MOLECULAR BIOLOGY AND DIAGNOSTICS

LESSON 1: INTRODUCTION TO MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY HERMANN J. MULLER

 Represents the intersection of
genetics, biochemistry and cell
 Recognized the “gene as a basis of
biology
life”, and so set out to investigate its
 Term coined by Warren Weaver,
structure
director of the Natural sciences
 Discovered the mutagenic effect of X-
section of the Rockefeller Foundation
rays on Drosophila, and utilized this
in 1938
phenomenon as a tool to explore the
 Branch of biology that studies the
size and nature of the gene
molecular basis of biological activity
 The study of the biochemical and ERWIN SCHROEDINGER
molecular processes within cells,
especially the processes of DNA
replication, RNA transcription, and  Proposed ways in which the principles
protein translation of quantum physics might account for
HISTORY OF MOLECULAR BIOLOGY the stability, yet mutability, of the
gene.
I. ORIGINS
NIELS BOHR
GREGOR MENDEL
 Expounded a principle of
 Father of Genetics complementarity between physics
 Pea plant experiment in 1857 and biology
 Nascent field of genetics was guided  In contrast to Schroedinger, Bohr (and
by Mendel’s Laws of segregation and subsequently Delbrueck) did not seek
independent assortment to reduce biology to physics; instead,
 The actual mechanisms of gene the goal was to understand how each
reproduction, mutation and discipline complemented the other
expression remained unknown
MAX DELBRUECK
THOMAS HUNT MORGAN and HIS
COLLEAGUES
 To investigate the self-reproductive
 Utilized the fruit fly, Drosophila characteristic of life, Delbrueck used
Melanogaster, as a model organism to bacteriophage, viruses that infect
study the relationship between the bacteria and the multiply very rapidly.
gene and the chromosomes in the  The establishment of “The Phage
hereditary process (Chromosome Group” in the early 1940s by
theory of heredity) Delbrueck and another physicist-
turned-biologist Salvador Luria
marked a critical point in the rise of
molecular biology

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MOLECULAR BIOLOGY AND DIAGNOSTICS

LESSON 1: INTRODUCTION TO MOLECULAR BIOLOGY AND DIAGNOSTICS

LINUS PAULING OSWALD AVERY, COLIN, MACLEOD, and

MACLYN MCCARTY

 Utilized his knowledge of structural  Isolated the S strain from the dead
chemistry to study macromolecular mice and isolated the protein and
structure nucleic acid (RNA and DNA) as these
 Contributed both theoretical work on were possible candidates for the
the nature of chemical bonds and molecule of heredity
experimental work using X-ray  Demonstrated the DNA was the
crystallography to discover the informational component transferred
physical structure of macromolecular during transformation
compounds
ALFRED HERSHEY AND MARTHA CHASE
FRIEDRICH MIESCHER
]
 Used phage viruses to confirm that
 First identified what he called the genetic material transmitted from
“nuclein” inside the nuclei of the generation to generation was DNA
human white blood cells and not proteins
 (The term “nuclein” was later
changed to “nucleic acid” and ERWIN CHARGAFF
eventually to “deoxyribonucleic acid”,
or “DNA”
 Noticed that DNA- whether taken
PHOEBUS LEVENE from a plant or animal- contained
equal amounts of adenine and
thymine and equal amounts of
 He was the first to discover: cytosine and guanine
o Order of the three major  He also found that amounts of
components of a single guanine, cytosine, adenine and
nucleotide (phosphate-sugar- thymine vary by species
base)  Chargaff’s rule
o Carbohydrate component of
RNA (ribose) ROSALIND FRANKLIN AND MAURICE WILKINS
o Carbohydrate component of
DNA (deoxyribose) the first to
 Sought a direct approach to solving
correctly identify the way RNA
the DNA structure using X-ray data.
and DNA molecules are put
The pattern of spots on the X-ray
together
photograph information that, in
 Proposed tetranucleotide structure
principle, could be used to
reconstruct a 3D image of the DNA
FREDERICK GRIFFITH
molecule.

 A scientist was working on a project in

1928 that formed the basis than DNA
was the molecule of inheritance
experiment involved mice and two
types of pneumonia

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 1: INTRODUCTION TO MOLECULAR BIOLOGY AND DIAGNOSTICS

II. CLASSICAL THE HUMAN GENOME PROJECT (HGP)

 Molecular Biology’s classical
 International, collaborative research
period began in 1953
program
 James Watson and Francis Crick’s
 GOAL: complete mapping and
discovery of the double helical
understanding of all the genes of
structure of DNA
human beings.
JAMES WATSON AND FRANCIS CRICK  In addition to introducing large-scale
approaches to biology, it has
produced all sort of new tools and
 Collaborated to build a model of the technologies that can be used by
double helical structure of DNA, with individual scientist to carry out
its two helical strands held together by smaller scale research in a much more
hydrogen-bonded base pairs effective manner.
LECTURE 2 FUTURE OF MEDICAL SCIENCE

