The document discusses various topics related to genetics and biotechnology including genetic engineering, polymerase chain reaction (PCR), DNA profiling, and genetically modified foods. It provides definitions and explanations of key terms and processes such as how PCR is used to amplify DNA, the steps involved in PCR including denaturation, annealing and elongation, and how gel electrophoresis can be used to analyze PCR products. It also summarizes techniques like DNA profiling that are used for forensic investigations and paternity testing.

2. What is Genetic Engineering?
• A process of inserting new genetic
information into existing cells in order to
modify a specific organism for the purpose
of changing one of its characteristics.
Or
• Altering the genetic material of cells or
organisms in order to make them capable of
making new substances or performing new
functions

3. What is Biotechnology?
• The use of living organisms (especially
microorganisms) in industrial, agricultural,
medical and other technological
applications; the application of the
principles and practices of engineering and
technology to the life sciences. or
• The branch of molecular biology that studies
the use of microorganisms to perform
specific industrial processes.

4. Now coming to the subject……

5. What is PCR ?
• A technique in molecular biology for creating multiple
copies of DNA from a sample; used in genetic fingerprinting
etc.
• Technique to amplify a target DNA sequence of nucleotides
by several hundred thousand fold.
• The polymerase chain reaction (PCR) is a technique to
amplify a single or few copies of a piece of DNA across
several orders of magnitude, generating thousands to
millions of copies of a particular DNA sequence.

6. What is Polymerase?
• An enzyme that catalyzes the formation of
new DNA and RNA from an existing strand
of DNA or RNA.
• An enzyme that creates genetic material,
either RNA or DNA, from building blocks.

7. Who developed this Process?
• Kary Banks Mullis is an
American biochemist and Nobel
laureate.
• Mullis received the prize for his
development of the Polymerase
Chain Reaction (PCR), a process
first described by Kjell Kleppe and
1968 Nobel laureate H. Gobind
Khorana that allows the
amplification of specific DNA
sequences
• The improvements provided by
Mullis have made PCR a central
technique in biochemistry and
molecular biology.Kary Banks Mullis

9. What are Requirements of PCR?
• DNA template that contains the DNA region
• Two primers that are complementary
• Taq polymerase or another DNA polymerase
with a temperature optimum at around 70 °C.
• Deoxynucleoside triphosphates (dNTPs; also
very commonly and erroneously called
deoxynucleotide triphosphates).

10. • Buffer solution, providing a suitable
chemical environment for optimum
activity and stability of the DNA
polymerase.
• Divalent cations, magnesium or
manganese ions.
• Monovalent cation potassium ions.

11. What is the Procedure of PCR?
• The PCR usually consists of a series of 20 to
40 repeated temperature changes called
cycles; each cycle typically consists of 2-3
discrete temperature steps.

12. They are six steps involved in this process.
As follows.
Initialization step.
Denaturation step.
Annealing step
Extension/elongation step
Final elongation.
Final hold.

17. What is Initialization ?
• The process of preparing something to
begin; an act of preparing something to
begin.

18. Initialization step
• This step consists of heating the reaction to
a temperature of 94–96 °C
• In which is held for 1–9 minutes. It is only
required for DNA polymerases that require
heat activation by hot-start PCR.

19. What is Denaturation?
• Denaturation is a process in which proteins
or nucleic acids lose their structure (tertiary
and secondary structure) by application of
some external stress or compound.

20. Denaturation step
• This step is the first regular cycling event
and consists of heating the reaction to 94–
98 °C for 20–30 seconds.
• It causes DNA melting of the DNA template
by disrupting the hydrogen bonds between
complementary bases, yielding single
strands of DNA.

21. What is Template Strand?
• The strand of the DNA double helix that is
copied by base pair complementarily to
make an RNA. The other, non-template
strand of the DNA duplex has a sequence
that is identical to the synthesized RNA.

22. Annealing step (hardening
something by heat treatment)
• The reaction temperature is lowered to 50–
65 °C for 20–40 seconds allowing annealing
of the primers to the single-stranded DNA
template.
• Typically the annealing temperature is
about 3-5 degrees.
• The polymerase binds to the primer-
template hybrid and begins DNA synthesis.

