MOLECULAR BIOLOGY AND DIAGNOSTICS LECTURE

INTRODUCTION TO CYTOGENETICS PRELIM

INTRODUCTION TO CYTOGENETICS Alfred Russel Wallace

HISTORY Against the theory of Natural Selection

● Natural selection was based on the observation that

● 8000 and 1000 B.C. - Selective breeding and populations tend to consist of more offspring than the
Domestication of horses, camels, oxen & various breeds of environment can support, leading to a struggle for survival
dogs among them.
● 5000 B.C. - Cultivation of maize, wheat, rice ● Less desirable traits must be preserved and flourished.
● Golden Age of Greek culture
Gregor Mendel (1860)
Hippocratic School of Medicine (500–400 B.C.)
● true starting point of our understanding of genetics
● (On the Seed) active “humors” in various parts of the male ● conducted a decade-long series of experiments using pea
body served as the bearers of hereditary traits
plants
● Gene depends on the fluids (semen) of the males. This
could be healthy or diseased and can be altered ● concluded that each trait in the plant is controlled by a pair
of genes and that during gamete formation members of a
Aristotle (384–322 B.C.) gene pair separate from each other
● Generative power of male semen resided in a “vital heat” ● forms the foundation for genetics, which is defined as the
contained within it branch of biology concerned with the study of heredity and
● Capacity to produce offspring of the same “form” as the variation
parent ● conducted his experiments before the structure and role
● Embryo (“miniature man”) is contained in the semen of
of chromosomes were known:
male.
● Heat cooked and shaped menstrual blood which was the ○ 20 years after his work was published,
“physical substance” that gave rise to an offspring. advances in microscopy allowed researchers to
● He claimed that semen + menstrual blood → offspring identify chromosomes
○ members of each species have a characteristic
William Harvey (1578–1657) number of chromosomes called the diploid
Theory of epigenesis number (2n)
○ Mitosis and meiosis were discovered on the late
● Organism is derived from substances present in the egg decades of 19th century
that differentiate into adult structures during embryonic
development
● Body organs are not initially present in the early embryo
but instead are formed de novo (anew)
● He claimed that no miniature adult is present from the
start. The organism develops slowly.

17th century
“Homunculus”
● Sex cells contain a complete, miniature adult, perfect in
every form
1830, Matthias Schleiden & Theodor Schwann

Cell theory

● All organisms are composed of basic units called cells,

which are derived from similar preexisting structures

Louis Pasteur

● Disapproved spontaneous generation (creation of living

organisms from nonliving components)
○ Living organisms were considered to be derived
from preexisting organisms and to consist of
cells.

Carolus Linnaeus (1707–1778)

Doctrine of fixity of species

● Animal and plant groups have remained unchanged in

form since the moment of their appearance on Earth

Charles Darwin
● The Origin of Species (1859) Human beings NORMALLY have 46 chromosomes and 92 chromatids.
● Theory of natural selection
○ “Matira matibay”
○ Causes of evolutionary change

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 Walter Sutton and Theodor Boveri Mutations

● noted that genes and chromosomes, as observed under ● defined as any heritable change
the microscope, have several properties in common ● source of all genetic variation
● behavior of chromosomes during meiosis is identical to ● not all mutations are bad, there are some which are
the presumed behavior of genes during gamete formation actually good, and those which did not have any effect at
described by Mendel all (e.g., null allele).
● independently proposed that genes are carried on
– Showed that the mutant trait could be traced to a single
chromosomes
chromosome, confirming the idea that genes are carried on
● basis of the chromosome theory of inheritance
chromosomes
○ inherited traits are controlled by genes residing
on chromosomes faithfully transmitted through
gametes, maintaining genetic continuity from CHEMICAL COMPONENT OF CHROMOSOMES
generation to generation.
1920s

● scientists were aware that proteins and DNA were the

major chemical components of chromosomes

1928, Frederick Griffith

● discovered the phenomenon of transformation, dead
bacteria could transfer genetic material to "transform"
other still-living bacteria.
1944, Oswald Theodore Avery, Collin Mcleod and Maclyn McCarty
● published experiments showing that DNA was the carrier
of genetic information in bacteria
1952 by Alfred Hershey and Martha Chase
● Scientists used to think that proteins carry the genetic
material.
★ Hershey-Chase Experiment
● showed that the DNA is the genetic material of the viruses
that infect bacteria, providing further evidence that the
DNA is the molecule responsible for inheritance.
● used radiolabeled bacteriophages to prove that DNA
carries genetic information (sulfur labeled protein capsule
has been washed out, meanwhile; phosphorus labeled
DNA remain
TERMINOLOGIES