MOLECULAR BIOLOGY  Genome-based research will

eventually enable medical science to
 Branch of biology that studies the develop highly effective diagnostic
molecular basis of biological activity tools, to better understand the health
 The study of the biochemical and needs of people based on their
molecular processes within cells, individual genetic make-ups, and to
especially the processes of DNA design new and highly effective
replication, RNA transcription, and treatments for disease
protein translation
GENETIC TESTING
DIAGNOSTICS
 Looks for changes, sometimes called
 Methods or systems for discovering the mutation or variants, in your DNA
cause of problem, illness, etc.  Also called DNA testing
DIAGNOSTIC MOLECULAR BIOLOGY REASONS FOR GENETIC TESTING
 Integration of the knowledge and  To learn whether you have a genetic
technology in molecular biology with condition that runs in your family
clinical laboratory techniques before you have symptoms
GENOME  To learn about the chance a current
or future pregnancy will have a
 An organism’s complete set of genetic condition
deoxyribonucleic acid (DNA), a chemical  To diagnose a genetic condition if you
compound that contains the genetic or your child has symptoms
instruction needed to develop and  To understand and guide your cancer
direct the activities of every organism prevention or treatment plan
 Human genome: ~3 billion base pairs,
which reside in the 23 pairs of NEWBORN SCREENING
chromosomes
 Identifies conditions that can affect a
 20,000-25,000 protein coding genes child’s long-term health or survival
 Early detection, diagnosis, and
intervention can prevent death or

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 disability and enable children to reach
their full potential

mRNA VACCINES

 Teach our cells how to make a protein

that will trigger an immune response
inside our bodies

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 2: NUCLEIC ACIDS

NUCLEIC ACIDS

 Received their name because they

were originally isolated from cell
nuclei
 Contain carbon, hydrogen, oxygen,
nitrogen and phosphorus
 Acidic character
 Found in all living beings
 Linear macromolecules formed by the
polymerization of units called
nucleotides

IMPORTANT FUNCTIONS IN THE CELL:

1. They are repository of the genetic Streptococcus pneumonia (S and R Strain)

information responsible for the Use of enzymes
transmission of inherited DNA as possible transforming principle
characteristics from parents to
children and from one cell to another HERSHEY AND CHASE’S EXPERIMENT
2. They guide cell protein synthesis and
are responsible for the correct
assembly of amino acids in defined
sequences
* morphological and functional
uniqueness of each living being are
determined by the information
contained in its nucleic acids

DNA AS GENETIC MATERIAL

Bacteriophage-Type of virus that infects bacteria

Centrifugation-separate bacterial cells and
 Bacterial Transformation bacteriophage
 Streptococcus pneumoniae (S and R S-35 and P-32 labelled
strain) Pellet- bacterial cells
 “Transforming principle” Supernatant- bacteriophage
DNA as genetic material

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 2: NUCLEIC ACIDS

HERSHEY AND CHASE’S EXPERIMENT

 Two strains of TMV (type A and Type

B)
 TMV-A (causes mottling of leaves) and
 Bacteriophage- type of virus that infects bacteria TMV-B (causes wrinkle pattern on
 S-35 and P-32 labelled leaves)
 Pellet- bacterial cells  In the absence of DNA, RNA acts as a
 Supernatant- bacteriophage genetic material
 DNA as genetic material

COMPONENTS OF NUCLEIC ACIDS

FRAENKEL- CONRAT’S EXPERIMENT

 Nitrogrenous base
 Phosphate group
 Pentose sugar

NITROGENOUS BASE

 Pyrimidines (Cytosine-Uracil-
Thymine) PYR-CUT)

 Aromatic nature, bases absorb

radiation in the ultraviolet (UV)
 Heinz L. Fraenkel-Conrat region of the spectrum, with a
 Tobacco mosaic virus (TMV)- spring- maximum at a wavelength of 260
like RNA encapsulated in a cylindrical nm
protein coat
 Protein and RNA components of TMV  SPECTROPHOTOMETRY: identify
used to infect tobacco leaf sample and estimate
concentration

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 2: NUCLEIC ACIDS

 Purines (Guanine-Adenine) PUR-

GA
o Derived from pyrimidine
with addition of imidazole
group

 Thymine (5-methyl-2,4-
dioxypyrimidine)
 Cytosine 92-oxo-4-aminopyrimidine)

PENTOSE SUGAR

 Aldopentose
 Aldehyde group at C-1
 Carbons 2 and 5 have the same  C-2’ position of the ribose sugar ring
number in both cycles  RNA- hydroxyl group
 DNA- hydrogen atom
‘

