MOLECULAR BIOLOGY AND DIAGNOSTICS combined influence on the growth and causing the transfer of the virulent strain

d causing the transfer of the virulent strain to the non-virulent

TOPIC 1: NUCLEIC ACIDS development of an organism strain
·
r
Lecturer: Ms. Praise Selah Dagoc, RMT the Human Genome Project was launched
▪ the project aims to map all the human genes
▪ due to this finding, they attracted attention again on DNA as a
possible contending molecule for carrying genetic information
INTENDED LEARNING OUTCOMES 1990
▪ define molecular biology and molecular diagnostics or all the base pairs in the human genome ▪ Austro-Hungarian-American biochemist
▪ discuss the scientific milestones that led to the discovery of the and was completed in April 2003 ▪ (1950) formulated the Chargaff’s Rules
DNA structure molecular biology goes post-genomic (specifically 1959)
▪ describe DNA and RNA: their composition, roles, and differences post-genomic ▪ his findings were fundamental in the
▪ diagram the structure of nitrogen bases and nucleotides ▪ utilizes the sequence of information provided discovery of DNA structure, because only
▪ list and describe types of RNA by genomics, but then situates it in an he was able to deduce the amount of the
analysis of all the other entities and activities Erwin Chargaff nucleotides and nitrogen bases found
LECTURE OUTLINE 2000s
involved in the mechanisms of transcription, inside the DNA but it wasn’t until
▪ introduction to mol bio and diagnostics
regulation, metabolism, and expression Rosalind Franklin came into the picture
▪ nucleic acids
▪ this does not only focus on the genes but also that the answer to DNA structure was
▪ DNA: molecular composition and structure
the proteins and all the processes finally becoming clear
▪ RNA: molecular composition, structure, and types
surrounding it CHARGAFF’S RULES
INTRODUCTION TO MOLECULAR BIOLOGY AND DIAGNOSTICS ▪ A, T, C, and G were not found in equal
▪ Swiss biochemist quantities
branch of biology that deals with the structure Friedrich ▪ (1868) first to isolate DNA: he called it
molecular ▪ amounts of nitrogen bases varied among
and function of the macromolecules (e.g. nucleic Miescher nuclein because he isolated it from the
biology species, but not between individuals of
acids and proteins) essential to life nucleus of blood cells, specifically the the same species
molecular the application of molecular biology in medical WBCs ▪ A=T, and G=C (amounts are
diagnostics testing ▪ based on his experiments, he noticed that approximately or almost equal to each
this component/molecule did not react other)
BRIEF HISTORY OF MOLECULAR BIOLOGY
according to how he expected it to react ▪ she was an expert in a technology called
Mendel’s Laws of Inheritance
with protein chemical reactions so he x-ray crystallography in which she used it
it wasn’t a while after he came up with it that the
th
19 century deduced that it was not protein to determine the structure of DNA
scientific field started to correlate the loss of
▪ Russian-American biochemist Rosalind Franklin ▪ the photograph
inheritance with DNA
▪ (1910) formulated the Tetranucleotide (diffraction image)
1938 ‘molecular biology’ as a term was introduced Hypothesis of DNA: shows an
James Watson and Francis Crick discovered the ▪ coined the term “nucleotide”: he was the “x” image that gives
DNA’s double helical structure first to use the term a clear clue to
significance: TETRANUCLEOTIDE HYPOTHESIS Watson and Crick
▪ the discovery of the DNA’s double helix Phoebus Levene ▪ nucleic acid was a structure of repeating that contrary to a previous study that
opened up so many opportunities in tetramer (four monomers) that is why it’s
1953 claimed that DNA was composed of three
molecular biology, not just in the discovery of tetranucleotide strands, this photo was a big clue to them
the structure of DNA itself ▪ however, the simplicity of the structure to deduce that DNA must be composed of
▪ opened so many advancements, not just in implied to them that nucleic acids were two strands instead of three
the medical field but in other fields also (e.g. too uniform contribute to complex ▪ for years, scientists have been trying to determine the
agricultural, biotechnology) genetic variation so, for them, it seemed structure of the DNA molecule and it was largely due to seeing
biological science goes molecular that this structure was just too simple to Franklin’s x-ray diffraction image of DNA along with Chargaff’s
▪ so many advancements and discoveries were contain genetic information rules that allowed Watson and Crick to come up with their 3D
1960s made ▪ hence, a lot of scientists back then model of the DNA double helix
▪ the PCR (polymerase chain reaction) was also believed that it was the protein that ▪ (1953) built the 3D model of DNA’s
discovered carried the genetic information so they double helical structure
molecular biology goes genomic focused on protein as the probable
James Watson ▪ for this discovery, Watson and Crick,
genomics: hereditary substance
and Francis Crick along with Wilkins (Franklin’s assistant),
▪ study of genes and their functions and TRANSFORMATION PRINCIPLE
1980s were awarded the Nobel Prize in 1962
related techniques ▪ in the 1920s up to the 1940s, several scientists conducted o since Rosalind Franklin already died,
▪ addresses all genes and their experiments using virulent and non-virulent strains of bacteria her assistant got the Nobel Prize
interrelationships in order to identify their and deduced through their experiments that DNA may be

