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LAPORAN TUTORIAL SKENARIO V : GENETIC ENGINEERING
OLEH :
1. DIAN NURHANI SAFITRI H1A 008 0052. RIFKA WIKAMTO H1A 008 0063. ARENTA MANTASARI H1A 008 0094. IKA NURFITRIA TAUHIDA H1A 008 0115. DINI HARIYATI M. S. H1A 008 0226. DANTE YUSTISIA H1A 008 0357. BQ. JATNA ATMAWATI H1A 008 0378. EVERT YANRI I. S. H1A 008 0399. SANGGITHA YUNINGTYAS H1A 008 045
TUTOR : dr. Nurhidayati, M. Kes
DEPARTEMEN PENDIDIKAN NASIONAL FAKULTAS KEDOKTERAN UNIVERSITAS MATARAM
NUSA TENGGARA BARATTAHUN AKADEMIK 2008/2009
0
PREFACE
First of all, the writer would like to thank God for blessing given our to finish
this paper with title of “ Genetic Engineering “ at the proper time. There were many
problems and difficulties that Writer faced in the process of writing this paper due to
the limited of knowledge and references. But those all could be overcome thought
hard work and lots of valuable advises given by the advisors
For the first, the Writer would like to express our special gratitude to the
tutor Dr. Nurhidayati, M.kes had to lead us on the making . On the second The
Writer would like to express gratitude all of my family, especially to my parents who
has supported me both financially as well as morally. To all my friends I can not
mention one by one for our friendship. I hope this paper can be useful for especially
for student in School of Medicine, Mataram University.
Tuesday, November 25th 2008
Writer
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CONTENTS
Preface .............................................................................................................. 1
Contents............................................................................................................. 2
Scenario ............................................................................................................ 3
Mindmapping ................................................................................................... 4
Learning Objectives ......................................................................................... 5
Gene Mutation .................................................................................................. 6
Genetic Engineering
Plasmid Recombinant Technique ....................................................... 12
Gene Therapy .................................................................................... 13
Human Cloning .................................................................................. 15
Hybridization ..................................................................................... 19
Polymerase Chain Reaction ............................................................... 20
Advantages and Disadvantages
Advantages of Biotechnology
In Plants .................................................................................. 23
In Pharmacogenomics ............................................................ 25
Advantages of Genetic Engineering Product ..................................... 26
Disadvantages of Genetic Engineering Products ................................ 27
Genetic Counseling ......................................................................................... 28
Relations with Ethics
Gene Therapy ..................................................................................... 32
Embryo Cloning ................................................................................. 32
Conclusions .................................................................................................... 35
Refferences ..................................................................................................... 36
2
SCENARIO
With the same kinds of recombinant DNA and PCR techniques used in basic
biological analysis, DNA molecular testing(involve DNA Mutation), gene cloning,
DNA typing, and gene therapy, Biotechnology and pharmaceutical companies
develop useful products. Many types of products are now available or are in
development, including pharmaceuticals (i.e Human Growth hormone, humulin, etc)
and vaccines for humans (i.e hepatitis B) and for animals and genetically engineered
organisms for improved production of important compounds in the food industry.
Recently, genetic engineering often use for cancer detection and therapy.
3
MINDMAPPING
BIOTEKNOLOGI ANALISIS DNA / PCR
MANFAAT
REKAYASA GENETIK
PROSES
TEKHNIK
KEUNTUNGAN
KERUGIAN
GEN MUTASI
PENYEBABMACAM
4MANFAAT
PROSES
TEKHNIKKERUGIAN
MANFAATMANFAAT
LEARNING OBJECTIVES
• Genetic engineering techniques
• Genetic engineering components
• Advantages & disadvantages of genetic engineering
• PCR techniques
• Gene mutation & mutagens
• Advantages of biotechnology
5
GENE MUTATION
Gen mutation is clasificated by eight type there are the substitution of that base
of nucleotyde, transperation of base nucleotyde, kind of polipeptyde, mutation 3 base
or codon, type of cell, according to the direct mutation, mutation base on the occur
and characteristic of cell.
1. Classification gen mutation according off subtitution base of nucleotyde
Substitution of base nucleotyde make a partener of the base is not
comfort, because of the not true of partener of the base, so make a change
of a codon or 3 base in the DNA. Classification off gen mutation is
transition and transvertion.
Transition
Transition is one kind of the gen mutation because of base of purin
(adenin;A and guanine;G) are change by a base of purin or base of
pirimidin(citocin;S and timin;T) are change by base of pirimidin. For
example adenin is change by guanine.
Or citosin is change by timin.
Transvertion
Transvertion : if base of purin is change by the different base like
pirimidin or base of pirimidin is change by a base of purin. For example
adenine is change by citosin or timin is change by a guaanin.
2. Tranferation of base nucleotide
Mutation transperation of base nucleotyde cause by deletion or
insertion nucleotyde at the DNA and then make a diferents struktur of the
mRNA molecules.
deletion
Deletion is a change of one nucleotide from the s patrand of gen
code, and make a changed reading frame, in mRNA. system of mecins that
do the translation isn’t know that a base have lossed because pungtuation
or reading code of in reading a codon. Because of the changed in the
structure of the amino acids that’s polimerysed.
Insertion
Insertion or the familiar with insert the one or more the base of
nucleotyde into a gen that will produce mRNA with the reading frame that
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mmpengaruhi from translation and effect in the insertion will look at
translation of mRNA. that may be will poduce the structur of amino acid in
the distal of insertion and produce of codon without names.
3. Type of polipeptyde or protein
Classification mutation according to the protein type there are trie
gen mutation, that’s missense mutation, nonsense mutation and silent
mutation.
Missense mutation
One of the nitrogen base that change the other nitrogen base so can
produce codon to code the amino acid their asalnya.so, that inflluence
metabolism body because of thats changes at amino acid, for example timin
changes by citosyn so the amino acid that produce is lisyn and in fact is a
guanine.