 Adenine (6-aminopurine)
 Guanine (2-amino-6-oxypurine)

MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 2: NUCLEIC ACIDS

PHOSPHATE GROUP

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  Nucleotides linked together by
phosphodiester bonds between 5’
and 3’ hydroxyl groups of the sugars
 Sugar phosphate backbone
 Nucleic acid sequences written in 5’ to
3’ direction

NUCLEIC ACIDS

 Nucleotides link to each other by ester

bonds, with the phosphate establishing
a bridge between carbon 5’ of the
pentose of one nucleotide and carbon
3’ of the pentose of another nucleotide
 5’ end= first nucleotide in the chain
 Nucleoside with its phosphate free
o Purine/Pyrimidine base +  3’ end= has the hydroxyl group of C3’
ribose/ deoxyribose sugar free
o Glycosidic bond’
 Nucleotide SUMMARY OF DNA AND RNA DIFFERENCES
o Nucleoside + phosphate
group 1. RNA contains ribose and DNA
o Phosphoester bond contains deoxyribose
2. Instead of thymine as in DNA, RNA
contains the nitrogenous base uracil
3. Usually, RNA consists of a single
strand and DNA consists of a double
strand
NUCLEIC ACID NITROGENOUS BASE=
Bases
Deoxyribonucleic acids Adenine (A), thymine
(DNA) (T), guanine (G),
cytosine (C)
Ribonucleic acids (RNA) Adenine (A), Uracil (U),
guanine (G), cytosine (C)

DEOXYRIBONUCLEIC ACID (DNA)

 Found almost entirely in the cell

nucleus, more precisely, in the
material forming the chromatin
 Small amount of DNA is also found in
mitochondria and chloroplasts.
 All somatic cells of individuals of the
same species have equal DNA content

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 2: NUCLEIC ACIDS

 HUMANS: amount of DNA per cell is 6

CHARGAFF’S RULE
x 10-6 µg (micrograms) or 6 pg
(picograms)
 Purine bases adenine (A) and guanine
(G) and pyrimidines thymine (T) and
Cytosine (C)

STRUCTURE OF DNA MOLECULE

1. Two long polynucleotide chains are

coiled around a central axis, forming a
right-handed double helix
2. The two DNA strand are
ANTIPARALLEL, that is, 5’ to 3’
orientation runs in opposite directions
3. The base of both chains lie
perpendicular to the axis, and they
are stacked on one another
4. The nitrogenous bases of opposite
chains are paired as the result of the
formation of a hydrogen bond in DNA
5. Each complete turn of helix is 34A
long
6. The double helix has a diameter of
20A
7. The amount of Adenine (A) residues is
proportional to the amount of
Thymine (T) residues in DNA. Also, the
amount of Guanine (G) residues is
proportional to the amount of
Cytosine (C)
8. The sum of the purines equal to the
sum of pyrimidine
9. The percentage of (G+C) is not
necessarily equal the percentage of
(A+T)

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 2: NUCLEIC ACIDS

PRIMARY STRUCTURE
 Sequence of their nucleotides
 In writing nucleotide sequence:
WRITE THE NUCLEOTIDES (USUALLY
USING THE ONE-LETTER
ABBREVIATIONS FOR THE BASES)
From the 5’ to 3’ end

SECONDARY STRUCTURE
 The connection of the two single
strands and the formation of a double
helix

TERTIARY STRUCTURE
 DNA double helix may be arranged in
space, in a tertiary arrangement of
the strands
 Two strand of DNA wind around each
other

QUATERNARY STRUCTURE
 Interactions between separate nucleic
acid molecules, or between nucleic
acid molecules and proteins

PHYSICAL PROPERTIES OF NUCLEIC ACIDS

 Important feature of DNA double

helix:
o DNA DENATURATION:
separate 2 strands
o DNA RENATURATION: base
pair the two strands together
 Complementary strands are held by
hydrogen bonds (lack of covalent
bonds makes it possible for DNA to
denature and renature without
affecting its properties)
 Hydrogen bonds can be disrupted by
high temperature, low salt
concentration, or high pH in vitro
 Melting temperature Tm – midpoint
of the temperature range over which
the DNA strands are half denatured
 Amount of denatured DNA- measured
by the increase in absorbance at 260
nm

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 3: DNA

 Bases were paired in a certain way; A

DNA STRUCTURE can form a Hydrogen bonds with T,
 Model structure: Double helix and G can form a hydrogen bond
 Several evidences for the conception specifically with C. This bonding is
of the model for the secondary known as base pairing and the two
structure of DNA by Watson and Crick strands are said to be complementary
in 1953  The sugar phosphate backbone sits on
 Each base pair is rotated ~36 with the outside while the bases lie on the
respect to the next inside, stacked in pairs per
 Helix has ten based pairs per turn and perpendicular to the axis of the helix
rises 0.34 nm in each turn  The hydrophobic interactions
 Twisting of the two DNA strands resulting from such stacking of the
around one another forms the double base pairs and the Van der Waals
helical structure, leaving gaps forces further stabilize the double
between each set of phosphate helix
backbones resulting in a minor
(narrow) groove (~12 A across) and a
major (wide) groove (~22 A across)
 Wide major groove is easily accessible
to DNA binding proteins
 The double helix is right-handed and
is the most favoured conformation of
DNA in the cellular environment
commonly referred to as the B-form
of DNA