=>
WITH LORRAINE M., ARA G., PATRICK Y. ,
GENEVIEVE R., & JOSHUA G.
AIRAH M.
 ▪ due to the amount of discoveries and NUCLEIC ACIDS PENTOSE SUGARS
previously published works of the ▪ so called because they were originally isolated from cell nuclei
scientists before them, they were able to and have acidic properties
connect the dots ▪ macromolecules containing carbon, hydrogen, oxygen, nitrogen,
▪ what differentiates the two from the and phosphorus
other scientists was that a lot of them a molecule that contains a very large
were focused on collecting the data of number of atoms (from that list of atoms
chemical experiments to come up with macromolecule
and elements, we can deduce that the
the structure of the DNA; on the other nucleic acid is a big molecule)
hand, although both of them were not as nucleotides building blocks of nucleic acids
knowledgeable or as skilled in the lab the two main naturally-occurring types of RNA DNA
experiments as the scientists, they nucleic acids ribose deoxyribose
approached the matter in a different DNA and RNA ▪ we also have artificial nucleic acids that has hydroxyl group in the
angle wherein they focused on building a only hydrogen in 2’carbon
are designed and synthesized by 2’Carbon
model of the DNA based on the findings
biochemists
of previous published works BASIC NUCLEOTIDE COMPONENTS
▪ since these works were already NUCLEIC ACIDS: NUCLEOTIDES The basic nucleotide component is composed of a nitrogenous
published, they have the right to COMPOSITION: base, pentose sugar and a phosphate group. So, based on the
interpret this data to build the 3D model ▪ nitrogenous base (pyrimidines and purines) image below, there is no oxygen attached to the 2’ carbon,
of DNA ▪ pentose sugar hence, this is your deoxyribose.
▪ although there were still controversy ▪ phosphate group
surrounding the discovery of the DNA
structure, it is undeniable that although I. NITROGENOUS BASE
they did not do the lab experiments pyrimidine purines
themselves, they were the ones who consist of fused 6-membered
managed to connect the dots between consist of 6-membered N-
and 5-memebered N-
various published works containing rings
containing rings
▪ through their careful analysis of these
studies, they were able to conclude that, PYRIMIDINE BASES
contrary to another recently released thymine cytosine uracil
study, DNA is composed of two in DNA in RNA
polynucleotide chains held together by
purine and pyrimidine bases through
hydrogen binding that the sugar in the
backbone was deoxyribose (instead of
ribose) that these two polynucleotide
chains ran antiparallel to each other
IMPLICATIONS OF WATSON AND CRICK’S DISCOVERY
▪ it revolutionized the field of biology into a global industry: it’s
not just used in the medical field but is also used in other fields
PURINE BASES
(e.g. zoology, biotechnology, agriculture, environmental
adenine guanine
science)
▪ it also enables scientists to access genetic information about
life that had not been previously available
o other examples of advancements through this discovery
are: genetically modified organisms (GMOs), cloning,
genome

