MODULE 2.

TECHNIQUES IN BIOTECHNOLOGY
I. TISSUE CULTURE
Tissue culture is gaining traction as an LEARNING OUTCOME
effective propagation method that embraces cell
technology to propagate new plants in artificial After completing this module, the students should be able to:
environments using tissue fragments from plant 1. Identify the different types of biotechnological
techniques;
media. The different types of tissue culture
2. Acquire and apply some techniques of biotechnology
methods are determined by the kind of plant
media used. The following are the most
commonly used types of tissue culture processes:
1. Seed Culture
Seeds are cultured in vitro to cultivate
healthy plants or seedlings.
2. Meristem Culture
Apical meristem from angiosperm and
gymnosperm shoots are cultured to produce
plants that are largely free from disease and
contamination. Contamination is one of the
greatest obstacles to effective tissue culture. For
this reason, meristem culture is often preferred as
the lack of vascular tissues ensures that contamination and spread of disease is limited. Most DIY tissue culture
and home tissue culture labs will use this method. Together with high-quality PPM™ and strict hygiene control,
plants propagated with meristem culture are least likely to carry diseases.
3. Callus Culture
A mass of cells gathered from any part of the plant material are cultured under in vitro conditions. No
specific section of the plant matter is used.
4. Bud Culture
Bud culture is separated into single node culture (stem node is used) and axillary bud method (where
axillary buds are separated from the leaf axils and placed in high cytokinin concentration).
5. Anther Culture
This type of plant tissue culture is known as haploid production and typically uses pollen culture for
production.
6. Cell Suspension Culture
This technique grows individual cells that have been gathered from any kind of plant material. The tissues
or callus are transferred into a liquid medium, instead of the commonly used gel substances.
The above types of tissue culture are just some of the different types of tissue culture. As you can see, there
are several different kinds of tissue culture processes, but the majority of people who have heard of tissue
culture have also heard of micropropagation and are probably wondering where propagation fits in.