1. X-ray diffraction studies

demonstrated that DNA was helical
and because the layer line spacing
was one-tenth of the pattern repeat,
there must be 10 nucleotides per turn
2. Data on the density of DNA fibers by
Linus Pauling suggested that the helix
must contain two strands
3. Discovery by Erwin Chargaff which
showed that irrespective of the actual
amounts of each base, certain pairs of
bases are always found in a 1:1 ratio.
(A)=(T) and (G) = (C)

The two polynucleotide strands in the double

helix:
 Aligned in opposite directions
(antiparallel)
 Stabilized by hydrogen bonding
between the nitrogenous bases on
the opposite strands

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 3: DNA
 Crucial aspect of the functionality of
FORMS OF DNA HELICAL STRUCTURE DNA: flexibility or the ability to
 B FORM change its organization under
o The structure of DNA in cells with physiological conditions.
the genome being majorly  Central property: The ability of the
organized in this conformation strands to separate without disrupting
 A FORM the covalent bonds, and reform into a
o The conformation that the double helix at rapid rates needed to
double-stranded RNA molecules sustain genetic function
adapt as do the DNA-RNA hybrid  FOR DNA REPLICATION
molecules. This may be due to the o The strands of parental DNA
presence of the 2-hydroxyl group need to unwind, with each
in RNA that poses a steric strand serving as the template
hindrance to organization in the B for the synthesis of a new
Form complementary strand
o Relatively compact A helix, the o The specificity of this is
bases lie farther to the outside determined by base pairing
and instead of lying flat, are and the resulting two double
strongly tilted with respect to the stranded DNA molecules are
axis, with more bases per turn an exact copy of the original
o Major groove in the A form is  This concept of self-replication via
much deeper and less accessible base pairing is crucial for the
in comparison to the B form transmission of genetic information
o Both A and B forms are right- during cell division
handed, but very different
 Z FORMS STABILITY OF SECONDARY AND TERTIARY
o Discovered under in vitro STRUCTURE OF DNA
conditions by Alexander Rich and
colleagues in 1979  Under physiological conditions, the A
o Different from the other classical and B forms of the helices confer
forms as in it is left-handed, has stability to the nucleic acid molecules
the most base pairs per turn, is  Double helix needs to unwind to
slender and least twisted and has perform important biochemical
a single groove with a higher functions such as replication and
density of negative charges in transcription
comparison to the other forms  The non-covalent forces that stabilize
o Got its name from the zigzag path the helix can be disrupted by heat or
that the sugar phosphate by exposure to lower salt
backbone follows along the helix concentrations, leading to the
o Most often found in separation of the two strands
polynucleotides with alternating  Such transition from helix to random
purines and pyrimidines in each coil is sharp and occurs over a very
strand narrow temperature range. It can be
monitored spectroscopically by
CENTRAL PROPERTY OF DNA HELICAL observing the absorbance of
STRUCTURE ultraviolet light at 260 nm

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MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 3: DNA

 The stacked bases in the double helix

conformation are able to absorb light
to a lesser degree in comparison to
when they are in a less constrained
environment, this phenomenon is
called hypochromism or the
hypochromic effect and results from
the interactions between the electron
systems of light absorbing
heterocyclic purine and pyrimidine
rings of the nucleotides
 Any departure from the helical
structure is reflected by a decline in
the hypochromic effect, leading to an
increase in optical density towards a
value characteristic of free bases

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 and the nearest emergency
phone. Knowledge of the
MOLECULAR BIOLOGY AND DIAGNOSTICS location of this apparatus may
help save your life and
WEEK 3: LABORATORY SAFETY AND BIOSAFETY prevent injury to others

