRanker.com ---

--- Content provided by FirstRanker.com ---

Point Mutations
The classic example is sickle cell disease, which is

--- Content provided by FirstRanker.com ---

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

--- Content provided by FirstRanker.com ---

corresponding to the sixth codon of the beta-

globin gene.

--- Content provided by FirstRanker.com ---

The altered codon specifies a different amino acid

i.e GLU to VAL

This causes a structural abnormality of the beta

--- Content provided by FirstRanker.com ---

globin molecule leading to hemoglobin

aggregation and red cell "sickling".

--- Content provided by FirstRanker.com ---

Other point mutations in and around Beta

--- Content provided by FirstRanker.com ---

globin gene result in decreased or, in some
instances, no production of beta globin
causing beta thalassemia

--- Content provided by FirstRanker.com ---

The thalassemias are characterized by

defects in the synthesis of haemoglobin
subunits, and so beta thalassemia results
when there is insufficient production of beta-

--- Content provided by FirstRanker.com ---

globin.


Deletions, Insertions and Rearrangements of DNA

--- Content provided by FirstRanker.com ---

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

--- Content provided by FirstRanker.com ---

genome via a process of DNA transposition.

The deletion of critical piece of DNA, the

--- Content provided by FirstRanker.com ---

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

--- Content provided by FirstRanker.com ---

changes in gene expression resulting in disease


Molecular analysis of thalassemias produces numerous example

--- Content provided by FirstRanker.com ---

of these processes-particularly deletions-as cause of disease

Deletions in the alpha-globin cluster, located on chromosome 16,

--- Content provided by FirstRanker.com ---

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

--- Content provided by FirstRanker.com ---

technique of RFLP can be used to pinpoint the lesion

Deletions or insertions of DNA larger than 50 bp can often be

--- Content provided by FirstRanker.com ---

detected by southern blotting while PCR can detect much smaller

change in DNA structure

--- Content provided by FirstRanker.com ---

Pedigree Analysis

Sickle cell disease again provides an excellent example of how

RDT can be applied to the study of human disease

--- Content provided by FirstRanker.com ---

The substitution of T for A in template strand of DNA in beta

globin gene changes the sequence in the region and destroys a

--- Content provided by FirstRanker.com ---

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

--- Content provided by FirstRanker.com ---

insertions or rare instances in which restriction endonuclease

cleavage site is affected

--- Content provided by FirstRanker.com ---

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.

--- Content provided by FirstRanker.com ---

Prenatal Diagnosis

--- Content provided by FirstRanker.com ---

If the genetic lesion is understood and a specific

probe is available, prenatal diagnosis is possible

DNA from cells collected from small volume of

--- Content provided by FirstRanker.com ---

amniotic fluid can be analyzed by Southern blot

transfer, and much smaller volume if PCR ?based

--- Content provided by FirstRanker.com ---

assays are used

Fetus with the restriction pattern AA is normal, if

with the SS pattern will develop the disease.

--- Content provided by FirstRanker.com ---

PCR is an in vitro method for amplifying a selected

--- Content provided by FirstRanker.com ---

DNA sequence

PCR permits the synthesis of millions of copies of a

specific nucleotide sequence in few hours

--- Content provided by FirstRanker.com ---

It can amplify the sequence, even when the targeted

sequence makes up less than one part in a million of

--- Content provided by FirstRanker.com ---

total initial sample

The method can be used to amplify DNA sequences

from any source, including viral,bacterial,plant or

--- Content provided by FirstRanker.com ---

animal

--- Content provided by FirstRanker.com ---

Procedure

1.

Constructing primer

--- Content provided by FirstRanker.com ---

It is not necessary to know the entire nucleotide sequence

of the target DNA in the PCR method.

--- Content provided by FirstRanker.com ---

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

--- Content provided by FirstRanker.com ---

construct two, single-stranded oligonucleotides,which are

complementary to the respective flanking sequences

--- Content provided by FirstRanker.com ---

The 3'OH end of each oligonucleotide points towards the

target sequence

--- Content provided by FirstRanker.com ---

2. Denaturing DNA
The target DNA to be amplified is heated to 95 degree

Celsius to separate dsDNA in to single strands

--- Content provided by FirstRanker.com ---

3. Annealing primers
The separated strands are cooled to 50 degree Celsius

and the two primers anneal to a complementary

--- Content provided by FirstRanker.com ---

sequence on the DNA.

4. Extending primers
DNA pol and DNTP are added to the mixture to initiate

--- Content provided by FirstRanker.com ---

the synthesis of two new strands which are

complementary to the original DNA strands.

--- Content provided by FirstRanker.com ---

Applications

1. Comparison of a normal gene to its mutant forms

--- Content provided by FirstRanker.com ---

Forensic analysis of DNA samples

Detection of low-abundance nucleic acid sequences

--- Content provided by FirstRanker.com ---

Prenatal diagnosis and carrier detection of cystic

fibrosis