Nonsense mutation
Stucture protein can produce from some of the amino acid. That amino
acid is product of translated codons. If in yhe gen mutation is fine the codon
stop before a prosess of making protein finish so syntesys protein will early
to finish, so protein is can not produce.
Silent mutation
Mutation isn’t make an some effect is a silent mutation it mean that’s
make to amino acid and make to polipeptyde in produce protein is not ,
Because the canged of structure base nukleotyde only in code of purin or
only in pirimidin but the amino acid that produce is same. For example in
Glutamin GTTT in glutamine will substitution with C but amino acid that
produce of protein is like before.
4. Mutation 3 base or codon
Mutation 3 base or codon is a mutation that the DNA get or lossed amino
acid.
5. According to the direct of the mutation
Forward Mutation : usually the reaction to changed from the normal cell
to abnormal cell.
Back mutation : usually the reaction to changed the cell from the
abnormal cell to the normal cell
6. Type of cell
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a. Mutation on cell somatics or body
Mutation in the cell somatic.
b. Mutation on cell germinal
Mutation in the cell germinal.
7. Characteristic of cell
a. Dominan mutation
Mutation ussually find in the heterozygot
b. Ressesif mutation
Mutation ussually find in the haployd cell and ussually caused by an
virus and bacterial.
8. mutation base on the occur
a. Spontan mutation
Mutation caused by pure from inside body like the dismetabolism not
only dismetabolism but also the radiation from the nature.
b. Induced mutation
Mutation that caused of the mutagen that from outside body. Mutagen
from outside body like chemical mutagen, biological mutagen, radiation
ionization mutagen, and ultraviolet ligh.
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A gene mutation is a permanent change in the DNA sequence that makes up a gene.
Mutations range in size from a single DNA building block (DNA base) to a large
segment of a chromosome.
Gene mutations occur in two ways: they can be inherited from a parent or acquired
during a person’s lifetime. Mutations that are passed from parent to child are called
hereditary mutations or germline mutations (because they are present in the egg and
sperm cells, which are also called germ cells). This type of mutation is present
throughout a person’s life in virtually every cell in the body.
Mutations that occur only in an egg or sperm cell, or those that occur just after
fertilization, are called new (de novo) mutations. De novo mutations may explain
genetic disorders in which an affected child has a mutation in every cell, but has no
family history of the disorder.
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Acquired (or somatic) mutations occur in the DNA of individual cells at some time
during a person’s life. These changes can be caused by environmental factors such as
ultraviolet radiation from the sun, or can occur if a mistake is made as DNA copies
itself during cell division. Acquired mutations in somatic cells (cells other than sperm
and egg cells) cannot be passed on to the next generation.
Mutations may also occur in a single cell within an early embryo. As all the cells
divide during growth and development, the individual will have some cells with the
mutation and some cells without the genetic change. This situation is called
mosaicism.
Some genetic changes are very rare; others are common in the population. Genetic
changes that occur in more than 1 percent of the population are called polymorphisms.
They are common enough to be considered a normal variation in the DNA.
Polymorphisms are responsible for many of the normal differences between people
such as eye color, hair color, and blood type. Although many polymorphisms have no
negative effects on a person’s health, some of these variations may influence the risk
of developing certain disorders.
Types of Gene Mutation :
1. Point Mutations is the mutation which change only one or a few base pairs. It
can be divided into two general categories :
Substitution Mutation
a. Transition Mutation is a mutation from one purine-pyrimidine
base pair to the other purine-pyrimidine base pair.
b. Transversion Mutation is a mutation from a purine-pyrimidine
base pair to a pyrimidine-purine base pair.
Insertions or Deletions
2. Missense Mutation is a gene mutation
when a base pair change in the DNA causes a change in codon so that a different
amino acid is inserted into the polypeptide.
3. Nonsense Mutation is a gene mutation
when a base pair change in the DNA changes codon for an amino acid to a stop
codon (UAG, UAA, or UGA).
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4. Silent Mutation is also a subset of
missense mutations that occurs when a base pair change in a gene alters a codon
such that the same amino acid is inserted in the protein.
5. Frameshift Mutation
Example :
Frameshift Mutation
Causes of Mutation :
1. The effects of natural radiation
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Transition : blue
GENETIC ENGINEERING
PLASMID RECOMBINANT TECHNIQUE
1. Isolate plasmid (vector) DNA and
human DNA.
Prepare two kinds of DNA: bacterial
plasmid as the vector and human DNA
containing the gene of interest. The
plasmid is taken from E. coli and
carrying two gene:
ampR: given résistance to ampicillin
antibiotic in E. coli
lacZ− : codes β-galaktosidase, that
breakdown lactose
2. Insert human DNA into plasmid:
a. Cut both DNAs with the same
restriction enzyme. Restriction
enzyme makes sticky ends at the
both DNA.
b. Mix the DNAs: they join by base
pairing, also this one, join with the
gene of interest.
c. Add DNA ligase to bond covalently. And then it makes DNA recombinant.
3. Put plasmids into lacZ− bacteria by transformation.
4. Clone cells:
a. Plate cells onto medium with ampicillin and x-gal.
Ampicillin: makes sure that only cell with DNA recombinant can grow,
because only this cell has ampR gene that resistance to ampicillin.
X-gal: makes easier to identify bacteria with DNA recombinant.
b. Identify clones of cells containing recombinant plasmids by their ability to
grow in presence of ampicillin and their white color
5. Identify clone carrying gene of interest.
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GENE THERAPY
Gene therapy involves the introduction of
genetic material into a cell to treat disease.
