Molecular genetics is the study of DNA structure, replication, and influence on organisms. Techniques of molecular genetics include gene therapy, amplification, polymerase chain reaction (PCR), cell culture, DNA cloning in bacteria, gel electrophoresis, and isolation of DNA. Gene therapy is an experimental technique that uses genes to treat or prevent disease. PCR amplifies a segment of DNA, allowing scientists to study small samples. Cell culture provides model systems to study cell physiology. DNA cloning makes copies of DNA fragments in bacteria. Gel electrophoresis separates DNA fragments by size. Isolation of DNA extracts free DNA molecules for further analysis. These techniques have applications in medicine, research, forensics, and molecular biology.

1. Molecular Genetics
TOPIC OF PRESENTATION : APPLICATIONS OF MOLECULAR GENETICS
PRESENTOR : SADIA MAZHAR
COLLEGE ROLL NO.880
UNIVERSITY ROLL NO.023809

2. Applications and techniques of Molecular
Genetics
 Molecular Genetics:
Molecular genetics is the study of the molecular structure of DNA, its cellular activities (including its replication),
and its influence in determining the overall makeup of an organism. Molecular genetics relies heavily
on genetic engineering (recombinant DNA technology), which in medicine has been used to mass-produce insulin,
human growth hormones, follistim (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors,
vaccines, and many other drugs. In research, organisms are genetically engineered to discover the functions of
certain genes.

3. A Simple Example-Translational Proces

4. Techniques of Molecular Genetics
 Gene Therapy
 Amplification
 Polymerase Chain Reaction(PCR)
 Cell Culture
 DNA Cloning in Bacteria
 Gel Electrophoresis
 Isolation of DNA

5.  Definition:
Gene therapy is an experimental technique that uses genes to treat or
prevent disease. In the future, this technique may allow doctors to treat a
disorder by inserting a gene into a patient's cells instead of using drugs or
surgery.
Gene Therapy

6.  Approaches of Gene Therapy
 Replacing a mutated gene that causes disease with a healthy copy of the gene.
 Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
 Introducing a new gene into the body to help fight a disease.

9.  Gene therapy is an experimental form of treatment that uses gene transfer of
genetic material into the cell of a patient to cure the disease. The idea is to modify
the genetic information of the cell of the patient that is responsible for a disease,
and then return that cell to normal conditions. Transfer of genetic material is done
commonly by using viral vectors that use their own biological capacities to enter
the cell and deposit the genetic material. Both inherited genetic diseases and
acquired disorders can be treated with gene therapy. Examples of these disorders
are primary immune deficiencies, where gene therapy has been able to fully
correct the presentation of patients, and/or cancer, where the gene therapy is still
at the experimental stage.

10. Diseases treated by Gene Therapy
 With its potential to eliminate and prevent hereditary diseases such as cystic fibrosis and
hemophilia and its use as a possible cure for heart disease, AIDS, and cancer, gene therapy is a
potential medical miracle-worker.
 Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve
your body's ability to fight disease. Gene therapy holds promise for treating a wide range of
diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS.

11. Amplification
In molecular biology, amplification is a process by which a nucleic acid molecule is enzymatically
copied to generate a progeny population with the same sequence as the parental one. The most widely
used amplification method is Polymerase Chain Reaction (PCR).
The result of a PCR amplification of a segment of DNA is called an “amplicon.” Nucleic
acids can also be amplified in an isothermal reaction involving a reverse transcriptase,
which copies RNA→DNA, and a DNA-dependent RNA polymerase, which transcribes
DNA→RNA. Isothermal amplification does not generate double-stranded DNA, and it
is mainly used for copying RNA. Ligase-based methods, including the so-called Ligase
Chain Reaction (LCR), can be also used for specific DNA or RNA amplification. A
fourth general method for nucleic acid amplification involves cloning the selected DNA
molecule into bacterial or eukaryotic cells, allowing them to reproduce, and collecting
the amplified DNA.

13. Importance of DNA Amplification
 DNA copies produced through PCR amplification can be used in a large number of medical and
forensic applications. It can likewise be used in the identification and detection of infectious
diseases and for a wide variety of research purposes in the field of molecular genetics. Genetic
testing.
 A single molecule of DNA is so small that it lies beyond the limits of detection of
most, if not all, assays. Even with sensitive radioisotopes, it would be difficult if not
impossible to detect a single molecule of DNA. The reason PCR was so important
and revolutionary to molecular biology is that one cold begin with a single
molecule of starting material and amplify it to an amount suitable for further
analysis, including but not limited to:

14. Polymerase Chain Reaction(PCR)
 Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to
billions of copies of a specific DNA sample, allowing scientists to take a very small
sample of DNA and amplify it to a large enough amount to study in detail.
 It is a technique used to make numerous copies of a specific segment
of DNA quickly and accurately. The polymerase chain reaction enables
investigators to obtain the large quantities of DNA that are required for
various experiments and procedures in molecular biology, forensic
analysis, evolutionary biology, and medical diagnostics.