23. Extension/Elongation step:
• At this step the DNA polymerase synthesizes
a new DNA strand complementary to the
DNA template strand by adding dNTPs.
• At its optimum temperature, the DNA
polymerase will polymerize a thousand
bases per minute.

24. Final elongation
• This single step is occasionally performed at
a temperature of 70–74 °C for 5–15 minutes
after the last PCR cycle to ensure that any
remaining single-stranded DNA is fully
extended.

25. Final hold
• This step at 4–15 °C for an indefinite time
may be employed for short-term storage of
the reaction.

26. • (1) Denaturing at 94–96 °C.
• (2) Annealing at ~65 °C
• (3) Elongation at 72 °C.
• Four cycles are shown here.
The blue lines represent the
DNA template to which
primers (red arrows) anneal
that are extended by the
DNA polymerase (light
green circles), to give shorter
DNA products (green lines),
which themselves are used as
templates as PCR progresses.

29. DNA bands in sample #2 and #3 indicate successful amplification of the target
sequence. The gel also shows a positive control, and a DNA ladder containing
DNA fragments of defined length for sizing the bands in the experimental
PCRs.

30. Applications of PCR
• Generating hybridization probes for Southern
or northern hybridization and DNA cloning.
• Bacterial colonies (E.coli) can be rapidly
screened by PCR for correct DNA vector
constructs.
• PCR may also be used for genetic
fingerprinting; a forensic technique used to
identify a person or organism by comparing
experimental DNAs through different PCR-
based methods.

31. • PCR can be used to analyze extremely small
amounts of sample. This is often critical for
forensic analysis, when only a trace amount
of DNA is available as evidence.
• PCR may also be used in the analysis of
ancient DNA that is tens of thousands of
years old. These PCR-based techniques have
been successfully used on animals, such as a
forty-thousand-year-old mammoth, and also
on human DNA

33. What is Electrophoresis?
Electrophoresis" refers to the electromotive
force (EMF) that is used to move the
molecules through the gel matrix.
By placing the molecules in wells in the gel
and applying an electric field, the molecules
will move through the matrix at different rates.

34. What is GEL?
The term "gel" in this instance refers to the
matrix used to contain, then separate the target
molecules.

35. What is Gel Electrophoresis?
A process for separating molecules by forcing
them to migrate through a gel under the
influence of an electric field.
or
A technique where DNA migrates through a
gel matrix in response to an applied electrical
current. Gel electrophoresis allows DNA to be
separated based on size.

38. A gel is prepared into thin layer and placed in
the container with small holes for placing
samples.
Then the equipment is conducted to electricity
for gel to conduct electricity.
Depending on charge the molecules attracted
more or less to other side.
DNA fragments are slightly negative and will
move towards positive charge.
Larger molecule will move slowly than smaller
molecules.

39. After the movements of molecules the electricity
is turned off and gel then stained to show the
spots of molecule movements.
The difference between the Gel & PCR here is :
PCR can be used to increase the size of a sample.
Gel electrophoresis can be used to compare the
section of DNA with the DNA of various
suspects in an investigation.

51. Here we are isolating DNA fragments from an
electroforesis gel.

58. What is PATERNITY PROFILING?
• Parental testing is the use of genetic
fingerprinting to determine whether two
individuals have a biological parent-child
relationship. A paternity test establishes
genetic proof whether a man is the biological
father of an individual, and a maternity test
establishes whether a woman is the biological
mother of an individual.

59. What is DNA PROFILING?
DNA profiling is also known as DNA finger
printing .It is also technique that compares
DNA from different sources without mapping
the entire genome.
The DNA profiling technique was first
reported in 1985 by Sir Alec Jeffreys at the
University of Leicester in England.

60. Sir Alec Jeffreys
9 January 1950 ,U.K

62. • It is easy to see in this example
that daughter 2 is the child from
the mother’s previous marriage
and son 2 is adopted. You can
see that both daughter 1 and son
1 share RFLPs with both the
mom and dad (coloured blue
and yellow respectively), while
daughter 2 has RFLPs of the
mom but not the dad, and son 2
does not have RFLPs from
either parent.