Many of the conditions treated in this way
are genetic diseases that occur when genes
malfunction. A common approach in gene
therapy is to identify a malfunctioning gene
and supply the patient with functioning
copies of that gene. Other approaches include
switching specific genes on or off,
introducing genes to kill cancer cells, to
suppress tumours by inhibiting the blood
supply, or to stimulate the immune system to
attack certain types of cells. Whichever
approach is used, the aim of gene therapy is
to introduce therapeutic material into the
target cells, for this to become active inside the patient and exert the intended
therapeutic effect. At present, gene therapy is still at the clinical research stage.
Gene therapy: somatic and germ-line gene therapy
Somatic Cell Gene Therapy
Many genetic diseases may be able to be treated by correcting the defective
genes, by gene therapy. Gene therapy is a therapeutic technique in which a
functioning gene is inserted into the cells of a patient to correct an inborn genetic
error or to provide a new function to the cell. It means the genetic modification of
DNA in the body cells of an individual patient, directed to alleviating disease in that
patient.
There have been several hundred human gene therapy clinical trials in many
countries (including USA, EU, Canada, China, Japan, New Zealand,etc), involving
over 6000 patients world-wide, for several different diseases including several
cancers.
Somatic cell gene therapy involves injection of 'healthy genes' into somatic
(body) cells of a patient. The DNA change is not inherited to children. The first
human gene therapy protocol began in September 1990 that successfully treated
adenosine deaminase deficiency (ADA) disease.
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From 1989 until September 1999 there were thousands of patients in trials and
no one died because of the experiments. 18 year-old Jesse Gelsinger died at the
University of Pennsylvania (USA) on 17 September 1999, four days after receiving a
relatively high dose of an experimental gene therapy. His death was the result of a
large immune reaction to the genetically engineered adenovirus that researchers had
infused into his liver. There was much review of the procedures for safety following
that case.
Gene therapy is still an experimental therapy, but if it is safe and effective, it
may prove to be a better approach to therapy than many current therapies, because
gene therapy cures the cause of the disease rather than merely treating the symptoms
of a disease. Also, many diseases are still incurable by other means, so the potential
benefit is saving life.
Germ-line gene therapy
At the present gene therapy is not inheritable. Germ cells are cells connected with
reproduction, found in the testis (males) and ovary (females), i.e. Egg and sperm cells
and the cells that give rise to them. Germ-line gene therapy targets the germ cells.
This type of therapy may also mean injecting DNA to correct, modify or add DNA
into the pronucleus of a fertilized egg. The latter technology would require that
fertilization would occur in vitro using the usual IVF procedures of super-ovulation
and fertilization of a number of egg cells prior to micromanipulation for DNA transfer
and then embryo transfer to a mother after checking the embryo's chromosomes.
How Does Gene Therapy Work
Gene therapy is designed to introduce genetic material into cells to
compensate for abnormal genes or to make a beneficial protein. If a mutated gene
causes a necessary protein to be faulty or missing, gene therapy may be able to
introduce a normal copy of the gene to restore the function of the protein.A gene that
is inserted directly into a cell usually does not function. Instead, a carrier called a
vector is genetically engineered to deliver the gene. Certain viruses are often used as
vectors because they can deliver the new gene by infecting the cell. The viruses are
modified so they can’t cause disease when used in people. Some types of virus, such
as retroviruses, integrate their genetic material (including the new gene) into a
chromosome in the human cell. Other viruses, such as adenoviruses, introduce their
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DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.
The vector can be injected or given intravenously (by IV) directly into a specific
tissue in the body, where it is taken up by individual cells. Alternately, a sample of the
patient’s cells can be removed and exposed to the vector in a laboratory setting. The
cells containing the vector are then returned to the patient. If the treatment is
successful, the new gene delivered by the vector will make a functioning protein.
Researchers must overcome many technical challenges before gene therapy
will be a practical approach to treating disease. For example, scientists must find
better ways to deliver genes and target them to particular cells. They must also ensure
that new genes are precisely controlled by the body.
A new gene is injected into an adenovirus vector, which is used to introduce the
modified DNA into a human cell. If the treatment is successful, the new gene will
make a functional protein.
HUMAN CLONING
In order to study the structure of a gene it is necessary to isolate it from other
genes. The most common method of achieving this is to make a gene library and
screen it for clones containing the gene of interest. Libraries can be made from the
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entire genome of the organism (genomic) or just from those genes that are expressed
(transcribed) in a particular tissue at a particular time (cDNA). DNA molecules may
be manipulated by using restriction endonucleases. These enzymes recognize specific
sequences in the DNA molecule and cut it wherever they occur. Fragments of DNA
may be joined together by DNA ligase. Fragments of DNA may be ligated into either
plasmids (autonomously replicating circular DNA molecules found in bacteria and
yeast) or bacteriophages (viruses that infect bacteria) and propagated by
transformation of Escherichia coli. The foreign DNA is faithfully replicated by the
host bacteria and individual clones may be selected by colony hybridization with a
suitable probe. Plasmid or phage DNA containing the fragment of interest can be
isolated from bacteria for sequencing or further manipulation.
Cloning is the process
of asexually producing a
group of cells (clones), all
genetically identical to the
original ancestor. The word
is also used in recombinant
DNA manipulation
procedures to produce
multiple copies of a single
gene or segment of DNA. It
is more commonly known as
the production of a cell or an
organism from a somatic cell of an organism with the same nuclear genomic (genetic)
characters - without fertilization. A clone is a collection of cells or organisms that are
genetically identical. Some vegetables are made this way, like asparagus, or flowers
like orchids.
Human reproductive cloning is the production of a human fetus from a single
cell by asexual reproduction. In 2001 a cloned embryo was reported made by nuclear
transfer, though in 1993 cloned embryos were made by splitting human embryos. In
the late 1990s reproductive cloning was used to produce clones of the adults of a
number of mammalian species, including sheep, mice and pigs. The most famous of
these was Dolly, the sheep. Many countries rushed to outlaw the possibility of
reproductive cloning in humans. Most mammalian embryos can only be split into 2-4
clones, after that the cells lack the ability to start development into a human being.