For further understanding on how the process of tissue culture done, watch the video by opening the link.
https://youtu.be/iFRnCt7iEik
MICROPROPAGATION AND TISSUE CULTURE
Tissue culture is a technique in which a small fragments of a plant (explant) are introduced into an
artificial, nutrient medium, which allows its functioning or growth.
Fragments of plant material, perhaps even just a couple of cells, are placed in an artificial growing
medium to develop into new plants. There is one significant difference between tissue culture and
micropropagation (although many people think the two are the same because they involve overlapping
techniques).
Micropropagation is defined as the propagation of multiple plants from a small amount of plant material,
whereas tissue culture is the first step in this process, when multiple the plantlets are cultured. It can also be said
that issue culture is an essential element of micropropagation, when multiple plantlets are produced. Although
both are separate techniques used to propagate thousands of identical plants, they often overlap in the
laboratory.
WHAT IS MICROPROPAGATION?
Micropropagation is the growth of new plants in tissue culture. Being an in vitro tissue culture
technique, micropropagation develops high-quality clone plants on a large scale. The shoot apex is traditionally
grown in agar, a nutrient-dense gelling agent. Because of the shoot apex’s undifferentiated meristematic tissue,
this technique could be considered a Meristem culture technique. Tissue culture is used in micropropagation to
develop new plants or seedlings from cells. Once they are well established, they will be introduced into the soil,
which is the second stage of micropropagation.
Micropropagation is used throughout industries across the globe, and here are three reasons why:
LARGE SCALE PRODUCTION
The key to this benefit is not only the quantity of plants produced, but also the fact that all new plants
carry the same genetic profile. There are very few cultivation methods that ensure such a high amount of
precision and identical plants can.
Disease free plants
In micropropagation, the meristem is used. Why is this related to disease-free plants? Well, the answer is
more straightforward than you might expect. Most plants spread disease, virus and other contamination through
vascular tissues while the meristem does not typically carry any contaminates.
CONSERVATION AND PRESERVATION
Micropropagation has the potential to aid conservation efforts as well. There are already multiple
projects going on around the world using micropropagation and tissue culture to conserve rare and endangered
plant species. Micropropagation can also be used to propagate plants that are difficult to grow or generate from
seed.
Although there are several different types of tissue culture, micropropagation is commonly used when
growers are striving for mass amounts of superb quality, identical plants. Many developing nations are
employing tissue culture techniques to meet demands for food and to benefit from exports. Palm oil, one
industry of constant conservational scrutiny, has also introduced tissue culture in the hopes of reducing its
reliance on natural resources. There are many ways that tissue culture can benefit modern-day society, where
we look for instant gratification and the consumer seems never to be satisfied. Tissue culture can help to reduce
the land area needed for crops, conserve delicate species, and even heighten and adjust gene profiles to ensure
excellent plants. From a business perspective, tissue culture may seem a costly endeavor, but it is an investment
that could offer rewards once you begin to reap what you have sewn.
II. DNA EXTRACTION
The Basics of DNA Extraction
You’ve probably heard of the Genetic Code or the Blueprint of Life; these terms refer to DNA. All
living things, including animals, plants, and bacteria, have DNA in their cells. DNA is a very long molecule
made up of a chain of nucleotides and the order of these nucleotides is what makes organisms similar to others
of their species and yet different as individuals. Genes are sections within this long DNA molecule.
In order to study DNA, you first have to get it out of the cell. In eukaryotic cells, such as human and
plant cells, DNA is organized as chromosomes in an organelle called the nucleus. Bacterial cells have no
nucleus. Their DNA is organized in rings or circular plasmids, which are in the cytoplasm. The DNA extraction
process frees DNA from the cell and then separates it from cellular fluid and proteins so you are left with pure
DNA.
The three basic steps of DNA extraction are 1) lysis, 2) precipitation, and 3) purification.
Step 1: Lysis
In this step, the cell and the nucleus are broken open to release the DNA inside and there are two ways
to do this. First, mechanical disruption breaks open the cells. This can be done with a tissue homogenizer (like
a small blender), with a mortar and pestle, or by cutting the tissue into small pieces. Mechanical disruption is
particularly important when using plant cells because they have a tough cell wall. Second, lysis uses detergents
and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins.
Step 2: Precipitation
When you complete the lysis step, the DNA has been freed from the nucleus, but it is now mixed with
mashed up cell parts. Precipitation separates DNA from this cellular debris. First, Na+ ions (sodium) neutralize
the negative charges on the DNA molecules, which makes them more stable and less water soluble. Next,
alcohol (such as ethanol or isopropanol) is added and causes the DNA to precipitate out of the aqueous solution
because it is not soluble in alcohol.
Step 3: Purification
Now that DNA has been separated from the aqueous phase, it can be rinsed with alcohol to remove any
remaining unwanted material and cellular debris. At this point the purified DNA is usually re-dissolved in water
for easy handling and storage.
III. DNA CLONING

DNA cloning is the starting point for many genetic engineering approaches to biotechnology research.
Large amounts of DNA are needed for genetic engineering. Multiple copies of a piece of DNA can be made
either by using polymerase chain reaction (PCR) or by cloning DNA in cells.

How is DNA cloned in cells?

To get multiple copies of a gene or other piece of DNA you must isolate, or ‘cut’, the DNA from its
source and then ‘paste’ it into a DNA vector that can replicate (or copy) itself.

The four main steps in DNA cloning are:

Step 1. The chosen piece of DNA is ‘cut’ from the source organism using restriction enzymes.

Step 2. The piece of DNA is ‘pasted’ into a vector and the ends of the DNA are joined with the vector DNA by
ligation.

Step 3. The vector is introduced into a host cell, often a bacterium or yeast, by a process called transformation.
The host cells copy the vector DNA along with their own DNA, creating multiple copies of the inserted DNA.

Step 4. The vector DNA is isolated (or separated) from the host cells’ DNA and purified.

DNA that has been ‘cut’ and ‘pasted’ from an organism into a vector is called recombinant DNA.
Because of this, DNA cloning is also called recombinant DNA technology.

What is cloned DNA used for?

DNA cloning is used to create a large number of copies of a gene or other piece of DNA. The cloned
DNA can be used to:

 Work out the function of the gene

 Investigate a gene’s characteristics (size, expression, tissue distribution)
 Look at how mutations may affect a gene’s function
 Make large concentrations of the protein coded for by the gene

What other types of cloning are there?

The term ‘cloning’ is also used to describe other laboratory processes:

 Reproductive cloning is the process of making a genetically identical copy of an organism.

 Therapeutic cloning is the process of making multiple copies of a cell to treat a disease.