● Genes
– unit of heredity
● Genome
– complete set of genetic instructions characteristic of an
organism
● Allele
– variants of genes
● Genotype
– alleles that are present ( underlying traits )
● Phenotype
– alleles that are expressed ( visible traits )
● Dominant alleles
– has an effect when present in just one copy
● Recessive alleles
– must be present in both to be expressed

CONTINUATION OF HISTORY

Genetic variation
● Drosophila melanogaster
● Variation produced by a mutation in one of the genes
controlling eye color.

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 ● Discovery of bacteria capable of protecting themselves
from viral infection by producing enzymes that cut viral
1953, James D. Watson and Francis Crick DNA at specific sites.
● Structure of the DNA molecule and mechanism by which
★ “Restriction enzymes”
information stored in it is expressed to produce phenotype
● Glycosidic Bond: the ether bond between sugar and the ● could be used to cut any organism’s DNA at specific
nitrogenous bases nucleotide sequences, producing a reproducible set of
● Phosphodiester Bond: the ether bond between sugar and fragments
phosphate groups ● Development of DNA cloning
● Hydrogen Bond: the bond between the nitrogenous bases ● In cloning, a vector and a DNA fragment produced by
(2 bonds between Adenine and Thymine, 3 bonds between cutting with a restriction enzyme are joined to produce a
Cytosine and Guanine) recombinant DNA molecule. The recombinant DNA is
transferred into a bacterial cell, where it is cloned into
Rosalind Franklin and Maurice Wilkins
many copies by replication of the recombinant molecule
● Double-helix model and by division of the bacterial cell.
● Structure showed that genetic information exists in the ○ Way of making large numbers of copies of DNA
sequence of nucleotides on each strand of DNA. sequences
● Although the structure of the DNA showed how
inheritance works, it was still not known how DNA
influences the behavior of cells.
○ DNA controls the process of protein production
○ Discovered that the cell uses DNA as a template
to create matching messenger RNA
○ Nucleotide sequence of messenger RNA
○ This translation between nucleotide and amino
acid sequence is known as the Genetic Code.

SOME GENETICALLY ALTERED TRAITS IN CROP PLANTS

HERBICIDE RESISTANCE
● Corn, soybeans, rice, cotton, sugarbeets, canola
INSECT RESISTANCE
● Corn, cotton, potato
VIRUS RESISTANCE
● Potato, yellow squash, papaya
NUTRITIONAL ENHANCEMENT
GENOTYPE TO PHENOTYPE ● Golden rice
ALTERED OIL CONTENT
Sickle Cell Anemia ● Soybeans, canola
● single-nucleotide change in the DNA encoding beta-globin DELAYED RIPENING
(CTC S CAC) leads to an altered mRNA codon (GAG S ● Tomato
GUG) and the insertion of a different amino acid (glu S
val), producing the altered version of the etab-globin
FIRST SUCCESSFUL CASE OF CLONING
protein that is responsible for sickle-cell anemia. (glutamic
acid to valine).
● Livestock such as sheep and cattle have been
commercially cloned for more than 30 years, mainly by a
method called embryo splitting.
● In 1996, Dolly the sheep was cloned by nuclear transfer, a
method in which the nucleus of an adult cell is transferred
into an egg that has had its nucleus removed.