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Therapeutic cloning is the cloning of embryos containing DNA from an
individual's own cell to generate a source of embryonic stem (ES) cell-progenitor
cells that can differentiate into the different cell types of the body. ES cells are
capable of generating all cell types, unlike multipotent adult-derived stem cells which
generate many but not all cell types. The aim is to produce healthy replacement tissue
that would be readily available. Since it is from the same body it is
immunocompatible so that the recipients would not have to take immunosuppressant
drugs for the rest of their lives, as they do if they receive an organ from another
person.
What is embryo cloning?
Cloning is the production of one or more individual plants or animals that are
genetically identical to another plant or animal.
Embryo cloning might be more accurately called "artificial twinning", because it
simulates the mechanism by which twins naturally develop. It involves removing one
or more cells from an embryo and encouraging the cell to develop into a separate
embryo with the same DNA as the original. It has been successfully carried out for
years on many species of animals. Some very limited experimentation has been done
on human embryos.
Nature itself is the greatest cloning agent. In about one of every 75 human
conceptions, the fertilized ovum splits for some unknown reason and produces
monozygotic (identical) twins. Each has an genetic makeup identical to the other. In
cloning, this same operation is done intentionally in a laboratory.
How human embryo cloning would be done
Human embryo cloning starts with a standard in vitro fertilization procedure.
Sperm and an egg cell are mixed together on a glass dish. After conception, the
zygote (fertilized egg) is allowed to develop into a blastula (a hollow mass of cells).
The zygote divides first into two cells, then four, then eight... A chemical is added to
the dish to remove the "zona pellucida" covering. This material provides nutrients to
the cells to promote cell division. With the covering removed, the blastula is divided
into individual cells which are deposited on individual dishes. They are then coated
with an artificial zona pellucida and allowed to divide and develop. The experiment
by Sillman et al. showed that the best results could be obtained by interrupting the
zygote at the two cell stage. Many of these pairs of zygotes were each able to develop
to the 32 cell stage, but stalled at that point. They might well have had the potential to
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develop further and even mature into a viable fetus, except that the original ovum was
defective and would have died anyway. For ethical reasons, the researchers had
selected embryos which had no possibility of ever maturing into fetuses, and thus
becoming newborn babies.
History of embryo cloning:
Cloning of embryos has been used in mice experiments since the late 1970's,
and in animal breeding since the late 1980's. The procedure splits a single fertilized
ovum into two or more clones, each of which is then implanted into the womb of a
receptive female.
However, research into cloning of human embryos has been restricted in the
United States and in some other countries. Pro-life groups which oppose free access to
abortion have had considerable political power. They were able to have all human
embryo research banned by the Regan and Bush Presidencies during most of the
1980's and into the early 1990's. During the first few days of President Clinton's
presidency, the ban on public funding of human embryo and fetal research was lifted.
We may not know the individual or team who first performed cloning of human
embryos. The methods used have been understood for many years and actually used
to clone embryos in cattle and sheep. It is likely that someone had successfully used
the method on a human embryo in secret. The first publicly announced human cloning
was done by Robert J. Stillman and his team at the George Washington Medical
Center in Washington D.C. They took 17 genetically flawed human embryos which
would have died within days no matter how they were treated. They were derived
from an ovum that had been fertilized by two sperm. This resulted in an extra set of
chromosomes which doomed the ovum's future. None could have developed into a
fetus. These ovum were successfully split in 1994-OCT, each producing one or more
clones. The main motive of the experiment seems to have been to trigger public
debate on the ethics of human cloning.
Dr. Steven Muller headed a panel in the US whose mandate was to produce
preliminary cloning guidelines. These would be used by the Federal National
Institutes of Health to decide which cloning research to fund. The panel recommended
that studies be normally limited to the use of preexisting, spare embryos - those that
developed during in vitro fertilization procedures that had been performed to assist
couples in conceiving. Generally about 20 or 24 ova are fertilized during these
procedures. Only three or four are implanted in the woman. The extra zygotes are
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either discarded or frozen for possible future use. New embryos would only be
prepared and used if needed for "compelling research." They further recommended
that any studies be normally terminated within 14 days of conception. Some
experiments might be authorized to continue until the 18th day, but no further. At that
gestational age, neural tube closure begins; this is the start of the development of a
nervous system. They recommended that certain procedures be banned, including
implanting human embryos in other species, implanting cloned embryos into humans,
the transfer of a nucleus from one embryo to another, and the use of embryos for sex
selection.
HYBRIDIZATION
Hybridization is based on the annealing properties of DNA
Double-standed DNA exists in life as a double helix, in which the two strands
are held together by hydrogen bonds between complementary bases (A with T, and G
with C).
Hybridization is a fundamental feature of DNA technology. It is a process by
which a piece of DNA or RNA of known nucleotide sequence, which can range in
size from as little as 15 bp to several hundred kilobases, is used to identify a region or
fragment of DNA containing complementary sequences. The first piece of DNA or
RNA is called a probe. Probe DNA will form complementary base pairings with
another strand of DNA, often termed the target, if the two strands are complementary,
and a sufficient number of hydrogen bonds is formed.
The principle of molecular hybridization
In molecular hybridization, it is essential that the probe and target are initially single-
strated.
Probes can vary in both their size and their nature. Hawever, one essential
feature of any hybridization reaction is that both the probe and the target mush be free
to base pair with one another. The process of separating the two strands of DNA is
called DNA denaturation or melting. Probe DNA is generally denatured by heating. If
the target DNA is double-stranded then it too mush be denatured. Either by heating or
treatment with alkali. (NB: alkali is not used to denature RNA because it leads to
hydrolysis of the polymer chain). Once both probe and target DNA are single-
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stranded mixing of the two will allow complementary bases to reassociate. This
process is called DNA annealing or reassociation.