IV. GENETIC CODE

The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA
sequences) is translated into proteins (amino acid sequences) by living cells.

Specifically, the code defines a mapping between tri-nucleotide sequences called codons and amino acids;
every triplet of nucleotides in a nucleic acid sequence specifies a single amino acid.

Because the vast majority of genes are encoded with exactly the same code, this particular code is often
referred to as the canonical or standard genetic code, or simply the genetic code, though in fact there are many
variant codes; thus, the canonical genetic code is not universal.

For example, in humans, protein synthesis in mitochondria relies on a genetic code that varies from the
canonical code.

The genome of an organism is inscribed in DNA, or in some viruses RNA.

 The portion of the genome that codes for a protein or an RNA is referred to as a gene.

Those genes that code for proteins are composed of tri-nucleotide units called codons, each coding for a
single amino acid.

Each nucleotide sub-unit consists of a phosphate, deoxyribose sugar and one of the 4 nitrogenous nucleotide
bases.

The purine bases adenine (A) and guanine (G) are larger and consist of two aromatic rings.

The pyrimidine bases cytosine (C) and thymine (T) are smaller and consist of only one aromatic ring.

In the double-helix configuration, two strands of DNA are joined to each other by hydrogen bonds in an
arrangement known as base pairing.

These bonds almost always form between an adenine base on one strand and a thymine on the other strand
and between a cytosine base on one strand and a guanine base on the other.

This means that the number of A and T residues will be the same in a given double helix as will the number
of G and C residues.

In RNA, thymine (T) is replaced by uracil (U), and the deoxyribose is substituted by ribose.

Table 1. The genetic code table.

 V. GENE GUN

Many methods of delivering extra DNA into the nucleus of plant cells have been tried, and several have
been successfully used to produce transgenic plants. The first to be discussed is the gene gun, also called
particle acceleration or microprojectile bombardment. While this method was used to create several commercial
events such as RoundupReady soybean, it has several limitations that make it less appealling than the use
of Agrobacterium as a transformation method. We will describe the Agrobacterium method in the next section.

The gene gun is a good example of a creative idea being developed into a practical technology.

Troy Weeks uses the gene gun to transform sorghum cells. (Image by T. Weeks)

The gene gun can be used on either tissue culture cells or seedlings (to make chimeric plants) of any
species. As the name implies, this method works by shooting DNA into the plant cells. As seen in these figures
from the animation, microscopic gold or tungsten particles are coated with hundreds of copies of the gene(s) to
be introduced.

Microscopic gold particles are used as ’bullets’ to deliver DNA into callus cells. (Image by P. Hain)

In earlier versions of the gene gun, DNA coated metal was loaded into a 22-caliber cartridge and then
shot into plant tissue culture cells. Current versions place the tissue culture cells in a vacuum chamber and then
propel the metal particles with a high pressure gas that is released in a sudden burst much like a popped balloon.
 The gold particles are coated with hundreds of copies of the gene of interest. (Image by P. Hain)

Gene gun transformation begins by growing cells in tissue culture, bombarding the cells with gene coated
gold particles in the gene gun, selecting out transgenic cells on selection media, and regenerating the
transgenic cells into plants. (Image by P. Hain)

Once inside the nucleus of a cell, the genes dissolve off of the gold particle and can potentially insert into
a chromosome. A major limitation of this method is that several copies of the transgene will often insert
into a chromosomal position. These high copy insertion events can be detected as DNA that should not be
expressed by the plant cell and the transgene copies are silenced. (Image by P. Hain)
 VI. BACKCROSS BREEDING

With all of the advances in molecular biology, it may seem surprising to find out that traditional plant
breeding methods are still needed in the development of plant varieties. Today, the backcross procedure is most
often used to move a transgene from a good tissue culture variety that was used in transformation to
an elite experimental line or variety. It turns out that for many crops, once the transgene is in the
crop species crossing is more efficient than transformation procedures. 

Backcrossing is more efficient than transforming the elite line because most transformation protocols

are optimized for a specific (often poorly adapted and lower yielding) laboratory line. Many elite lines (which
are high yielding) are not amenable for transformation. Hence genetic engineers transform their lab line and
breeders backcross the trangene from the lab line into the elite line.  