DEVELOPMENT OF RECOMBINANT DNA

Early 1970s

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 8 Werner Syndrome
Premature aging

9 Melanoma
FIRST PATENTED GENETICALLY ALTERED ORGANISM
Tumors originating in the skin

● First genetically altered organism to be patented, the onc 10 Multiple Endocrine Neoplasia, Type 2
strain of mouse, genetically engineered to be susceptible Tumors in endocrine gland and other tissues
to many forms of cancer. These mice were created for
studying cancer development and the design of new 11 Sickle-Cell Anemia
Chronic inherited anemia, In which red blood cells sickle,
anticancer drugs.
clogging arterioles and capillaries
● Researchers have transferred human genes into animals —
so-called transgenic animals — that as adults, produce
12 Phenylketonuria (PKU)
human proteins in their milk. By selecting and cloning An inborn error of metabolism;
such animals, biopharmaceutical companies can produce If untreated, results in mental retardation
a herd with uniformly high rates of protein production.
Human proteins from transgenic animals are now being 13 Retinoblastoma
tested as drug treatments for diseases such as Childhood tumor of the eye
emphysema. If successful, these proteins will soon be
commercially available. 14 Alzheimer Disease
Degenerative brain disorder marked by premature senility

15 Tay-Sachs Disease
Fatal hereditary disorder
Involving lipid metabolism often occurring in Ashkenazi jews

16 Polycystic Kidney Disease

Cysts resulting in enlarged kidneys and renal failure

17 Neurofibromatosis (NF1) Breast Cancer

Benign tumors of nerve tissue below the 5% of all cases
skin

HUMAN CHROMOSOME NUMBER 18 Amyloidosis

Accumulation in the tissues of an insoluble fibrillar protein

19 Familial Myotonic Dystrophy

X Hemophilia A Muscular Adrenoleukodys Hypercholesterole Form of adult muscular dystrophy
Clotting deficiency Dystrophy trophy (ALD) mia
Progressive Fatal nerve Extremely high
deterioration of disease cholesterol
the muscle
20 ADA Immune Deficiency
Y Azoospermia First hereditary condition treated by gene therapy
Absence of sperm in semen
21 Amyotrophic Lateral Sclerosis
1 Gaucher Disease (ALS) Late-onset lethal degenerative nerve disease
A chronic enzyme deficiency occurring frequency among
Ashkenazi jews
22 Glucose-Galactose Malabsorption Syndrome
Potentially fatal digestive disorder
2 Ehlers-Danlos Syndrome
Connective tissue disease

3 Retinitis Pigmentosa
Progressive degeneration of the retina

4 Huntington Disease
Lethal, late-onset, nerve degenerative disease

5 Familial Adenomatous Polyposis (FAP)

Intestinal polyps leading to colon cancer

6 Hemochromatosis Spinocerebellar Ataxia

Abnormally high Destroys nerves in the brain and
absorption of iron spinal cord, resulting in loss of muscle
from the diet control

7 Cystic Syndrome
Mucus in lungs, interfering with breathing

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 ●

● The molecular basis for hundreds of genetic disorders is 1860s Mendel’s work published
now known (Figure 1–13).
● Genes for sickle-cell anemia, cystic fibrosis, hemophilia, 1890s, 1900s, 1910s, Chromosome theory of inheritance
muscular dystrophy, phenylketonuria, and many other 1920s, 1930s, 1940s proposed.
metabolic disorders have been cloned and are used for the Transmission genetics evolved
prenatal detection of affected fetuses. In addition, tests
1900s Mendel’s work rediscovered, correlated
are now available to inform parents of their status as
behavior in meiosis
“carriers” of a large number of inherited disorders.
● The combination of genetic testing and genetic counseling
1940s, 1950s DNA shown to carry genetic information.
gives couples objective information on which they can Watson-Crick model of DNA
base decisions about childbearing.
● At present, genetic testing is available for several hundred 1950s, 1960s Era of molecular genetics.
inherited disorders, and this number will grow as more Gene expression, regulation understood
genes are identified, isolated, and cloned.
● The use of genetic testing and other technologies, 1970s, 1980s Recombinant DNA technology developed.
including gene therapy, raises ethical concerns that have DNAcloning begins
yet to be resolved.
1990s Genomics begins.
Human Genome Project initiated

2000s Application of genomics begins

● A timeline showing the development of genetics from

Gregor Mendel’s work on pea plants to the current era of
genomics and its many applications in research, medicine,
and society.
● Having a sense of the history of discovery in genetics
should provide you with a useful framework as you
proceed through this textbook.