GENERAL PRECAUTIONS o Never eat or drink in the laboratory

o Proper clothing should be worn in area
the laboratory at all times o Contamination of food or
o Long sleeve shirts, long pants drink can take place without
and full shoes are the best your knowledge. Dropping a
choice. Skin protection will be reagent bottle on the lab floor
at a maximum if such may cause a toxic substance
garments are covered with a to spatter into your food or
full lab coat/apron. drink. If you must eat or drink
o Wear safety goggles in the laboratory something during the
o Recently, the American laboratory period, leave the
Chemical Society has food or drink outside the
approved the use of contact laboratory
lenses but only when worn in o Prior to the laboratory period, the
combination with safety student should read the experimental
goggles. The contact/goggle protocol thoroughly, noting any
combination is important questions for the instructor.
because modern eyewash o When preparing reagents for your use
stations are not able to from a common stock solution, be
remove chemicals trapped sure to return the stock solution to its
behind contacts proper storage place
o Make use of the fume hoods for o May chemicals used in
handling volatile and/or hazardous biochemistry experiments
chemicals degrade rapidly below 4C. If
o When handling the chemicals, you are preparing a mixture
gloves should be worn to from a purchased chemical
protect the hands stock, check the label on the
o Dispose of solid and liquid waste in reagent bottle for the proper
containers, which are properly storage procedures
labelled o After using common laboratory
o Notify the instructor if any of equipment, micropipettes, distilled
the waste containers are full water bottles, timers, etc., return
or damaged. If you are not them to their proper place
sure where to dispose of the immediately after cleaning
waste ask your instructor for o Use only as much of the chemicals
help. supplied to you as you need
o Get acquainted with the layout of the o Never insert the tip of your
laboratory pipet/micropipette into a common
o Pay special attention to the reagent bottle
fire extinguishers, emergency o This practice can easily spread
eyewash stations, first aid kits contamination throughout

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 laboratory, thus ruining many
hours of work by yourself and
other students
MOLECULAR BIOLOGY AND DIAGNOSTICS

WEEK 3: LABORATORY SAFETY AND BIOSAFETY

o After each lab period, clean your work

HIGH RISK MICROBES
area and any glassware that you used
o After cleaning used glassware BSL
and rinsing with tap water, 4

rinse the glassware with BSL 3

distilled water and place it in
a strainer to dry overnight. LOW RISK MICROBES
BSL 2

BIOSAFETY LEVELS
BIOSAFETY
BSL 1
 It includes the set of safety precautions
which reduce a laboratorian’s risk of
exposure to potentially hazardous
chemicals and infectious microbes. BSL-1 LABORATORY
Additionally, it limits the contamination  Microbes are not known to
of the work environment and, consistently cause disease in
ultimately, the community. healthy adults and present
BIOSAFETY LEVELS minimal potential hazard to
 Four biosafety levels consisting of laboratorians and the
measures for containment of microbes environment
and biological agents  Example: NON-PATHOGENIC
 Levels are determined by infectivity, STRAIN OF E. COLI
severity of disease, and transmissibility Laboratory practices
of microbes and biological samples  Standard microbiological practices are
 Also include: the nature of the work followed
conducted, origin of the microbe, the  Work can be performed on an open
agent and route of exposure lab bench or table (A)
 Each biosafety level builds on the Safety Equipment
controls of the level below it  Personal protective equipment (B),
 Every microbiology laboratory, (Lab coats, gloves, eye protection) are
regardless of biosafety level, follows worn as needed
standard microbiological practices, such Facility Construction
as no mouth pipetting, labelling all the  A sink must be available for hand
reagents, washing hands, sterilizing washing
equipment, disinfecting the work areas  The lab should have doors to separate
and so on. the working space from the rest of the
facility

BSL-2 LABORATORY
 BSL-2 builds upon BSL-1 for a lab that
works with microbes that pose

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 moderate hazards to laboratorians  Access to the laboratory is restricted
and the environment and controlled at all times
 Microbes
MOLECULAR BIOLOGY ANDare typically indigenous and
DIAGNOSTICS Safety Equipment
associated with diseases of varying  Appropriate PPE must be worn, and
WEEK 3: LABORATORY
severity SAFETY AND BIOSAFETY respirators might be acquired (A)
 Example: STAPHYLOCOCCUS AUREUS  All work with microbes must be
Laboratory practices performed within an appropriate
 Access to the laboratory is biological safety cabinet or BSC (B)
restricted when work is being
conducted Facility construction
Safety Equipment  A hands-free sink and eyewash are
 Appropriate personal protective available near the exit.
equipment (PPE) (A) is worn,  Exhaust air cannot be recirculated,
including lab coats and gloves. Eye and the laboratory must have
protection and face shields can sustained directional airflow by
also be worn, as needed drawing air into the laboratory from
 All procedures that can cause clean areas toward potentially
infection for aerosols or splashes contaminated areas
are performed within a biological  Entrance to the lab is through two
safety cabinet (BSC) (B) sets of self-closing and locking doors
 An autoclave or an alternative (C)
method of decontamination is
available for proper disposals BSL-4 LABORATORY
Facility construction  BSL-4 builds upon the containment
 The laboratory has self-closing doors requirements of BSL-3 and is the
 A sink and eyewash are readily highest level of biological safety
available  The microbes in a BSL-4 lab are
dangerous and exotic, posing a high-
BSL-3 LABORATORY risk of aerosol-transmitted infections
 BSL-3 builds upon the containment caused by these microbes are
requirements of BSL-2 for working frequently fatal and without
with the microbes that can be either treatment or vaccines
indigenous or exotic, and that can  Example: EBOLA and MARBURG
cause serious or potentially lethal VIRUSES
disease through respiratory
transmission Laboratory Practices
 Respiratory transmission is the  Change clothing before entering
inhalation route of exposure  Shower upon exiting
 Example: MYCOBATERIUM  Decontaminate all materials before
TUBERCULOSIS, SARS-CoV-2 exiting
Safety equipment
Laboratory Practices  All work with the microbe must be
 Laboratorians are under medical performed within an appropriate
surveillance and might receive Class III BSC, or by wearing a full body,
immunizations for microbes they air-supplied, positive pressure (A) suit
work with Facility Construction