In this lesson the focus will be on the backcross method, which is a form of recurrent hybridization
(repeated crossing to a single variety) where a superior characteristic may be added to an otherwise desirable
variety. In this method the breeder has considerable control of the genetic variation in the segregating
population in which selections are to be made. The backcross method has been used extensively for
transferring qualitative characters (characters with clear phenotypes that are easy to identify in cross progeny)
such as disease resistance. It is effective in both self and cross pollinated crop species. To better understand the
applications of backcrossing, the gene for leaf rust resistance in wheat will be used as an example. Figure 1
shows the visible symptoms of leaf rust in susceptible wheat. The left picture leaves are resistant, they have a
small amount of rust. The plant on the right is covered with rust, it is susceptible.

Fig. 1. Two examples of wheat, the one on the left is resistant to rust, the one on the right is
susceptible to rust.

Figure 2 illustrates how the backcross procedure can be used to move leaf rust resistance (RR, Rr) from
one variety to a susceptible variety (rr). The actual procedure for back crossing is almost self-explanatory. In
back crossing you have a donor parent (has a gene of interest) and a recurrent parent (an elite line that could be
made better by adding the gene of interest). The donor parent is crossed to the recurrent parent.
The progeny of this cross is then crossed to the recurrent parent (it is 'crossed back' to the recurrent parent,
hence the term back cross). The progeny of this cross is selected for the trait of interest and then crossed back
to the recurrent parent. This process is repeated for as many back crosses as are needed to create a line that is
the recurrent parent with the gene of interest from the donor parent. The goal of backcrossing is to obtain a line
as identical as possible to the recurrent parent with the addition of the gene of interest that has been added
through breeding.
 Fig. 2. Backcross breeding with a dominant trait.

In the end, you want to keep only the individuals homozygous for the resistance gene. To obtain them,
self Rr plants from BC4. The resulting offspring will be 1RR : 2Rr : 1rr. Progeny testing would be needed to
identify RR from Rr plants. 
Progeny testing is where the genotype of a parent plant is determined by genotypes of the line’s progeny. In the
case of an RR plant, the progeny will all be RR (no segregation for the gene/trait). However in the case of an Rr
plant, the progeny will segregate 1/4 RR : 1/2 Rr : 1/4 rr. Therefore, the progeny of RR plants will be uniformly
resistant to leaf rust, while the progeny of Rr plants will segregate for resistance and susceptibility.
In contrast, if the genes for rust resistance had been recessive (i.e., ss = resistant) rather than dominant,
then the introduced resistant gene is only carried in the heterozygote and would not be detected throughout the
backcross program. After each backcross, one would have to self the heterozygote (Ss) in order to produce
resistant plants (ss) in the progeny. These resistant plants (ss) are then backcrossed to the recurrent parent (SS).
See Figure 3.
When working with recessive traits, such as this example, Allard (1960) suggests advancing the
1  backcross to the F2 generation followed by selection for the desirable character from the donor parent (ss) and
st

the general features of the recurrent parent. The 2nd and 3rd backcrosses are then made in succession after which
the inbreeding with selection phase for ss is repeated. This is followed by the 4th and 5th backcrosses in
succession. The BC5F2 that are resistant (SS) are crossed to recurrent parent (SS) for the BC6F1 which is Ss. 
 Fig. 3. Backcross breeding with a recessive trait.

The BC7F1 is selfed to get in the BC6F2 : 1/2 SS (susceptible) : 1/2 Ss (susceptible) : 1/2 ss (resistant)
backcross with intense selection for both the desired character (ss) and the recurrent parent plant phenotype.
You have successfully transfered the gene.(If interested, see Allard, p. 156-157, for further description and
rationale for this approach.)

WATCH THIS VIDEO FOR FURTHER UNDERSTANDING. https://mediahub.unl.edu/uploads/0ae2c95a-debc-11e8-