Organism Human Diseases

E. coli Colon cancer and other cancers

S. cerevisiae Cancer, Wermer syndrome

D. melanogaster Disorders of the nervous system,

cancer

C. elegans Diabetes

D. rerio Cardiovascular disease

M. musculus Lesch-Nyhan disease, cystic fibrosis,

fragile-X syndrome, and many other
diseases

Genetic Studies Rely on the Use of Model Organisms

1st gen model organisms-mus musculus, drosophila melanogaster;

● Microbes- Saccharomyces cerevisiae, E.coli

● Nematode- Caenorhabditis elegans
● Dario rerio- zebra fish

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 MOLECULAR BIOLOGY AND DIAGNOSTICS LECTURE
I PRELIM

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 MOLECULAR BIOLOGY AND DIAGNOSTICS LECTURE
BIOSAFETY AND BIOSECURITY PRELIM
infection, confirming a bacteriological inoculation. Stevens
reported the first signs of illness arose on September 27,
BIOSAFETY 2001 during a business trip in North Carolina. Researchers
established that his death was the first incident with the
The discipline addresses the safe handling and containment of lung form of anthrax in the USA since 1976
infectious microorganisms and hazardous biological materials.

GUIDELINE ON LABORATORY BIOSAFETY AND

BIOSECURITY BIOSECURITY

Describes protection, control and accountability for valuable CEN WORKSHOP AGREEMENT 15793
biological materials within laboratories, in order to prevent their loss,
theft, misuse, diversion of, unauthorized access or intentional ● Focuses on laboratory boorish management
release whether or not the biorisk(s) is acceptable. ● Published by European Committee for Standardization
GUIDELINE ON LABORATORY BIOSAFETY AND BIOS

BRIEF HISTORY LABORATORY SAFETY MANUAL

● The origins of biosafety is rooted in the US biological ● Published by WHO

weapons program which began in 1943, as ordered by ● Includes information on different levels of containment
then US President Franklin Roosevelt and was active laboratories, types of biological safety cabinets, good
during the Cold War. microbiological techniques, disinfection and sterilization
○ It was eventually terminated by President of equipment
Richard Nixon in 1969
CARTAGENA PROTOCOL ON BIOSAFETY
● Ira L. Baldwin - first scientific director of Camp Detrick
● International regulatory framework to ensure and
○ He established a biological weapons program
adequate level of protection in the field of safe transfer,
for defensive purposes.
handling and use of living modified organism
● Newell A. Johnson - designed modifications at biosafety
of Detrick.
○ He developed Class III safety cabinets and NATIONAL COMMITTEE ON
laminar flow hoods to address specific risks. BIOSAFETY OF THE PHILIPPINES

● Established under E.O 430 series of 1990

● At the height of increasing mortality and morbidity due to ● Focuses on the organizational structure for biosafety:
smallpox in 1967, WHO aggressively pursued the procedures for evaluation of proposals with biosafety
eradication of the virus. concerns; procedures and guidelines on the introduction,
● It was also during this time that serious concerns about movement, and field release of regulated materials; and
biosafety practices worldwide were raised contributing procedures on physico-chemical and biological
directly to the decision of the World Health Assembly to containment.
consolidate the remaining virus stocks into two locations:
CDC and State Research Center of Virology and
Biotechnology Vector in Russia. NATIONAL BIOSAFETY FRAMEWORK
● In 1974, CDC published the Classification of Etiological
Agents on the Basis of Hazard, that introduced the
concept of establish ascending levels of containment ● Combination of policy, legal, administrative, and technical
associated with risks in handling groups of infectious instruments developed to attain the objective of Cartagena
microorganisms that present similar characteristics. Protocol on Biosafety
● Arnold Wedum - director of Industrial Health and Safety at
the US Army Biological Research Laboratories was
recognized as one of the pioneers of biosafety that DA A.O. No 8
provided the foundation for evaluating risks of handling
infectious microorganisms.
● Policies on the importation and release of plants and plant
● Anthrax is a disease caused products derived from modern biotechnology
by Bacillus anthracis, a germ
that lives in soil. Many people
know about it from the 2001 DOH A.O 2007-0027
bioterror attacks. In the
attacks, someone purposely
spread anthrax through the ● Requires clinical laboratories to ensure policy guidelines
U.S. mail. This killed five on laboratory biosafety and biosecurity
people and made 22 sick.
● The first documented incident of illness associated with
the circulation of anthrax was registered on October 2, ORGANIZATIONS IN THE FIELD OF BIOSAFETY
2001 in Florida.
● Photo-publisher Robert Stevens, age 63, was hospitalized 1. American Biological Safety Association
with the diagnosis of meningitis and subsequently died on 2. Asia-Pacific Biosafety Association
October 5, 2001. 3. European Biological Safety Association
● The autopsy showed that the death was caused by 4. Philippine Biosafety and Biosecurity Association
symptoms that had the characteristics of an anthrax 5. Biological Risk Association Philippines
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 CLASSIFICATION OF MICROORGANISM ACCORDING TO RISK GROUPS