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 
The laboratory is in separate building
or in an isolated and restricted zone
of the building. The laboratory has
dedicated
MOLECULAR BIOLOGY ANDsupply and exhaust air, as
DIAGNOSTICS
well as vacuum lines and
WEEK 3: LABORATORY SAFETY
decontamination AND BIOSAFETY
systems.

RISK GROUPS AND BIOSAFETY LEVELS

RISK GROUP 1
 Agents are not associated with
disease in healthy adult humans
RISK GROUP 2
 Agents are associated with human
disease which is rarely serious and for
which preventative or therapeutic
interventions are often available
RISK GROUP 3
 Agents are associated with serious or
lethal human disease for which
preventative or therapeutic
interventions may be available
RISK GROUP 4
 Agents are likely to cause serious or
lethal human disease for which
preventative or therapeutic
interventions are usually not available

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  When a linear DNA is free in solution,
it assumes a pitch that contains 10.4
base pairs per turn. As the result, the
MOLECULAR BIOLOGY AND DIAGNOSTICS DNA is less tightly wound than 10.0
base pairs per turn in the Watson and
WEEK 4: DNA TOPOLOGY Crick B-Form DNA
 In order to understand the origin of
TOPOLOGY supercoiling; imagine a linear DNA of
 A branch of mathematics concerned 4,200 base pairs in length. If the DNA
with those properties of geometric were in the B-form, one would expect
configurations (Such as point sets) the two strands of the helix to be
which are unaltered by elastic wrapped around each other 400 times
deformations (such as stretching or (4,200 bp/10.4 bp/turn). Imagine a
twisting) that are homeomorphisms linear DNA in which the two ends
(continuous transformations) become connected to form an open
 Topological characteristics of DNA and circle. This is referred to as RELAXED
specifically DNA supercoiling influence CIRCULAR DNA
all major DNA processes in living cells  On the other hand, if the linear DNA
were unwound 10%, say 40 turns,
TERTIARY STRUCTURE OF DNA before its ends were joined, then the
 Many naturally occurring DNA DNA molecule would be under stress.
molecules are circular, with no free 5’ When the molecule is free in solution,
or 3’ end (ex. Prokaryotic DNA is it will coil about itself in space, as the
circular, and this DNA forms a two strands simultaneously twist
supercoil) about each other in order to return to
 Rotating the DNA end changes the equilibrium value of 10.4 base pairs
helical twist of the DNA from its per turn.
preferred state containing about 10.4
base pairs per turn
 When the DNA is ligated (meaning
joined together), the untwisted
portion of DNA will tend to spring
back to adopt its favoured state, with
10.4 base pairs per turn. This,
however, causes the plasmid to wrap
around itself, and the closed DNA
circle is now supercoiled
 In 1965, Vinograd et al. discovered
that the circular DNA chromosomes
isolated from small viruses such as
SV40 or polyoma virus were in a
highly compact or folded
conformation.
 The supercooling or writhing of the
circular DNA was a result of the DNAs
being under wound with respect to
the relaxed form of DNA

REGIDOR  MT 3B
 strand and then rejoining the broken
strand
 Lk is always an integer since two
strands must always be wound about
MOLECULAR BIOLOGY AND DIAGNOSTICS each other an integral number of
times upon closure
WEEK 4: DNA TOPOLOGY  The Lk of a covalently closed circular
DNA can be resolved into two
THE TOPOLOGY OF SUPERCOILED DNA components called ‘the twists (Tws)
 The DNA that is ‘underwound is and the (Wrs)’
referred to as negatively supercoiled’. o LK = Tw + Wr
The DNA in this case forms the right-  In a relaxed circular DNA duplex of
handed double helices 500 bp, L is 50 (assuming that 10 bp
 The DNA that is ‘overwound is per turn in the B-DNA)
referred to as positively supercoiled
DNA helix and is a left-handed helix

LINKING NUMBER (LK)