A single-stranded probe and target can potentially anneal in a variety of ways:
Formation of probe-probe complementary strands (when using a ds probe)
Reannealing of the complementary strands of the target. So-called homoduplexes
Formation of probe-target heteroduplexes.
POLYMERASE CHAIN REACTION (PCR)
Generating large numbers of identical copies of DNA by the construction and cloning
of DNA molecules was made possible in the 1970s. Recombinant DNA techniques
molecular genetics by making it possible to analyze genes and their function in new
ways. However, cloning DNA is the time consuming. In the mid-1980s, the
polymerase chain reaction (PCR) was developed, and this has resulted in a new
revolution in gene analysis. In a process called amplification, PCR produces an
extremely large number of copies of a specific DNA sequence from a DNA mixture
without having to clone the sequence in a host organism. The ampilfied PCR products
are called amplimers. PCR has become one of the most important tools in modern
molecular biology. Kary Mullis, who developed the technique, shared the Nobel Prize
in Chemistry 1993. (the other recipient, M. Smith, received the prize for the other
work.
PCR Steps
PCR begin with the double stranded DNA containing the sequence to be amplified
and a pair of oligonucleotide primers which flank that DNA. The primers usually are
20 or more nucleotides long and are made synthetically, so a limitation of PCR is that
information must be available about the sequence of interest. In brief, PCR is done as
follows:
a. denature the double stranded DNA to single strands by heating at 94-95o C
b. cool the solution, and anneal the primers at 37-65o C, depending on how well
the base sequences of the primers complement the base sequence of the DNA.
The two primers are designed so that they anneal to the opposite strands of the
template DNA flanking the sequence to be amplified. As the result, the 3’ ends
of the primers face each other
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c. extend the primers with DNA polymerase at 70-75o C. For this, a special heat-
resistant DNA polymerase, such as Taq DNA Polymerase, is used. )this
particular enzyme is the DNA polymerase of a thermophilic bacterium,
Thermus aquaticus)
d. repeat the heating cycle to denature the DNA to single strands, and cool the
solution to anneal the primers again
e. repeat the extension of the primer with Taq DNA polymerase. In each of the
two doble stranded molecules produced in the figure, one strand is a unit
length; that is, it is the length of DNA between the 5’ end of primer A and the
5’ end of primer B—the length of the target DNA. The other strand in both
molecules is longer than the unit length
f. repeat the denaturation of DNA and the annealing of new primers. (for
simplification, the further amplification of those strands which are longer than
unit length is omitted in the rest of the figure)
g. repeat the extension of the primer with Taq DNA polumerase. This produce
unit length, double stranded DNA. Note that it took three cycles to produce the
two molecules of unit length DNA. Repeated denaturation, aneealing, and
extension cycle result in a geometric increase in the amount of unit length
DNA
With PCR, the amount of new DNA generated increases geometrically. Strating with
1 molecules DNA, 1 cycle of PCR produce 2 molecules, 2 cycles produce 4
molecules, 3 cycles produce 8 molecules, 2 of which are the target DNA. A further 10
cycles produce 1.024 copies (210) of the target DNA, and in 20 cycles there will be
1.048.576 copies (220) of the target DNA. The procedure is rapid, each cycle taking
only a few minutes in a thermal cycler, a machine that automatically cycles the
reaction through programmed temperature changes.
Advantages and Limitation of PCR
PCR is a powerfull techniques for amplifying segments of DNA. Such amplification
is similar to cloning DNA using vectors. However, PCR is much sensitive and quicker
technique than cloning. Specifically, PCR can produce million of copis of DNA
segment, starting with just one DNA molecule, in only a few hours. By contrast,
cloning requires a significant amount of starting DNA for restriction digestion, and
then at least a week is needed to go through all the cloning steps. There are two major
limitations of PCR, however. First, PCR requires the use of specific primers, so
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sequence information on the DNA that to be amplified must be available in order for
primers to be designed. Second, the length of DNA that can be amplified by PCR is
limited by the enzime and surrounding conditions to about 20 kb. A further issue with
PCR is that Taq polymerase has no proofreading activity; accordingly, base pair
mismatches that occur during replication go uncorrected in this in vitro procedure.
Since PCR involves a geometric increase in the number of DNA molecules, the lack
of error correction is more or less serious depending on when in the amplification
process in error is introduced. That is, on the one hand, if an erros is introduced in the
first round of PCR, then all derivative DNA molecules will have the error. On the
other hand, and increasingly lower fraction of the DNA molecules will have the error
the later in the rounds of PCR it is introduced. Some alternative enzymes are available
for PCR that do have proofreading activity, and these enzymes significantly decrease
the error frequency. One such enzyme is Vent polymerase, which was originally
extracted from a bacterium growing around high temperature deep-sea oceanic vents.
Finally, the excellent sensitivity of PCR is liability in some applications. Because
PCR can produce many copies from a single DNA molecule, great care has to be
taken that the right DNA molecules are amlified. In forensic applications, for
example, it is crucial that DNA used for evidence have no chance of being
contaminated by DNA from the investigators or researchers.
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ADVANTAGES AND DISADVANTAGES
ADVANTAGES OF BIOTECHNOLOGY
The advantages of biotechnology, today and in the future, are nearly limitless.
Plant biotechnology offers the potential to produce crops that not only taste better but
are also healthier.
Agronomic or "input" traits create value by giving plants the ability to do
things that increase production or reduce the need for other inputs such as chemical
pesticides or fertilisers. Our current products with input traits include potatoes, corn
and soybeans that produce better yields with fewer costly inputs through better control
of pests and weeds. Already, farmers in Romania are growing potatoes that use 40%
less chemical insecticides than would be possible using traditional techniques.