63. DNA profiling is used to compare the DNA of a
suspect from blood ,salvia,testes etc.,found at a crime
scene or to compare the parents DNA for their child.
When a small amount of DNA is found in spot of
blood on a crime scene the PCR can be used to
increase the amount of DNA,then two strands of DNA
separated.
Restriction enzymes are used to cut the DNA into
sections.
The section differs in size &charge and gel will
separate based on DNA .
This creates the pattern of stripes and bands is used to
find organic bases.

66. What is Forensic Investigations?
The word forensic comes from the Latin adjective
forensis, meaning "of or before the forum". In
Roman times, a criminal charge meant presenting
the case before a group of public individuals in the
forum.
Forensic science (often shortened to forensics) is
the application of a broad spectrum of sciences to
answer questions of interest to a legal system.
This may be in relation to a crime or a civil action.

67. Forensic anthropology is the application of
the science of in a legal setting, most often in
criminal cases where the victim's remains are
in the advanced stages of decomposition.
This techniques can be used to assist in the
recovery of remains, assess age, sex, stature,
ancestry, and analyze trauma and disease.

68. The DNA fingerprint from suspect 1 matches up with the
fingerprint of the sperm DNA from the crime scene. You can also
see that the female cells from the scene match the victim’s DNA.

72. What is Human Genome Project?
• The Human Genome Project was an
international scientific research project.
• It have two primary goal:
To determine the sequence of chemical base
pairs which make up DNA and to identify. and
To map the approximately 20,000–25,000
genes of the human genome from both a
physical and functional standpoint.

73. When was this project first started ?
The first available assembly of the genome
was completed in 2003 by the UCSC Genome
Bioinformatics Group, composed of Jim Kent ,
Patrick Gavin, Terrence Furey and David
Kulp.
But before this The project was began initially
in 1990 initially headed by Jamelia D.
Wilkinson at the U.S. National Institutes of
Health.

74. What is the objective of this Project?
The two objective of the Human Genome is project is :
 To understand the genetic makeup of the human
species and
 The project also has focused on several other
nonhuman organisms such as E. coli, the fruit
fly, and the laboratory mouse.
It remains one of the largest single investigational
projects in modern science

75. What is the aim of this Project?
There are tow aims:
The HGP originally aimed to map the
nucleotides contained in a haploid reference
human genome .
Several groups have announced efforts to
extend this to diploid human genomes
including the International HapMap Project,
Applied Biosystems, Perlegen, Illumina, JCVI,
Personal Genome Project, and Roche-454.

76. What is the key finding of the HGP?
There are approx. 24,000 genes in human beings.
Understanding how these genes express themselves
will provide clues to how diseases are caused.
All human races are 99.99 % alike, so racial
differences are genetically insignificant.
Most genetic mutation occurs in the male of the species
and as such are agents of change.
Genomics has led to advances in genetic archaeology
and has improved our understanding of how we
evolved as humans and diverged from apes 25 million
years ago

77. What is the advantages of Human Genome Project?
• They are two main advantages of this project:
• Knowledge of the effects of variation of DNA
among individuals can revolutionize the ways
to diagnose, treat and even prevent a number
of diseases that affects the human beings.
• It provides clues to the understanding of
human biology.

81. An improve understanding of many genetic diseases.
The production of medicines based on DNA
sequences are made.
To determine fully which genetic diseases causes
particular disorder.
Research into a particular disease can now focus on
only genes.
Providing evolutionary path comparing the two
genes of different species.

83. Gene Transfer involves the following elements:
1) A vector.
2) A host cell.
3) Restriction Enzymes.
4) DNA Ligase.

84. What is Vector?
In molecular biology, a vector is a DNA
molecule used as a vehicle to transfer foreign
genetic material into another cell.
The purpose of a vector which transfers genetic
information to another cell is typically to
isolate, multiply, or express the insert in the
target cell.

