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

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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

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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.

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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

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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

2. Errors in replication

3. Virus

4. Chemical agents (HNO2 and NH2OH)

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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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REFFERENCES

Baynes, John. W, Dominiczak, Marek. H. 2005. Medical Biochemistry, edisi 2. Elsevier Mosby: London

Campbell, Reece & Mitchell. Biologi, ed.5, jilid 1. Erlangga: Jakarta

Whitmann D.B. 2000. Genetically Modified Foods : Harmful or Helpful?http://www.sca.co 25 April 2008.

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