Quality traits -- or "output" traits -- help create value for consumers by enhancing the
quality of the food and fibre produced by the plant. Likely future offerings include
potatoes that will absorb less oil when fried, corn and soya beans with an increased
protein content, tomatoes with a fresher flavour and corn and sweet potatoes that
contain high levels of amino acids, such as lysine.
Someday, seeds will become the ultimate energy-efficient, environmentally
friendly production facilities that can manufacture products which are today made
from nonrenewable resources. An oilseed rape plant, for example, could serve as a
factory to add beta carotene to canola oil to alleviate the nutritional deficiency that
causes night blindness.
GM plants could nevertheless provide a means of significantly improving
human health, first of all by supplying better quality food. Plants could be deprived of
their most harmful ingredients (such as lipids which are bad for cholesterol) or
enriched with molecules of nutritional benefit, the latter of particular benefit to
southern countries. European laboratories recently developed a 'golden rice' enriched
with carotene. This molecule is a precursor of vitamin A and could therefore help
correct the nutritional deficiencies affecting millions of people. Another example is
research aimed at increasing the lycopene content of tomatoes. This molecule has
beneficial anti-oxidising effects which reduce the risk of prostate tumours
The table below will explain more about the plants that was produced from
tissue culture methods with their advantages.
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Plants Product Usefull
Papaver somniferm
Digitalis sp
Jasminum sp
Menta piperita
Chinchona ledgeriana
Chrysanthemun sp
Catharantus roseus
Captis japonica
Derris elliptica
Panax ginseng
Kodein
Digoksin
Jasmine
Mentol
Kina
Pirethrin
Indol alkaloid
Berberin
Rotenon
Saponin
Analgesic
Cardiac disease therapy
Perfume
Food aromatic
Malaria (Medicine)
Insectiside
Anti-leukimia
Antiseptic
Insectiside
insectiside
The advantages of tissue culture for agricultural research on higher plants
included:
1. Virus elimination and production of pathogen-free plants
2. Rapid multiplication of superior plants
3. Production of haploids and homozygous diploid plants by anther or pollen culture
4. Production of interspecific hybrids by test tube fertilization and embryo rescue
5. Production of secondary metabolites via callus or cell suspension culture
6. Production of new genetic resources by somaclonal variation
7. Preservation and international movement of germplasm
8. Protoplast technology and gene transfer.
9. genetically identical, virus – indexed, germplasm maintenance, hybrid production
for incompatible species haploid, plants year round production difficult to
propagate species, new variant and research tool
Another kind of biotechnology is cloning. This method has advantage to
replace infected organs (such as cancer) with new cloned organs and create more
population if needed. In medical, biotechnology gives more advantages for improving
the health quality. Biotechnology can use for produce the monoclonal antibody and
gen therapy. It is already developed by researches.
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Pharmacogenomics
Pharmacogenomics is the study of how an individual's genetic inheritance
affects the body's response to drugs. The term comes from the words pharmacology
and genomics and is thus the intersection of pharmaceuticals and genetics.
Pharmacogenomics holds the promise that drugs might one day be tailor-made for
individuals and adapted to each person's own genetic makeup. Environment, diet, age,
lifestyle, and state of health all can influence a person's response to medicines, but
understanding an individual's genetic makeup is thought to be the key to creating
personalized drugs with greater efficacy and safety. Pharmacogenomics combines
traditional pharmaceutical sciences such as biochemistry with annotated knowledge of
genes, proteins, and single nucleotide polymorphisms.
One can anticipate the benefits of Pharmacogenomics, which are as follows:
• More Powerful Medicines
Pharmaceutical companies will be able to create drugs based on the proteins,
enzymes, and RNA molecules associated with genes and diseases. This will facilitate
drug discovery and allow drug makers to produce a therapy more targeted to specific
diseases. This accuracy not only will maximize therapeutic effects but also decrease
damage to nearby healthy cells.
• Better, Safer Drugs the First Time
Instead of the standard trial-and-error method of matching patients with the
right drugs, doctors will be able to analyze a patient's genetic profile and prescribe the
best available drug therapy from the beginning. Not only will this take the guesswork
out of finding the right drug, it will speed recovery time and increase safety as the
likelihood of adverse reactions is eliminated. Pharmacogenomics has the potential to
dramatically reduce the estimated 100,000 deaths and 2 million hospitalizations that
occur each year in the United States as the result of adverse drug response (1).
• More Accurate Methods of Determining Appropriate Drug Dosages
Current methods of basing dosages on weight and age will be replaced with
dosages based on a person's genetics --how well the body processes the medicine and
the time it takes to metabolize it. This will maximize the therapy's value and decrease
the likelihood of overdose.
• Advanced Screening for Disease
Knowing one's genetic code will allow a person to make adequate lifestyle and
environmental changes at an early age so as to avoid or lessen the severity of a genetic
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disease. Likewise, advance knowledge of particular disease susceptibility will allow
careful monitoring, and treatments can be introduced at the most appropriate stage to
maximize their therapy.
• Better Vaccines
Vaccines made of genetic material, either DNA or RNA; promise all the
benefits of existing vaccines without all the risks. They will activate the immune
system but will be unable to cause infections. They will be inexpensive, stable, easy
to store, and capable of being engineered to carry several strains of a pathogen at
once.
ADVANTAGE GENETIC ENGINEERING PRODUCT (PGR)
1. which is resistant to pest attack and crop disease.
PRG has given advantage to farmer that is with depressing expenditure of expense of
pesticide purchasing. Besides, PRG also lessens loss of market as result of rejection of
consumer to impure commodity by pesticide, and can depress its(the breakdown area
as result of usage of abundant pesticide in operation of pest and disease. Result of
research indicates that cultivation Bt. corn earns manifestly depress the application of
pesticide and lessens loss of expense of operation OPT.