WITH LORRAINE M., ARA G., PATRICK Y. ,

GENEVIEVE R., & JOSHUA G.
AIRAH M.
 DNA (DEOXYRIBONUCLEIC ACID) formation of hydrogen bonds between o made possible by the two antiparallel ssDNA polymers twisting
hybridization
▪ major function: Storage of genetic information for most two complementary strands of DNA around the same axis
organisms phosphodiester bond between the sugars and o this allows H bonds to form between A&T and C&G
▪ long, double-stranded polymeric (many monomers) molecule bonds phosphorous groups o as long as the bases are in complementary order, the strands
(dsDNA) How do we know which one is 5’ and which one is 3’? of the helix have a ladder-like structure with rungs (base pairs)
▪ predominantly exists in a right-handed double helix form also Remember OrgChem and BioChem in numbering Carbons. It is of consistent size
known as the alpha-helix based on the carbons found in the ribose or deoxyribose sugar. ▪ flexibility of the carbon-oxygen linkages in the phosphodiester
bond allows the ladder to twist forming a regular helix
COMPOSITION the deoxyribose has 5 carbons:
o planar base pairs are stacked on top of each other: no room for
1 nitrogenous bases: adenine, cytosine, guanine, thymine ▪ 1’ is attached to the nitrogenous base
water molecules in between
2 pentose sugar ▪ 2’ contains the hydroxyl group and a ribose for RNA, while in DNA,
▪ helical dsDNA is stable at pH 4-9
3 phosphate group it does not contain a hydroxyl group that is why it’s called
▪ melting point (Tm of the DNA)
deoxyribose since it only has hydrogen
Nitrogenous bases are attached to the deoxyribose sugar, where o temperature at which 50% of dsDNA is converted to ssDNA
nitrogenous bases attach to and forms a polymer with deoxyribose ▪ hence, to know if it is a ribose or a deoxyribose, just look at the 2’
(stability broken in the presence of high temperature or
sugars of other nucleotides through a phosphodiester bond. carbon if it contains an H (which is DNA) or an OH (which is RNA)
extreme pH or destabilizing agents)
▪ the 5th one (5’) is located outside the ring connected to the
o depends on the relative G-C vs. A-T base pair content
phosphate above it (left side) or below it (right side)
⇨ the 3 hydrogen bonds of G-C base pairs require more energy
5’ to 3’ 3’ to 5’ to disrupt than the 2 hydrogen bonds of A-T base pairs
top bottom top bottom ⇨ hence, the DNA with higher G-C base pairs will take longer
ends in 5’ ends in 3’ ends in 3’ ends in 5’ to melt since it will have a higher Tm compared to the DNA
NOTE: this is based on the left side, the right side will have the with more A-T base pairs
opposite ends of the left side “We knew that DNA was important. We knew it was an important
▪ polarity: 5’ -> 3’ molecule. And we knew that its shape was likely to be important.
▪ antiparallel strands But we didn’t realize just how important it would be. We didn’t
▪ complementary base pairing (A-T, C-G) realize that the shape would give us a clue to the replication
mechanism. And this turned out to be really an unexpected dividend
TWO TYPES OF BONDS HOLDING DNA TOGETHER
phosphodiester bonds hydrogen bonds from finding out what the shape was.”
phosphate group + ribose / connect the nitrogenous base - Francis Crick
deoxyribose pairs RNA: RIBONUCLEIC ACID
DNA ▪ has ribose sugar, containing a hydroxyl group at the 2’
▪ stable, only loses its normal conformational structure at extreme ▪ uracil substitutes with thymine instead of Adenine. (A-U, C-G)
heat, pH, or in the presence of destabilizing agents ▪ predominantly single-stranded but it’s not always 100% the case
▪ double stranded helix: most energetically favorable state for o exceptions such as through internal homologies, RNA can fold
DNA molecular structure and loop among themselves to take on a double stranded
DNA
please view the video in this part from 25:25 to 34:07 character important for their function
backbone bases o RNA can also pair with complementary single strands of DNA
backbone
steps of the ladder hydrophilic hydrophobic or another RNA to form a double helix
(sides of the ladder)
forms stable H bonds with
phosphate group and non-soluble in water at ▪ much shorter than DNA
nitrogenous bases surrounding water molecules
deoxyribose sugar neutral pH ▪ transient and less stable
in solution
o not just because of its ss structure but also due to its
purines pyrimidines o in vivo, we have water everywhere, so your backbone can form susceptibility to alkaline hydrolysis via the 2’ hydroxyl group of
adenine and guanine cytosine and thymine
hydrogen bonds with surrounding solutions due to their the ribose moiety
the “blue links” between the base pairs hydrophilic nature o because it has a hydroxyl group sa 2-5, naka”siwil” ra iyahang
are hydrogen bonds, which is a stable hydroxyl group which is why it easily gets hydrolyzed via
▪ for a DNA molecule to remain stable, the bases must not be in
hydrogen bonds bond that connects the nitrogenous bases alkaline hydrolysis
contact with water because it is hydrophobic, hence, its
(the bond can only be broken by high ▪ copied, or transcribed, from DNA
temperatures) placement is in the middle for protection

WITH LORRAINE M., ARA G., PATRICK Y. ,

GENEVIEVE R., & JOSHUA G.
AIRAH M.
 TYPES OF RNA REFERENCES
▪ largest component (80-90%) of cellular
ribosomal RNA RNA
(rRNA) ▪ important in the structural and functional
part of the ribosomes (protein synthesis)
▪ initial connection between the
information stored in DNA and the
messenger RNA translation apparatus that will produce the
(mRNA) protein products
▪ amount in the cell depends on its
requirement for its final product
▪ short, singe-stranded polynucleotides of
73-93 bases in length
transfer RNA
▪ key link between transcribing and
(tRNA)
translating RNA into protein
▪ at least one tRNA for each amino acid
small nuclear ▪ functions in splicing in eukaryotes
RNA (snRNA) ▪ stays in the nucleus after its transcription
COMPARISON OF KEY FEATURES OF DNA AND RNA
FEATURE DNA RNA
sugar* deoxyribose ribose
thymine-adenine uracil-adenine
base pairs
cytosine-guanine cytosine-guanine
double-stranded variable, determined
3D structure
alpha helix by base sequence
subject to base
stable
stability hydrolysis
degraded by DNase degraded by RNase
maintains genetic carries genetic
function information in information to
nucleus cytoplasm
*basis of the name differences between the two

STUDY QUESTIONS
1. What is the function of the DNA in the cell?
2. Compare the structure of the nitrogenous bases. How do purines
and pyrimidines differ?
3. Which of the ribose car bonds participate in the phosphodiester
bond?
4. Write the complementary sequence to the following:
5’ AGGTCACGTCTAGCTAGCTAGA 3’

WITH LORRAINE M., ARA G., PATRICK Y. ,

GENEVIEVE R., & JOSHUA G.
AIRAH M.