15.  The PCR technique is based on the natural processes a cell uses to replicate a new DNA
strand. Only a few biological ingredients are needed for PCR. The integral component
is the template DNA—i.e., the DNA that contains the region to be copied, such as
a gene. As little as one DNA molecule can serve as a template. The only information
needed for this fragment to be replicated is the sequence of two short regions
of nucleotides (the subunits of DNA) at either end of the region of interest. These two
short template sequences must be known so that two primers—short stretches of
nucleotides that correspond to the template sequences—can be synthesized. The
primers bind, or anneal, to the template at their complementary sites and serve as the
starting point for copying. DNA synthesis at one primer is directed toward the other,
resulting in replication of the desired intervening sequence. Also needed are free
nucleotides used to build the new DNA strands and a DNA polymerase, an enzyme that
does the building by sequentially adding on free nucleotides according to the
instructions of the template.

16. Steps of PCR
 PCR is a three-step process that is carried out in repeated cycles. The initial step is
the Denaturation , or separation, of the two strands of the DNA molecule. This
is accomplished by heating the starting material to temperatures of about 95 °C
(203 °F). Each strand is a template on which a new strand is built. In the second
step the Reduction in temprature to about 55 °C (131 °F) so that the primers
can anneal to the template. In the third step the Increase in temprature to
about 72 °C (162 °F), and the DNA polymerase begins adding nucleotides onto the
ends of the annealed primers. At the end of the cycle, which lasts about five
minutes, the temperature is raised and the process begins again. The number of
copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient
amount of DNA

19. Importance of PCR
 The Polymerase Chain Reaction (PCR) is an important tool for many applications. For example,
it can be used to amplify a sample of DNA when there isn't enough to analyze (e.g. a sample of
DNA from a crime scene, archeological samples), as a method of identifying a gene of interest, or to
test for disease.
 Polymerase chain reaction (PCR) is often considered as one of the most
important scientific advances in the field of molecular biology. With this
revolutionary yet inexpensive biochemical technology, it’s possible to generate
millions of DNA copies from a single strand of DNA.
 As a result of this, PCR is considered to be one of the most indispensable
techniques used in medical and biochemical research laboratories.

20. Applications of PCR
 Polymerase chain reaction, or molecular photocopying as it is lovingly
called by some people, can be used in a variety of applications. DNA
copies produced through PCR amplification can be used in a large number
of medical and forensic applications. It can likewise be used in the
identification and detection of infectious diseases and for a wide variety of
research purposes in the field of molecular genetics.
 Medical Applications
 Genetic testing. PCR was first used to analyze the presence of genetic
disease mutations.
 Tissue typing prior to organ transplantation.
 Formulation of individualized cancer therapy treatments.

21.  Forensic Applications
 Genetic fingerprinting. PCR can be used to incriminate or rule out suspects in a crime
investigation.
 Parental testing. PCR can be used to confirm the biological parents of an adopted
child and/or identify the remains of an unidentified body.
 Infectious Disease Detection and Identification
 Detection of the Human Immunodeficiency Virus (HIV), one of the most difficult viruses
to detect, and other disease organisms such as those that cause middle ear infection,
tuberculosis and Lyme disease.
 Early detection of several forms of cancer including leukemia and lymphoma.
 Detection of viral DNA and virulent sub-types, including those that caused earlier
epidemics.

22.  Applications in Molecular Biology Research
 DNA sequencing, DNA cloning and gene expression. PCR can be used to
produce huge amounts of pure DNA samples from a limited source.
 Production of hybridization probes for both northern and southern blot
hybridization.
 Analysis of DNA from ancient sources.

23. Cell Culture
 Cell culture is one of the major tools used in cellular and molecular biology, providing excellent
model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic
studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and
carcinogenesis.
 Cell culture is the process by which cells are grown under controlled conditions, generally outside
their natural environment. After the cells of interest have been isolated from living tissue, they can
subsequently be maintained under carefully controlled conditions.