2. PRG tolerant to herbicide type.
This PRG gives advantage of expense of in overcoming weed because farmer doesn't
require usage of herbicide in gross with various herbicide types. Crop result of the
genetic engineering resistant to herbicide type, its(the example strain kedele result of
genetic engineering Mosanto which is not has negative effect if the application of type
herbicide Roundup.
3. PRG tolerant to chilling.
Antifreez gene from cool water fish has been diintroduksi to some crops between of
tobacco and tomato, so that crop earns mentolelir to cool temperature which at
ordinary crop can result damage at malting process.
4. PRG tolerant to dryness or salinity.
This PRG can stay at condition of dry area and soil containing high salt.
5. PRG in addition nutrition. PRG can assist adds lacking of certain vitamin type,
like strain golden rice which is variety PRG paddy added by vitellarium can prevent
blindness at resident in nations grows.
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6. PRG as drug or vaccine. Vaccine inserted at crop product like at tomato crop or
potato is more memeudahkan in process of delivery and storage, compared to
hypodermic vaccine.
7. PRG as phytoremediation. PRG plant can be exploited to lessen pollution of
subterranean heavy metal.
8. Determination of fingerprint by using DNA test in criminal case.
DISADVANTAGE GENETIC ENGINEERING PRODUCT
1. Death of organism is not target.
Result of research of laboratory indicates that corn variety Bt. has caused high death
at ” monarc butterfly caterpillars” though this insect corn crop nonaggression. This
thing is because pollen corn Bt brought by wind to crop milkweed which is host ”
monarc butterfly caterpillars”.
2. Degradation of effectivity from pesticide.
Usage of PRG resistant plant to pest continually earns stimulate pest genes
appearance which resistant to some pesticide types.
3. Gene transfer to species that is is not become target.
Appearance case ” superweeds” a real resistant to herbicide as result of usage PRG
(soybean roundup). This thing happened caused by gene transfer from PRG plant to
weed.
4. Allergy.
Some food products coming from PRG to generate allergic impact to man. Intoduksi
gentertentu like gene kacagkacangan into soy crop can generate reaction of allergi is
having an effect on to body resilience.
5. Usage of PRG is assessed not economic and harms farmer
Because to yield PRG requires high cost and hereinafter this PRG usually patented by
its(the creator. Expense of research and patent right PRG will be charged upon
consumer (farmer) through sale of PRG which expensive. Besides, PRG in general
didn't yield descendant and applied only once plants. This condition it is of course
generates high dependency of farmer to seed PRG by producer company PRG.
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GENETIC COUNSELING
Important points that contain in Genetic Counseling :
• Genetics services includes clinical genetics (genetic counselling), laboratory and
education services.
• Genetic counselling provides
– Information
– Supportive counselling regarding the diagnosis and risk for a genetic condition in
the family
– Diagnostic, carrier, predictive and presymptomatic genetic testing where
appropriate
– Management of conditions in some cases
• The health professional team providing genetic counselling may consist of clinical
geneticists or other medical specialists,
genetic counsellors and social workers
• Genetic counselling is provided as part of a comprehensive genetics service whose
elements include clinical, laboratory and
education
• The availability of genetics services varies throughout Australia and New Zealand
Genetic counselling is provided by a team of health professionals who work
together to provide an individual or family with current information and supportive
counselling (advice or guidance) regarding problems in growth, development and
health that may have a genetic basis. This can assist families and individuals to
understand and adjust to the diagnosis of a genetic condition.
What happens in genetic counselling?
The consultation
During the consultation:
• A family health history is collected to provide information about the health of family
members
• A diagnosis of a genetic condition may be made or confirmed in a pregnancy, after
birth, in childhood or later in life. The diagnosis may be made on the basis of clinical
features, biochemical tests or genetic tests . This diagnosis may mean that other
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family members are at risk. On the other hand, a family member may be reassured to
find that he/she does not have, or is unlikely to develop, the particular condition
Where there is a genetic condition in a family, the genetic counselling team may:
• Estimate the risks that other family members, or future children, will be affected by
the condition. Often, however, a person is reassured following genetic counselling to
find out
that a condition is unlikely to recur in their family
• Discuss the impact and possible effects on the individual and their family in a
supportive atmosphere. Management strategies can be developed and referral
provided to
appropriate community resources, including support groups. Both verbal and written
information about the condition and its impact is provided to assist people in dealing
with some of the issues that may arise from the diagnosis of a genetic condition
• Discuss if appropriate prenatal testing and other reproductive options to ensure that
any decision is made on an informedAssociate Genetic Counsellors are graduate
health professionals in the process of completing the requirements of the HGSA to
become a certified genetic counsellor. Genetic Counsellors and Associate Genetic
Counsellors provide genetic counselling as part of a multi-disciplinary team. Some
work in `outreach’ and are linked to a major genetics unit
• Social workers with a special interest in genetics and particular genetic conditions,
work closely with clinical geneticists, genetic counsellors and support groups. When
should genetic counselling be sought.
Picture about genetic counseling
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There are a number of reasons for which genetic counselling is appropriate. These
include:
• When there is a condition that runs in a family and individuals are concerned that
they or their children will develop the condition
• Where a previous child is affected by a serious problem in growth, development or
health
• Where one or more family members (blood relatives not related by marriage) have
unusual features, or a serious health problem
• Where a woman is in her mid 30s or older and is either planning a pregnancy or is
already pregnant
• When a couple are blood relatives Information in this Fact
basis. Many genetic conditions can be diagnosed before birth
– If a genetic condition is identified by prenatal diagnosis, genetic counselling is the
means by which current information and support is provided so that an informed
decision can be made regarding the continuation of the pregnancy.
– Where there has been exposure to a potential teratogen (chemical, drugs,
medications, radiation or other environmental agents which can cause birth defects),
genetic counselling provides an opportunity to obtain current information and support
and discuss strategies and options.