GROUP 1 GROUP 2 GROUP 3 GROUP 4

Microorganism that are Microorganism that are Microorganism that cause Microorganism that produce
unlikely to cause human or unlikely to be a significant serious diseases to human life-threatening diseases to
animal disease risk to laboratory workers, or animals and may present humans or animals
community, livestock or a significant risk to
environment laboratory workers

Low individual Risk Moderate individual risk High individual risk High individual risk

Low Community Risk Limited community risk Limited to moderate High community risk
community risk

BIOSAFETY LEVELS

BSL Agents Practices Primary Barriers and (Secondary Barriers)

Safety Equipment

1 Not known to consistently Standard microbiological ● No primary barriers Laboratory bench and
cause diseases in healthy practices required. sink
adults ● PPE: laboratory coats and required
gloves eye, face
protection, as needed

2 ● Agents associated with BSL-1 practice plus: Primary barriers: BSL-1 plus
human disease ● Limited access ● BSCS or other physical ● Autoclave available
● Routes of transmission ● Biohazard warning signs containment devices used for
include per- cutaneous ● "Sharps" precautions all manipulations of agents
injury, ingestion, mucous ● Biosafety manual defining that cause splashes or
membrane exposure any needed waste aerosols of infectious
decontamination or medical materials
surveillance policies ● PPE Laboratory coats,
gloves, face and eye
protection, as needed

3 Indigenous or exotic agents BSL-2 practice plus: Primary barriers: BSL-2 plus
that may cause serious or ● Controlled access ● BSCs or other physical ● Physical separation
potentially lethal disease ● Decontamination of all waste containment devices used for from access condors
through the inhalation route ● Decontamination of all open manipulations of ● Self-closing,
of exposure laboratory clothing before agents double-door access
laundering ● PPE: Protective laboratory ● Exhausted air not
clothing. gloves, face, eye recirculated
and respiratory protection, as ● Negative airflow into
needed laboratory
● Entry through airlock
or anteroom
● Hand washing sink
near laboratory exit

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 4 ● Dangerous/exotic agents BSL-3 practices plus: Primary barriers: BSL-3 plus
which post high individual ● Clothing change before ● All procedures conducted ● Separate building or
risk of aerosol-trans- mitted entering in Class Ill BSCs or Class I or isolated zone
laboratory infections that ● Shower on exit II BSCs in combination with ● Dedicated supply and
are frequently fatal, for ● All material decontaminated full-body, air-supplied positive exhaust. vacuum, and
which there are no vaccines on exit from facility pressure suit decontamination
or treatments systems
● Agents with a close or ● Other requirements
identical antigenic outlined in the text
relationship to an agent
requiring BSL-4 until data
are available to redesignate
the level
● Related agents with
unknown risk of
transmission

BIOSAFETY CABINETS

SIDE–BY–SIDE CLASS COMPARISON

Classes of Personnel Product Environmental Use

Biosafety Protection Protection Protection
Cabinets

Class I Yes No Yes ● Not in use today for

Inward airflow Unfiltered room air is drawn Exhaust air is HEPA-filtered bioagents
through sash across work surface ● May be used to
opening enclose equipment or
procedures with
aerosol potential

Class II Yes Yes Yes ● Most common class

A1, A2, B1, B2 Inward airflow By HEPA filtered air drawn Exhaust air is HEPA-filtered of BSC used today, esp.
through sash down onto work surface & Type A2
opening room air kept away ● Used to handle
specimen material,
biological toxins, cell
tissue culture,
biohazardous agents

Class III Yes Yes Yes ● Provides the highest

(Glove Box) Complete HEPA filtered air is supplied to Exhaust air is double level of containment for
containment of work surface; total containment HEPA-filtered handling the most
interior work area keeps room air out dangerous
microorganisms

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