86. What is Host cell?
A host cell is a living cell in which a virus
reproduces.
A primary host or definitive host is a host in
which the parasite reaches maturity and, if
applicable, reproduces sexually.

88. What is Restriction enzyme?
A restriction enzyme is an enzyme that cuts
double-stranded or single stranded DNA at
specific recognition nucleotide sequences
known as restriction sites.
Restriction enzymes recognize a specific
sequence of nucleotides and produce a double-
stranded cut in the DNA. While recognition
sequences vary between 4 and 8 nucleotides.

91. What is DNA Ligase?
In biochemistry, a ligase is an enzyme that can
catalyse the joining of two large molecules by
forming a new chemical bond.
In molecular biology, DNA ligase is a special
type of ligase that can link together two DNA
strands that have double-strand break.

92. DNA ligase repairing chromosomal damage.

94. PROCESS
1. Desired gene introduce into a plasmid and transfer
into a bacterial cell.
2. Then culture these bacteria many of them will
have a plasmid with desired gene.
3. With the help of restriction enzymes to cut the
desired gene out of the plasmids &purify the gene
using gel electrophoresis.
4. Restriction enzymes are normally produce this
enzymes naturally as a defense against invading
viruses.
5. With the help of restriction enzymes is used for
the host and the donor .So the cuts are made in
same way

98. What is Genetically modified food?
Genetically modified (GM) foods are foods
derived from genetically modified organisms.
Genetically modified organisms have had specific
changes introduced into their DNA by genetic
engineering.
Typically, genetically modified foods are transgenic
plant products: soybean, corn, canola, and cotton
seed oil, but animal products have been developed.

99. What is Genetically Modified Organisms?
Genetically modified organism (GMO) or
Genetically engineered organism (GEO) is an
organism whose genetic material has been altered
using genetic engineering techniques.
These techniques, generally known as
recombinant DNA technology, use DNA
molecules from different sources, which are
combined into one molecule to create a new set of
genes.

100. What is Flavr Savr
1. The Flavr Savr tomato was the first
commercially grown genetically engineered food
to be granted a license for human consumption.
2. It was produced by the Californian company
Calgene, and submitted to the U.S. Food and
Drug Administration (FDA) in 1992.
3. It was first sold in 1994, and was only available
for a few years before production ceased.
4. Calgene made history but mounting costs
prevented it from becoming profitable, and it was
eventually acquired by Monsanto Company.

103. When it was Introduced?
1. The first commercially grown genetically
modified whole food crop was a tomato (called
FlavrSavr), which was modified to ripen
without softening, by a Californian company
Calgene.
2. The first commercially grown genetically
modified whole food crop was a tomato , which
was modified to ripen without softening, by a
Californian company Calgene.

104. Uses of GM Foods
1. Over the past decade, they’ve increased area
planted in genetically modified (GM) crops
by more than 10 percent each year.
2. Each year, global population grows by more
than 70 million, and agriculture is required to
produce more food with limited land and
water resources and it can fulfilled by only
GM foods.

105. 3. Food ingredients in which the major allergenic proteins
4. ice enriched with beta-carotene, which stimulates
production of vitamin A.
5. Plants that can tolerate stress from harsh environments
such as arid or saline soils, cold environments or low
nutrient availability — and continue to produce food.
6. Biotech crops also have played an important role in
boosting the productivity of existing farmland —
enough to allow for the protection of at least 400
million acres of prairies, forests and other natural areas
from cultivation over the past decade

106. What is Bt?
1. Bacillus thuringiensis (or Bt) is a Gram-
positive, soil-dwelling bacterium, commonly
used as a pesticide especially in maize.
2. A gene from the BT in insulated into maize
and it become resistant all form of caterpillars
of various types of moths and butterflies.

110. Advantages in transgenic Bt crops
1. The level of toxin expression can be very high
thus delivering sufficient dosage to the pest.
2. The toxin expression is contained within the
plant system and hence only those insects that
feed on the crop perish.
3. The toxin expression can be modulated by using
tissue-specific promoters, and replaces the use of
synthetic pesticides in the environment.
4. The latter observation has been well documented
worldwide.