• Discuss and arrange appropriate genetic testing, including carrier, predictive and
presymptomatic testing, where available
Follow up
After the initial consultation an opportunity may be provided to go over the
information and offer on-going support as families and individuals learn about the
condition. It is very common for people to think of many questions after the genetic
counselling session, and new questions also arise as a condition develops. Follow-up
is provided in further consultations, if geographically possible, or by telephone.
A letter summarising the consultation(s) is also provided.
Who provides genetic counselling?
Genetic counselling is provided by a multi-disciplinary team of
professionals that may include:
• Clinical geneticists and other specialist medical practitioners with expertise in the
genetics of their field of medicine eg oncologists (cancer genetics) and neurologists
(eg
Huntington disease and Alzheimer disease)
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• Genetic counsellors are graduate health professionals with specialist training and
certified by the Human Genetics Society of Australasia (HGSA) to provide genetic
counselling. Where an individual or their partner has some concerns about a condition
in themselves or their family being passed on to their children.
• When a fetal abnormality is detected during pregnancy
• When there is concern about exposure to some environmental agent such as drugs,
medications, chemicals or radiation that might cause birth defects.
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RELATIONS WITH ETHICS
THE ETHICAL ISSUES SURROUNDING GENE THERAPY
Because gene therapy involves making changes to the body’s set of basic
instructions, it raises many unique ethical concerns. The ethical questions surrounding
gene therapy include:
How can “good” and “bad” uses of gene therapy be distinguished?
Who decides which traits are normal and which constitute a disability or
disorder?
Will the high costs of gene therapy make it available only to the wealthy?
Could the widespread use of gene therapy make society less accepting of
people who are different?
Should people be allowed to use gene therapy to enhance basic human traits
such as height, intelligence, or athletic ability?
Current gene therapy research has focused on treating individuals by targeting
the therapy to body cells such as bone marrow or blood cells. This type of gene
therapy cannot be passed on to a person’s children. Gene therapy could be targeted to
egg and sperm cells (germ cells), however, which would allow the inserted gene to be
passed on to future generations. This approach is known as germline gene therapy.
The idea of germline gene therapy is controversial. While it could spare future
generations in a family from having a particular genetic disorder, it might affect the
development of a fetus in unexpected ways or have long-term side effects that are not
yet known. Because people who would be affected by germline gene therapy are not
yet born, they can’t choose whether to have the treatment.
IS EMBRYO CLONING MORAL?
Cloning of animals seems to have a number of potentially positive results:
Scientists are attempting to create transgenic pigs which have human genes.
Their heart, liver or kidneys might be useable as organ transplants in
humans. This would save many lives; thousands of people die each year
waiting for available human organs. Once achieved, transgenic animals
could be cloned to produce as many organs as are needed.
Experience gained in cloning may add to our understanding of genetics.
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Researchers have produced transgenic animals. These are genetically
altered, typically in order to produce human hormones or proteins in its
milk. These materials can be separated from the milk and used to heal
humans. Cloning would produce as many genetically altered animals as are
needed. The alternative is to simply allow them to mate; this would produce
many offspring that had lost the inserted human gene and thus would be
unable to produce the medication.
Embryo cloning of humans: Some scientists believe that embryo cloning and related
research is moral and might eventually lead to very positive results:
It might produce greater understanding of the causes of miscarriages; this
might lead to a treatment to prevent spontaneous abortions. This would be of
immense help for women who cannot bring a fetus to term.
It might lead to an understanding of the mechanisms by which a morula (a
mass of cells that has developed from a blastula) attaches itself to the wall of
the uterus. This might generate new, effective contraceptives that exhibit very
few side effects.
The rapid growth of the human morula is similar to the rate at which cancer
cells propagate. Cancer researchers believe that if a method is found to stop
the division of a human ovum then a technique for terminating the growth of a
cancer might be found.
Parents who are known to be at risk of passing a genetic defect to a child
could make use of cloning. A fertilized ovum could be cloned, and the
duplicate tested for the disease or disorder. If the clone was free of genetic
defects, then the other clone would be as well. The latter could be implanted
in the woman and allowed to mature to term.
In conventional in vitro fertilization, doctors attempt to start with many ova,
fertilize each with sperm and implant all of them in the woman's womb in the
hope that one will result in pregnancy. But some women can only supply a
single egg; her chances of becoming pregnant are slim. Through the use of
embryo cloning, that egg might be divisible into, say, 8 zygotes for
implanting. The chance of those women becoming pregnant would be much
greater.
Cloning could produce a reservoir of "spare parts". Fertilized ova could be
cloned into multiple zygotes; one could be implanted in the woman and
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allowed to develop into a normal baby; the other zygotes could be frozen for
future use. In the event that the child required a bone marrow transplant, one
of the zygotes could be taken out of storage, implanted, allowed to mature to a
baby and then contribute some of its spare bone marrow to its (earlier)
identical twin. Bone marrow can be harvested from a person without injuring
them.
A woman could prefer to have one set of identical twins, rather than go
through two separate pregnancies. She might prefer this for a number of
reasons:
to minimize disruption to her career.
to make a normal vaginal delivery possible (twin fetuses are smaller than a
single fetus; delivery of a larger, single fetus might be impossible because of
her shape.
she might prefer to only have to endure the discomfort of a single
pregnancy.
she might wish to have children that could contribute a kidney to their
sibling, if needed.
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CONCLUSIONS
There are many techniques used in genetic engineering, such as: recombinant
DNA, gene therapy, hybridization, human cloning, and polymerase chain reaction
(PCR), that it has its advantages and disadvantages in many aspect. All of this
tehniques has a relation with ethics, which it can same or different.
Genetic engineering is connected with gene mutation. If someone want to do a
research of genetic engineering, he must mutated the gene, typed induced mutation.
Not all of gene mutation has a disadvantages. Some of it has an advantages that has a
great effect in genetic engineering developing.
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