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BY, DR.M.L Kulkarni DR - Gayathri.K Davanagere. Reviewed by DR - Subramanya.N.K Bangalore

This document provides an overview of newer advances in genetic technologies including DNA structure, gene structure, gene action, recombinant DNA technology, polymerase chain reaction (PCR), Southern blot hybridization, fluorescence in situ hybridization (FISH), DNA microarray technique, linkage analysis, enzyme replacement therapy, gene therapy, and gene therapy using adenovirus vectors. Key genetic technologies discussed include PCR, which amplifies DNA segments, Southern blot hybridization for identifying DNA fragments, and microarrays for studying gene expression of many genes at once. The document also reviews several applications of these genetic technologies.

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74 views

BY, DR.M.L Kulkarni DR - Gayathri.K Davanagere. Reviewed by DR - Subramanya.N.K Bangalore

This document provides an overview of newer advances in genetic technologies including DNA structure, gene structure, gene action, recombinant DNA technology, polymerase chain reaction (PCR), Southern blot hybridization, fluorescence in situ hybridization (FISH), DNA microarray technique, linkage analysis, enzyme replacement therapy, gene therapy, and gene therapy using adenovirus vectors. Key genetic technologies discussed include PCR, which amplifies DNA segments, Southern blot hybridization for identifying DNA fragments, and microarrays for studying gene expression of many genes at once. The document also reviews several applications of these genetic technologies.

Uploaded by

garlic100
Copyright
© © All Rights Reserved
Available Formats
Download as PPS, PDF, TXT or read online on Scribd
You are on page 1/ 34

NEWER ADVANCES IN GENETIC

BY,
DR.M.L KULKARNI
DR.GAYATHRI.K
DAVANAGERE.
REVIEWED BY
DR.SUBRAMANYA.N.K
BANGALORE

DNA BASICS
All genetic material (DNA) contained in a
single germ cell (sperm and ovum) is known
as human genome, that contains all
information of life.
Structure:
DNA exists as a double stranded helix.
The anology of rope ladder can be given to
the structure of DNA .Each side of the
ladder is made up of chains of sugar(s) and
phosphates(P).The step of ladder is made
up of 2 nitrogenous bases from 2 poles
projecting inward and 2 bases are joined by
hydrogen bond.

The nitrogenous bases consist of the


purines(adenine-A, guanine-G)
Pyrimidines(cytosine-C, thymine-T).
A always pairs with T and G with C.
Each pair of complementary
nitrogenous base is termed a base
pair.
A nucleotide is defined as a unit
consisting of phosphate, sugar and
nitrogenous pair.
The order of base on one single, strand
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is known as DNA sequence. The DNA
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GENE STRUCTURE

Genes are segments of DNA stretches


and are transcribed into RNA and later
transcribed into proteins.
Genes are composed of exons (the
coding region ) and introns(the non
coding region ).

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GENE STRUCTURE

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GENE ACTION

Transcription: one strand of structural


gene DNA forms a template for the
synthesis of RNA known as messenger
RNA(m RNA).
Translation: A triplet of bases called
codon codes for synthesis of particular
aminoacid, mRNA becomes the template
in the ribosome for translation.
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GENE ACTION

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RECOMBINANT DNA TECHNOLOGY

The process of
manipulating genetic
material, in which DNA is
artificially cut, joined and
synthesized is termed as
DNA recombinant
technology or Genetic M.L.K
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USES OF DNA RECOMBINANT


TECHNOLOGY

i.Gene structure/Mapping/Function.
ii. Clinical Genetics
iii. .Preimplantation genetic diagnosis.
. Prenatal diagnosis.
.Presymptomatic diagnosis.
. Carrier detection.
iii. Diagnosis and pathogenesis of disease
.Genetic
.Infective
. Malignant.
iv. Biosynthesis
.Recombinant products
.Vaccines
v. Treatment of genetic diseases
vi. Gene therapy.
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DNA RECOMBINANT TECHNOLOG

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POLYMERASE CHAIN REACTION

PCR is a powerful technology that is


used to amplify the DNA segment of
interest i.e,molecular photocopying.
STEPS:
Target DNA is denatured by heat and
double standed DNA is seperated into
two single strands.
Primers are added to DNA strands,
these primers hybridize to
complementary fragments of now
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single stranded target DNA.

Then bases and tag polymerase are


added, while temperature is
increased.
The bases are used as building block
for synthesis of DNA strands and
polymerase begins to assemble them.
Such cycles are repeated several
times automatically and in few hours,
the target DNA segment can be
amplified to one million fold.
The produced DNA can be analysed
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using labelled probes, sequencing
or
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direcct vision with U.V light.