 The total number of times one strand
of the DNA helix is linked with the
other in a covalently closed circular  The Lk for a relaxed DNA is usually
molecule. taken as the reference parameter and
m, . is written as Lo
 By convention, the Lk is defined as
positive for right-handed helix and
negative for left-handed helix. Since
left-handed Z-DNA occurs very rarely
negative Lks are not encountered in
all DNA studies for practical purpose

TWISTS AND WRITHES

 Twists (Tw)
SALIENT FEATURES ABOUT LINKING NUMBER o The number of times the two
 The Lk is only defined for a covalently strands of DNA are twisted
closed DNA and its value is fixed about each other
provided the molecule remains  Writhes (Wr)
covalently closed. The Lk does not o The number of times that the
change whether the covalently closed DNA helix is coiled about itself
circle is forced to lie in a plane in a in three-dimensional space
stressed conformation or it is allowed o Tw and Wr are geometric
to supercoil about itself freely in rather than topological
space properties.
 The Lk of a circular DNA can only be o The Tw and the Wr are not
changed by breaking a necessarily integers. It is just
phosphodiester bond in one of the their sum, the Lk that is an
two strands, allowing the intact Integer
strand to pass through the broken

REGIDOR  MT 3B
  If we use an SV40 DNA molecule, for  Lk/LK= = the superhelical density
example, which is precisely 5,243  SUPERHELIX DENSITY OR SPECIFIC
base pairs in length, we would find LINKING DIFFERENCE
that: o The difference between the
o LK= Tw + Wr Lk of a DNA in the supercoiled
o Lk= 5,243/10.4= 504.13 form and the Lk in the relaxed
 The DNA length and its pitch in form. In a cell, it’s value is
solution determine the Tw of DNA usually of 5-7%
o Tw= length (bp)/pitch o Example:
(bp/turn)  0.1 means that 10%
 The Tw and the Lk determine the of the helical turns in
value of the Wr a sample of DNA (in
o Wr= Lk- Tw its B configuration)
o =504.13- 24.13 have been removed.
o = 480 This underwinding
 Unlike the Tw and the Lk, the Wr of a results in negative
DNA only depends on the path the supercoiling.
helix axis take in space. If the path of
the DNA is in a plane, the Wr is always MEASUREMENT OF SUPERHELICAL DENSITY
zero.  The superhelical density of a circular
 In addition, if the path of the Dan DNA can be observed and measured
helix were on the surface of a sphere, in several ways:
then the total Wr can also be shown o Electron microscopy
to be zero. o Sedimentation velocity
 Since supercoiled
WRITHES molecules are more
 Wrs can come in different forms. If a compact, they sediment
DNA molecule wrap around itself, faster in a centrifuge
then the Wrs are known as than when they are
supertwists relaxed.
 If a DNA molecule wrap around o Electrophoresis
something else (another molecule for  In a agarose gel, a
instance), then the Wrs are known as supercoiled DNA
‘solenoidial’ Wrs. migrates much more
 In solution, the Wrs can isomerize rapidly than does a
between the supertwists and relaxed molecule of the
solenoidal forms. same length. The DNA
separates into discrete
MEASURING SUPERCOILS bands depending on the
 The topoisomerases change the Lk Lk
(some directly and some indirectly).  Since the DNAs resolved
 The change in the Lk (Lk) is a in this way differ from
measure of the supercoiling each other only in their
 If the Lk in a supercoiled DNA and the topology, they are
Lk in the relaxed state (both of which referred to as
must be integers) is compared, then ‘topological isomers or
the ratio would be: topoisomers’.

REGIDOR  MT 3B
  Molecules that differ by
one unit Lk can be
separated by
electrophoresis in
agarose due to the
difference in their Wr
(that is due to difference
in folding)

TOPOISOMERASES  Type 2 Topoisomerase

 Are enzymes that change the Lk of a o These enzymes act through a
circular wound double-stranded DNA mechanism, in which both the
 The change in Lk changes the Wr. The phospodiester backbone
variation in Wr subsequently changes chanins are broken
the state of the compaction of the simultaneously. As a result,
DNA molecule. The naturally the Lk changes by two.
occurring DNA is underwound or o Some type 2 enzymes can use
negatively supercoiled ATP to introduce the
 This is advantageous because it superhelical turns into the
permits the DNA to be transiently and DNA. The best-characterised
locally melted to permit the enzymes member of this class is E.coli
of the DNA replication and Topoisomerase II- better
transcription to copy and synthesize known as DNA gyrase
new DNA and RNA o E.coli DNA gyrase is a
tetrameric protein consisting
TYPES OF TOPOISOMERASES of two subunits (875 aas) and
 Type 1 Topoisomerase two B subunits (804 aas).
o These enzymes remove Depending on the DNA
supercoils by breaking only substrates, this enzymes can
one of the two strands of the change positive supercoils
DNA. As a result, these into negatove supercoils or
enzymes change the Lk by 1 increase the number of
each time negative supercoils by 2.
o The best-characterized o Type II topoisomerases
member of this chlass in E.coli catalyse catenation and
is Topoisomerase I. this decatenation, i.e., linking and
enzyime is 864 amino acids in unlinking of two different
length and is monomeric; it is DNA duplexes. The enzyme
encoded by the topA gene also introduces negative
supercoils at or near the Ori C
site in the DNA template. DNA
gyrase also removes the
positive supercoils that are
formed ahead of the growing
for during replication