ba2d-005056832e99/media.mp4
 VII. CLONING
Molecular Cloning
In general, the word “cloning” means the creation of a perfect replica; however, in biology, the re-
creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone
an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process
that is referred to as molecular cloning.
Cloning small fragments of the genome allows for the manipulation and study of specific genes (and
their protein products), or noncoding regions in isolation. A plasmid (also called a vector) is a small circular
DNA molecule that replicates independently of the chromosomal DNA of microorganisms such as E. coli. In
cloning, the plasmid molecules can be used to provide a “folder” in which to insert a desired DNA fragment.
Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of
DNA from the human genome (or the genome of another organism that is being studied) is referred to
as foreign DNA, or a transgene, to differentiate it from the DNA of the bacterium, which is called the host
DNA.
Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can
contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by
antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale
production of important reagents, such as insulin and human growth hormone. An important feature of plasmid
vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS).
The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly available
restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a
predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA.
Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2-
or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary
overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the
DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction
endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same
restriction endonuclease (Figure 1).
Recombinant DNA Molecules
Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they
are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of
different parts of the molecules can be traced back to different species of biological organisms or even to
chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant
proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be
moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be
engineered to express proteins only when stimulated by certain environmental factors, so that scientists can
control the expression of the recombinant proteins.
View an animation of recombination in cloning from the DNA Learning Center.
Cellular Cloning
Unicellular organisms, such as bacteria and yeast, naturally produce clones of themselves when they
replicate asexually by binary fission; this is known as cellular cloning. The nuclear DNA duplicates by the
process of mitosis, which creates an exact replica of the genetic material.
PRACTICE QUESTION

Figure 1. This diagram shows the steps involved in molecular cloning.

You are working in a molecular biology lab and, unbeknownst to you, your lab partner left the foreign
genomic DNA that you are planning to clone on the lab bench overnight instead of storing it in the freezer. As a
result, it was degraded by nucleases, but still used in the experiment. The plasmid, on the other hand, is fine.
What results would you expect from your molecular cloning experiment?
a. There will be no colonies on the bacterial plate.
b. There will be blue colonies only.
c. There will be blue and white colonies.
d. The will be white colonies only.
Reproductive Cloning
Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular
organism. Most multicellular organisms undergo reproduction by sexual means, which involves genetic
hybridization of two individuals (parents), making it impossible for generation of an identical copy or a clone of
either parent. Recent advances in biotechnology have made it possible to artificially induce asexual
reproduction of mammals in the laboratory.
Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization
of the egg occurring; this is a form of asexual reproduction. An example of parthenogenesis occurs in species in
which the female lays an egg and if the egg is fertilized, it is a diploid egg and the individual develops into a
female; if the egg is not fertilized, it remains a haploid egg and develops into a male. The unfertilized egg is
called a parthenogenic, or virgin, egg. Some insects and reptiles lay parthenogenic eggs that can develop into
adults.
Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote
results. The zygote nucleus contains the genetic information to produce a new individual. However, early
embryonic development requires the cytoplasmic material contained in the egg cell. This idea forms the basis
for reproductive cloning. Therefore, if the haploid nucleus of an egg cell is replaced with a diploid nucleus from
the cell of any individual of the same species (called a donor), it will become a zygote that is genetically
identical to the donor. Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an
enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.
The first cloned animal was Dolly, a sheep who was born in 1996. The success rate of reproductive
cloning at the time was very low. Dolly lived for seven years and died of respiratory complications (Figure 2).
There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect
the life expectancy of a cloned individual. Since Dolly, several animals such as horses, bulls, and goats have
been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities. There
have been attempts at producing cloned human embryos as sources of embryonic stem cells, sometimes referred
to as cloning for therapeutic purposes. Therapeutic cloning produces stem cells to attempt to remedy detrimental
diseases or defects (unlike reproductive cloning, which aims to reproduce an organism). Still, therapeutic
cloning efforts have met with resistance because of bioethical considerations.

PRACTICE QUESTION
REFER TO FIGURE 2. To create Dolly, the nucleus was removed from a donor egg cell. The nucleus from a
second sheep was then introduced into the cell, which was allowed to divide to the blastocyst stage before being
implanted in a surrogate mother. (credit: modification of work by “Squidonius”/Wikimedia Commons)
QUESTION: Do you think Dolly was a Finn-Dorset or a Scottish Blackface sheep?
 Figure 2. Dolly the sheep was the first mammal to be cloned

EXERCISE NO. 1.
Scientists have been able to isolate diploid cells from a species of dinosaur and would like to attempt
to clone this dinosaur. What type of cloning method would they need to use to do this and how would they
create a viable zygote?
A. They would use cellular cloning and would create a zygote by taking the cell obtained from the
dinosaur and replicating it via asexual reproduction.
B. They would need to use molecular cloning and would create a zygote by inserting the DNA obtained
from the dinosaur cell into a plasmid and allowing it to replicate.
C. They would use reproductive cloning and create a zygote by combining female and male gametes
from the species to create the clone.
D. They would use reproductive cloning and would create zygote by taking the diploid cell of the
dinosaur and inserting it into an enucleated egg cell to create zygote

Answers to practice problem:

Cellular cloning: Answer b. The experiment would result in blue colonies only.
Reproductive cloning: answer: Dolly was a Finn-Dorset sheep because even though the original cell came from
a Scottish blackface sheep and the surrogate mother was a Scottish blackface, the DNA came from a Finn-
Dorset.