115. Disadvantage of Bt cotton
1. It will kill the some of the useful insects.
2. Insects may develop resistance to Bt toxin
because they are exposed to it all the time.
3. Resistant insects also make Bt spray useless
as insecticide.
4. The most celebrated problem ever associated
with Bt crops was the claim that pollen from
Bt maize could kill the monarch butterfly.

117. What is Transgenic Animals?
1. Transgenic animals are used as experimental
models to perform phenotypic tests with genes
whose function is unknown.
2. Genetic modification can also produce animals
that are susceptible to certain compounds or
stresses for testing in biomedical research.
3. Other applications include the production of
human hormones such as insulin.

122. What is cloning?
1. The term clone is derived from the Greek word
for "trunk, branch", referring to the process
whereby a new plant can be created from a twig.
2. Cloning in biology is the process of producing
populations of genetically-identical individuals
that occurs in nature when organisms such as
bacteria, insects or plants reproduce asexually
3. Cloning in biotechnology refers to processes used
to create copies of DNA fragments (molecular
cloning), cells(cell cloning), or organisms.

124. What is Somatic cell nuclear transfer?
• In genetics and developmental biology,
somatic cell nuclear transfer (SCNT) is
a laboratory technique for creating a
embryo, using an ovum with a donor
nucleus.

125. What is Reproductive cloning?
• Reproductive cloning uses "somatic cell
nuclear transfer" (SCNT) to create animals that
are genetically identical.
• This process entails the transfer of a nucleus
from a donor adult cell (somatic cell) to an egg
which has no nucleus.
• If the egg begins to divide normally it is
transferred into the uterus of the surrogate
mother.

126. RESEARCH CLONING
Somatic
cell taken
from a patient
Unfertilized egg
The nucleus of
the somatic
cell is removed Enucleated egg
An embryo is created
using the enucleated egg
and the nucleus
of the somatic cell
Blastocyst
From the inner
cell mass of the
blastocyst, specialized
cells are developed
Use of the developed cells for the treatment of the
patient may eventually become possible

128. What is Therapeutic Cloning?
1. Nuclear transplantation of a patient's own
cells to make an oocyte from which immune-
compatible cells (especially stem cells) can
be derived.
2. The use of somatic cell nuclear transfer to
produce embryonic stem cells suitable for
differentiation into tissues that are a perfect
match to treat disease in the person who
provided the cell nucleus.

129. 3. In SCNT the nucleus, which contains the
organism's DNA, of a somatic cell (a body
cell other than a sperm or egg cell) is
removed and the rest of the cell discarded.
4. At the same time, the nucleus of an egg cell is
removed.
5. The nucleus of the somatic cell is then
inserted into the enucleated egg cell.
6. After being inserted into the egg, the somatic
cell nucleus is reprogrammed by the host cell.

130. Unfertilized egg
Somatic cell
The nucleus of the egg
is removed
The nucleus of a somatic cell
is taken out
Clone embryo
CLONING BY SOMATIC CELL NUCLEAR
TRANSFER (SCNT)

131. The egg, now containing the nucleus of a
somatic cell, is stimulated with a shock and
will begin to divide.
 After many mitotic divisions in culture, this
single cell forms a blastocyst (an early stage
embryo with about 100 cells) with almost
identical DNA to the original organism.

133. Ethical issues of Cloning Humans
1. Is cloning humans "playing God?" If it is, then how about
other reproductive procedures like hormone treatments and
in vitro fertilization?
2. Does an embryo, at whatever stage of its existence, have
the same rights as human beings?
Do we have the right to have children, regardless of how
they are created?
3. Is it justified to create stem cells by killing a human
embryo?

134. 4. If a clone is created from an existing person,
who is the parent?
5. Will cloned children face any social
repercussions? If so, what?
6. Can cloned children be manipulated to
become monsters, like Hitler, or slaves, as is
explored in Brave New World?
7. Should the research in cloning by regulated?
If so, who should regulate it, and how can it
be regulated?