APPLICATIONS OF PCR
Genetic disorder caused by single base pair
changes, small deletion or insertion.(Eg:
Thalassemia with base pair change, DMD with
DNA deletion.)
Diagnosis of infectious diseases.
Diagnosis and follow up of cancers.
Study of bone marrow engraftment.
DNA fingerprinting for paternity cases and
forensic purposes.
HLA typing for bone marrow and kidney
transplantation.
Study of gene linkage and gene mapping.
Study of evolution.
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POLYMERASE CHAIN REACTION


TECHNIQUE

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SOUTHERN BLOT HYBRIDIZATIO


In this technique, first the DNA is cut in to
fragments by the use of bacterial restriction
enzymes, these pieces are then subjected to
electrophoresis in an agarose gel, smaller
fragments move faster than larger ones.
The agarose gel with DNA tracks is
transferred for a permanent record by
placing a nitro-cellulose paper on top of the
agarose gel,retaining the alignment of
fragments(blotting).
The record is then washed in a solution
containing radiolabelled DNA probe with
phosphorous
32.The
probe
finds
its
complementary fragments of DNA, unites
with it (Hybridization).
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This hybridized fragment can be identified
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SOUTHERN BLOT
HYBRIDIZATION

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SOUTHERN BLOT
HYBRIDIZATION

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APPLICATIONS

To find the presence of a


particular gene or a part of the
fragment in the sample DNA.
To know whether the size of the
DNA fragment carrying the gene is
normal, increased or decreased.
To identify presence of large
deletions, duplications or large
gene arrangements.
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FLUOROSCENCE INSITU HYBRIDIZATION(F

The DNA of the subject is first


denatured by heat into two separate
strands.
The fluorescet probe of interest is
added to the denatured DNA
sample,which then hybridizes(unites)
with the complementary segment if
present and then reforms into double
helix(re anneals)
The recombinant DNA is then seen
through a fluorescent
microscope,which detects the M.L.K
prescence or absence of the G.K

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DNA microarray technique


This technique is used to study the expression of
many genes at a time and that too on a single slide
known as DNA CHIP.
DNA Chip is a glass slide in which several miniature
wells are arranged in an order i.e microarray.
In these oligonucleotide probes are placed.
The m RNA is extracted from sample to be tested.
This m RNA is converted in to c DNA, which is then
labelled with a fluorescent dye.
The m RNA from normal cells is also extracted and
from this c DNA are then mixed with the
oligonucleotide probes that are placed in wells
(micro array).
Laser beams are then used to excite the
fluorochromes. Exciting patterns depend on
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relative amount of normal or abnormal cDNA.
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Analysis is done by capturing the


digital image and later in analyzing in
computer.
This is gene expression study.
(FUNCTIONAL GENOMICS).
APPLICATIONS:
Drug designing, pharmacogenetics,
cancer research, analyzing several
mutations in single go.
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DNA MICROARRAY
TECHNIQUE

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LINKAGE ANALYSIS :
If the disease causing mutation,cannot
be identified or difficult to test, DNA
markers situated in or near the gene
can be used to trace the mutated gene
in the family.
APPLICATIONS:
Prenatal diagnosis.
Carrier detection.
D.M.D
Hemophilia
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ENZYME REPLACEMENT THERAPY

This therapy is a potential


treatment for certain genetic
diseases.This method of treatment
is dependent on the delivery of the
intravenously delivered enzyme ,to
the cell surface receptor where the
enzyme is deficient.
The first successful therapy of
enzyme replacement was done in
Gauchers disease.
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REPLACED

DISEASE

ENZYME

Gauchers disease
Imiglucerase
Fabry disease
Agalsidase
Mucopolysaccharidosis1
Aldurazyme
Mucopolysaccharidosis v1
Galsulfase
Mucopolysaccharidosis 11
Idrsulfase

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GENE THERAPY
Gene therapy was conceptualized
in early 1970s and has subjected
to many clinical trials since then.
Gene therapy is a novel approach
to treat, cure, or ultimately
prevent a diseaseby changing the
expression of persons genes.
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APPROACHES TO GENE
THERAPY

Ex vivo approach: The target cells are


removed from the body and new
gene is introduced invitro by one of
the
gene delivery methods.
In vivo approach: (Direct gene
transfer): The theraupetic gene is
introduced directly into the affected
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tissue without removing cells from
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TYPES OF GENE THERAPY


Somatic cell therapy: this involves
insertion of a theraupetic gene into
somatic cells such as
fibroblasts,myoblasts,epithelial
cells,endothelial cells,nervous,glial
cells,etc.
Germline therapy: This involves the
introduction of a foreign gene into germ
cells like sperm,ovum,or fertilized
egg,resulting in their expression in both
somatic as well as germ cells of the
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offspring.
offspring
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METHODS OF GENE DELIVERY


Physical
.Physical injection
.Micro injection
.Aerosol
.Gene gun
Chemical
.Calcium phosphate
.DEAE-dextran
.Liposomes
Biological
. Viral vectors
. Retro virus
.Adeno virus
.Herpes simplex virus
Neo-organ implants
Tissue transplantation
Human artificial chromosome
Others
Receptor mediated delivery
Virally directed enzyme prodrug therapy.

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GENE THERAPY USING ADENOVIRUS


VECTOR

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Following are the disorders under trial for gene


therapy:

Cystic fibrosis
Alpha-1 antitrypsin deficiency
Hemophilia A and B
Gauchers disease
Beta-hemoglobinopathies
Familial hypercholesterolemia
Phenylketonuria.

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Thank you

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