VIII. DNA SEQUENCING

Definition
DNA sequencing is a method used to determine the precise order of the four nucleotide bases – adenine,
guanine, cytosine and thymine - that make up a strand of DNA. These bases provide the underlying genetic
basis (the genotype) for telling a cell what to do, where to go and what kind of cell to become (the phenotype).
Nucleotides are not the only determinants of phenotypes, but are essential to their formation. Each individual
and organism has a specific nucleotide base sequence.
The two scientists in the photograph are reading the genetic code for a DNA sample on a highlighted
light board. Such analysis is usually done by a computer. Credit: National Cancer Institute.

Importance
DNA sequencing played a pivotal role in mapping out the human genome, completed in 2003, and is an
essential tool for many basic and applied research applications today. It has for example provided an important
tool for determining the thousands of nucleotide variations associated with specific genetic diseases, like
Huntington's, which may help to better understand these diseases and advance treatment.
DNA sequencing also underpins pharmacogenomics. This is a relatively new field which is leading the
way to more personalised medicine. Pharmacogenomics looks at how a person's individual genome variations
affect their response to a drug. Such data is being used to determine which drug gives the best outcome in
particular patients. Over 140 drugs approved by the FDA now include pharmacogenomic information in their
labelling. Such labelling is not only important in terms of matching patients to their most appropriate drug, but
also for working out what their drug dose should be and their level of risk in terms of adverse events. Individual
genetic profiling is already being used routinely to prescribe therapies for patients with HIV, breast cancer,
lymphoblastic leukaemia and colon cancer and in the future will be used to tailor treatments for cardiovascular
disease, cancer, asthma, Alzheimer's disease and depression. Drug developers are also using pharmacogenomic
data to design drugs which can be targeted at subgroups of patients with specific genetic profiles.
Discovery
Although scientists established DNA had a double helix structure in 1953, it was to be many more years
before they could analyse DNA fragments. In part this reflected the fact that small DNA molecules contain
several thousands of nucleotides and it was difficult to obtain large quantities of homogeneous DNA. Scientists
also lacked the means to degrade DNA which they needed for sequence analysis.
A new chapter opened up in the 1960s with the emergence of techniques to sequence ribonucleic acid
(RNA)s. Ray Wu, a Chinese American biologist based at Cornell University, published one of the first methods
for sequencing DNA in 1970. Using highly labelled deoxynucleotides (single units of DNA) and DNA
polymerase he found a way to sequence the terminal region of a DNA molecule. Critically, Wu's approach
broke the DNA sequence down into several different components for analysis, thereby circumventing the need
for large quantities of homogeneous DNA. Subsequently, in 1971, Wu demonstrated his method could sequence
the ends of DNA in lambda phage, and two years later that it had the capacity to determine the sequence of any
DNA.
Over the course of the 1970s Wu's method was modified by Fred Sanger at the Laboratory of Molecular
Biology in Cambridge, UK. In 1975 Sanger, together with Alan Coulson, published what became known as the
'Plus and Minus' technique. This enabled the sequencing of up to 80 nucleotides in one go. Three years later, in
1977, Sanger and his colleagues announced another technique called the 'Sanger method' or 'dideoxy
sequencing'. This made it possible to sequence much longer stretches of DNA very rapidly. Their approach
appeared alongside the reporting of another technique by Allan Maxam and Walter Gilbert at Harvard
University.
While the Maxam-Gilbert method initially proved the most popular, it soon fell out of favour because it
necessitated the use of hazardous chemicals and radioisotopes. Added to this, the method it was difficult to
scale-up and could not be used in standard molecular biology kits because of its technical complexity. By
contrast, the Sanger method gained popularity because it was easier to use and more reliable. It was also
amenable to automation, paving the way to the first generation of automated DNA sequencers. The first
automated DNA sequencer was devised in 1986 by Leroy Hood and colleagues at the California Institute of
Technology together with a team including Lloyd Smith and Michael and Tim Hunkapiller. These machines
used capillary electrophoresis rather than gel electrophoresis using slabs.
Several new DNA sequencing methods and machines have been developed since the 1990s. These were
built following the introduction of microfluidic separation devices which improved sample injection and
speeded up separation times. Such innovations improved both the efficiency and accuracy of sequencing,
allowing for high-throughput sequencing, and radically lowered the cost. Between 2001 and 2011 the cost of
sequencing a genome shrank from $100 million to $10,000.
Application
DNA sequencing provides the means to know how nucleotide bases are arranged in a piece of DNA.
Several methods have been developed for this process. These have four key steps. In the first instance DNA is
removed from the cell. This can be done either mechanically or chemically. The second phase involves breaking
up the DNA and inserting its pieces into vectors, cells that indefinitely self-replicate, for cloning. In the third
phase the DNA clones are placed with a dye-labelled primer (a short stretch of DNA that promotes replication)
into a thermal cycler, a machine which automatically raises and lowers the temperature to catalyse replication.
The final phase consists of electrophoresis, whereby the DNA segments are placed in a gel and subjected to an
electrical current which moves them. Originally the gel was placed on a slab, but today it is inserted into a very
thin glass tube known as a capillary. When subjected to an electrical current the smaller nucleotides in the DNA
move faster than the larger ones. Electrophoresis thus helps sort out the DNA fragments by their size. The
different nucleotide bases in the DNA fragments are identified by their dyes which are activated when they pass
through a laser beam. All the information is fed into a computer and the DNA sequence displayed on a screen
for analysis.
The method developed by Sanger was pivotal to the international Human Genome Project. Costing over
US$3 billion and taking 13 years to complete, this project provided the first complete Human DNA sequence in
2003. Data from the project provided the first means to map out the genetic mutations that underlie specific
genetic diseases. It also opened up a path to more personalised medicine, enabling scientists to examine the
extent to which a patient's response to a drug is determined by their genetic profile. The genetic profile of a
patient's tumour, for example, can now be used to work out what is the most effective treatment for an
individual. It is also hoped that in the future that knowing the sequence of a person’s genome will help work out
a person's predisposition to certain diseases, such as heart disease, cancer and type II diabetes, which could pave
the way to better preventative care.
Data from the the Human Genome Project has also helped fuel the development of gene therapy, a type
of treatment designed to replace defective genes in certain genetic disorders. In addition, it has provided a
means to design drugs that can target specific genes that cause disease.
Beyond medicine, DNA sequencing is now used for genetic testing for paternity and other family
relationships. It also helps identify crime suspects and victims involved in catastrophes. The technique is also
vital to detecting bacteria and other organisms that may pollute air, water, soil and food. In addition the method
is important to the study of the evolution of different population groups and their migratory patterns as well as
determining pedigree for seed or livestock.