REGIDOR  MT 3B
 MEDICAL APPLICATIONS
 Topoisomerases are essential
enzymes
 The mutations of any of the genes
coding for topoisomerases are usually
lethal. They are, therefore, the targets
for the antibiotics and other drugs.
 Bacteria can be killed by novobiocin
or nalidixic acid. Both of these inhibit
DNA gyrase
 NOVOBIOCIN blocks ATP binding and
NALIDIXIC ACID blocks the breakage
and rejoining mechanism. These
antibiotics do not inhibit eukaryotic
topoisomerases and can be used to
eradicate bacterial infections. Some
bacteria, however, are now resistant
to novobiocin. Eukaryotic
topoisomerase inhibitors, such as
doxorubicin and etoposide, are used
as chemotherapeutic agents.

REGIDOR  MT 3B
 The Mitochondrial DNA (mtDNA)
 Mitochondrial DNA (mtDNA) is the
DNA located in mitochondria and is
circular in shape.
 In mammals, each double-stranded
circular mtDNA molecule consists of
15,000-17,000 base pairs
 The nuclear and mitochondrial DNAs
TYPES OF DNA are believed to be of separate
evolutionary origin. The mtDNAs are
The chromosomal DNA thought to be derived from the
 The DNA molecule may be circular or circular genomes of the bacteria that
linear and can be composed of were engulfed by the early ancestors
100,000-10,000,000,000 nucleotides of today’s eukaryotic cells
in a long chain.  Each mitochondrion is estimated to
 Typically, eukaryotic cells (cells with contain 2-10 mtDNA copies
nuclei) have large linear  About 100-10,000 separate copies of
chromosomes and prokaryotic cells mtDNA are usually present per cell
(cells without defined nuclei) have (egg and sperm cells are exceptions)
smaller circular chromosomes, in humans (and probably in
although there are many exceptions metazoans in general)
to this rule. In addition, cells may  In most multi-cellular organisms, the
contain more than one type of mtDNA is inherited from the mother
chromosome; for example, (maternally inherited)
mitochondria in most eukaryotes and  Both males and females have the
choroplasts in plants have their own mtDNA
small chromosomes.
The Chloroplast DNA
The autosomal DNA  Chloroplast genomes are relatively
 Most of the DNAs are large, usually ~140 kb in higher plants
autosomal/chromosomal DNAs and <200 kb in lower eukaryotes. This
 Hald of the autosomal/chromosomal is comparable to the size of large
DNAs are from each of the parents. bacteriophage, e.g., T4 of ~65 kb
This is the DNA that can uniquely  There are multiple copies of the
identify a specific individual genome per organelle, typically 20-40
 The autosomal DNA contains almost in a higher plant
all of our health/medical information  The chloroplast genome codes for all
 The autosomal DNA is used in the rRNA and tRNA species needed
maternity/paternity tests and for for protein synthesis. The ribosomes
forensic/crime purposes. include two small rRNAs in addition to
the major species. The tRNA set
The Y DNA resembles that of mitochondria.
 Only males have the Y chromosomes,
Y-DNA, which rarely changes (mutates The Plasmid DNA
slowly) and is passed down to sons  Is a small DNA molecule that is
from the father’s direct paternal/male separate from and can replicate
line.

REGIDOR  MT 3B
 independently of the chromosomal
DNA
 They are double-stranded and, in
many cases, circular
 Plasmids usually occur naturally in
bacteria; however, sometimes, they
are found in eukaryotes (e.g., the 2-
µm-ring in Saccharomyces cerevisiae)
 Plasmid sizes vary from 1 to over
1,000 kb pairs
 Plasmids are capable of autonomous
replication within a suitable host.

TYPES OF RNA
There are 4 types of RNA, each encoded by its
own type of gene:
1. mRNA- messenger RNA
a. encodes amino acid sequence
of a polypeptide
2. tRNA- transfer RNA
a. brings amino acids to
ribosomes during the
translatio.
3. rRNA- Ribosomal RNA
a. with ribosomal proteins
makes up the ribosomes, the
organelles that translate the
mRNA
4. snRNA- small nuclear RNA
a. with proteins forms
complexes that are used in
RNA processing in eukaryotes
(not found in prokaryotes)

REGIDOR  MT 3B