26. Importance of Cell Culture in Molecular
Genetics
 Cell culture is one of the major tools used in cellular and molecular biology, providing
excellent model systems for studying the normal physiology and biochemistry of cells (e.g.,
metabolic studies, aging), the effects of drugs and toxic compounds on the cells and
mutagenesis and carcinogenesis
 Cell cultures are an extremely important tool for healthcare scientists. They
provide a model system for physiology and biochemistry of selected cells to
be studied. By examining their physiology their aging pathway can be
studied and their biochemistry allows processes such as metabolic rate to
be observed. The cells interaction with drugs could also be observed which
proves a useful tool for drug screening programs, clinical trials and
pharmaceutical companies. Whatever the purpose for using cell cultures, it
is an extremely consistent and reliable process that has good reproducibility
of results that can be obtained using a batch of clonal cells.

28. DNA Cloning in bacteria
 DNA cloning is a molecular biology technique that makes many
identical copies of a piece of DNA, such as a gene. ... Bacteria with the
correct plasmid are used to make more plasmid DNA or, in some cases,
induced to express the gene and make protein.
 Molecular cloning is a set of experimental methods in molecular biology
that are used to assemble recombinant DNA molecules and to direct their
replication within host organisms.

31.  One of the most important contributions of DNA cloning and genetic engineering to cell
biology is that they have made it possible to produce any of the cell's proteins in nearly
unlimited amounts. Large amounts of a desired protein are produced in living cells by using
expression vectors
 Uses of DNA cloning
 Biopharmaceuticals. DNA cloning can be used to make human proteins with
biomedical applications, such as the insulin mentioned above. ...
 Gene therapy. In some genetic disorders, patients lack the functional form of a particular
gene. ...
 Gene analysis.

32. Gel Electrophoresis
 Gel electrophoresis is a method for separation and analysis of macromolecules and their
fragments, based on their size and charge.
 Gel electrophoresis is a technique used to separate DNA fragments
according to their size.
 DNA samples are loaded into wells (indentations) at one end of a gel, and
an electric current is applied to pull them through the gel.
 DNA fragments are negatively charged, so they move towards the positive
electrode. Because all DNA fragments have the same amount of charge
per mass, small fragments move through the gel faster than large ones.
 When a gel is stained with a DNA-binding dye, the DNA fragments can be
seen as bands, each representing a group of same-sized DNA fragments.

33.  What is a gel?
 As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gels for
DNA separation are often made out of a polysaccharide called agarose, which comes as dry,
powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed
to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose
molecules that are held together by hydrogen bonds and form tiny pores.
 At one end, the gel has pocket-like indentations called wells, which are where the DNA samples
will be placed:
 Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is
hooked to a positive electrode, while the other end is hooked to a negative electrode. The main
body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can
conduct current. Although you may not be able to see in the image above (thanks to my amazing
artistic skills), the buffer fills the gel box to a level where it just barely covers the gel.
 The end of the gel with the wells is positioned towards the negative electrode. The end without
wells (towards which the DNA fragments will migrate) is positioned towards the positive electrode.

35.  DNA fingerprinting uses gel electrophoresis to distinguish between samples of the
genetic material. The human DNA molecules are treated with enzymes that chop
them at certain characteristic points, thereby reducing the DNA to a collection of
more manageably sized pieces. The DNA fragments are loaded into a gel and
placed in an electrical field, which electrophoretically sorts the DNA fragments
into various bands. These bands can be colored with a radioactive dye to make
them visible to imaging techniques

36.  Applications of gel electrophoresis
 In the separation of DNA fragments for DNA fingerprinting to
investigate crime scenes.
 To analyze results of polymerase chain reaction.
 To analyze genes associated with a particular illness.
 In DNA profiling for taxonomy studies to distinguish different
species.

37. Isolation of DNA
 The free DNA molecules are subsequently isolated by one of several methods. ... A
small pellet of DNA can be collected by centrifugation, and after removal of the
ethanol, the DNA pellet can be dissolved in water (usually with a small amount of
EDTA and a pH buffer) for the use in other reactions.
 The first isolation of DNA was done in 1869 by Friedrich
Miescher.[1] Currently it is a routine procedure in molecular
biology or forensic analyses. For the chemical method, there are many
different kits used for extraction, and selecting the correct one will save
time on kit optimization and extraction procedures. PCR sensitivity
detection is considered to show the variation between the commercial
kits.

38.  DNA extraction is one of the most modern of the biological sciences. Scientists and doctors use
DNA extraction to diagnose many medical conditions to genetically engineer both plants and
animals. DNA extraction can also be used to gather evidence in a crime investigation.
 Application. The ability to extract DNA is of primary importance to studying the genetic causes of
disease and for the development of diagnostics and drugs. It is also essential for carrying out
forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for
determining paternity.

39. That,s all for now!
Thanks for tolerating!
Any question?