LEARNING ACTIVITIES

Group activity:
Individual activity:
Natural
Famous Cloning Riddle
Cloned Animals
Get thisGroup yourselves
activity started byinto 10, 4 member
researching some ofeach
the group and answer
most famous theanimals:
cloned riddle below, direct
Dolly the message
sheep, your
Cumulina
answer to ROGER
the mouse, POLICARPIO
or the cows via messenger.
Noto and Kaga. These areThe
justfirst group
a few to guess,
examples of wins the game.
the animals that you can choose.
Each ofMost
you cloning is done artificially,
should research each clonedasanimal
the work
and of scientists
take who
notes on howintentionally create organisms
scientists performed with
the cloning.
the
Youexact same
should DNA. the cloning process in diagrams. You should determine if something turned out
replicate
unexpectedly, and if so, how that may have happened.
The riddle: There is one type of cloning that can happen naturally. What is it?
Hint: I S
Discussion forum. Via messenger group
Cloning Debate
In the 1990s, cloning was a very controversial concept - so controversial that President Clinton
imposed a ban that prevented federal money from being used to clone humans. Discuss the reasons people
may have been wary of cloning and explain the benefits of cloning, such as the potential to develop
important medicine. Decide where you belong, either pro-cloning or anti-cloning, and have a research and
develop reasons for your chosen position. Through the group chat, express your reasoning and attempt to
persuade the rest of the class to your side.