Lecture 1

Molecular Biology
Dr.Duraid Qassim Alshareef

Consultant in Microbiology and Immunology

Introduction:

The beginning of molecular biology had remote roots since the work of the early
pioneers in that science.

 Gregor Mendel (1850): in an Austrian monastery, there lived a monk

named Mendel, Gregor Mendel. Monks had a lot of time on there hands and
Mendel spent his time crossing pea plants. As he did this over & over again,
he noticed some patterns to the inheritance of traits from one set of pea
plants to the next. By carefully analyzing his pea plant numbers (he was
really good at mathematics), he discovered three laws of inheritance.

Mendel's Laws are as follows:

the Law of Dominance :

 law stating that when two alleles of an inherited pair is heterozygous, then,
the allele that is expressed is dominant whereas the allele that is not
expressed is recessive.

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the Law of Segregation:

 When an organism makes gametes, each gamete receives just one gene
copy, which is selected randomly.

the Law of Independent Assortment:

different genes were inherited independently of one another.

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 Chromosomes were first observed in plant cells by Swiss botanist Karl

Wilhelm von Nägeli in 1842, and independently in Ascaris worms by
Belgian scientist Edouard Van Beneden (1846-1910). The use of basophilic
aniline dyes was a fundamentally new technique for effectively staining the
chromatin material in the nucleus.
 The molecule now known as DNA was first identified in the 1860s by a
Swiss chemist called Johann Friedrich Miescher. Johann set out to
research the key components of white blood cells, part of our body’s
immune system. The main source of these cells? was pus-coated bandages
collected from a nearby medical clinic. “Johann called this mysterious
substance ‘nuclein’. Unbeknown to him, Johann had discovered the
molecular basis of all life – DNA.
Johann carried out experiments using salt solutions to understand more
about what makes up white blood cells. He noticed that, when he added acid
to a solution of the cells, a substance separated from the solution. This
substance then dissolved again when an alkali was added. When
investigating this substance he realized that it had unexpected properties
different to those of the other proteins? he was familiar with. Johann called

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this mysterious substance ‘nuclein’, because he believed it had come from

the cell nucleus. Unbeknown to him, Johann had discovered the molecular
basis of all life – DNA. He then set about finding ways to extract it in its
pure form.
 Their behavior in animal (salamander) cells was later described in detail by
German anatomist Walther Flemming, the discoverer of mitosis, in 1882.
The name was invented later by another German anatomist, Heinrich von
Waldeyer."Altman 1889: create a method for isolating protein free nucleic
acid.
 F. Griffith 1928: Discovered type specific transformation using streptococci
The pneumococcus bacterium occurs naturally in two forms with
distinctively different characteristics. The virulent (S-strain) form has a
smooth polysaccharide capsule that is essential for infection. The non-
virulent (R-strain) lacks the polysaccharide capsule, giving it a rough
appearance. Mice injected with S-strain of the pneumococcus bacteria die
from pneumonic infection within a few days, while mice injected with the R-
strain bacteria continue to live. Injection with heat-killed S-strain bacteria
also results in the mice surviving.

Griffith was surprised to find in his experiments that mice injected with a
mixture of heat-killed S-strain and live but non-virulent R-strain produced
lethal results. In fact, Griffith discovered living forms of the S-strain bacteria
in the infected mice .

He hypothesize that the R-strain bacteria had somehow been transformed by

the heat-killed S-strain bacteria. Some "transforming principle", transferred
from the heat-killed S-strain, had enabled the R-strain to synthesize a
smooth polysaccharide coat and become virulent.

 The discovery in 1953 of the double helix, the twisted-ladderstructure of

deoxyribonucleic acid (DNA), by James Watson and Francis Crick marked a
milestone in the history of science and gave rise to modern molecular
biology, which is largely concerned with understanding how genes control
the chemical processes within.

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 In the mid-1950s, Arthur Kornberg began to study the mechanism of DNA

replication. By 1957 he has identified the first DNA polymerase. The
enzyme was limited, creating DNA in just one direction and requiring an
existing primer to initiate copying of the template strand.
 In 1969 Thomas D. Brock reported the isolation of a new species of
bacterium from a hot spring in Yellowstone National Park. Thermus
aquaticus (Taq), became a standard source of enzymes able to withstand
higher temperatures than those from E. Coli.
 Also in 1971, Cetus Corporation was founded in Berkeley, California by
Ronald Cape, Peter Farley, and Donald Glaser. Initially the company
screened for microorganisms capable of producing components used in the
manufacture of food, chemicals, vaccines, or pharmaceuticals. After moving
to nearby Emeryville, they began projects involving the *, primarily the
cloning and expression of human genes, but also the development of
diagnostic tests for genetic mutations.
 In 1976 a DNA polymerase was isolated from T. aquaticus. It was found to
retain its activity at temperatures above 75 °C.

 PCR Discovery:
Polymerase chain reaction (PCR) is a technique used in molecular biology to
amplify a single copy or a few copies of a segment of DNA across several orders of
magnitude, generating thousands to millions of copies of a particular DNA
sequence.

The Evolution and Revolution of PCR

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In 1983, Kary Mullis, PhD, a scientist at the Cetus Corporation, conceived of PCR as
a method to copy DNA and synthesize large amounts of a specific target DNA. Over
the next two years, a team of Cetus scientists that recognized the potential impact
PCR could have on molecular biology, researched, refined and made the theoretical
process a reality.

The team presented for the first time in 1985 at the American Society for Human
Genetics annual meeting.2 Later that year, Science, a journal of the American
Association for the Advancement of Science 3, reported the results in the first-ever
publication of the process.

Two significant advances have enabled PCR to become the technology it is today
– Taq polymerase and the thermal cycler.

In 1986, Cetus scientists isolated the Taq polymerase from Thermus aquaticus, a

bacterium found in hot springs. Because Taq could withstand high temperatures,
it removed the need for human intervention during the reaction, streamlining and
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shortening the process. Without a heat-resistant enzyme like Taq polymerase,

PCR could not be used on a large scale as the process would have been too
cumbersome.

Prior to Taq, DNA polymerase from E. coli, an enzyme that could not withstand
rapid heating and cooling, was used in the second step of PCR. Using E. Coli, the
polymerase was manually replaced at each step of the reaction as it degraded
from the heat.

In 1987, PerkinElmer, another US-based biotech company, launched a thermal

cycler, an instrument that is programmed to regulate the temperature of a
reaction, heating or cooling the samples as needed. Once again, this advance
minimized human interaction in the reaction, leading to an elegant, efficient and
streamlined process.

Real time PCR:

A real-time polymerase chain reaction (Real-Time PCR), also known as

quantitative polymerase chain reaction (qPCR), is a laboratory technique of
molecular biology based on the polymerase chain reaction (PCR).

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