Molecular Biology

Department of Medical Technology

Far Eastern University
The Nucleic Acids (DNA and RNA) Reviewer
Nucleic acids: naturally occurring chemical compound that is capable of being broken down to
yield phosphoric acid, sugars, and a mixture of organic bases (purines and pyrimidines).

DNA is Genetic Material

• Frederick Griffith
o used Streptococcus pneumoniae virulent and avirulent strains to demonstrate
“transformation principle”.
Griffith used two related strains of bacteria known as R and S
• R strain (rough or non-encapsulated avirulent S. pneumoniae): did
not cause sickness
• S strain(smooth or encapsulated virulent strain): causes sicknes
o Transformation in Bacteria
Mechanism of a horizontal gene transfer in which a DNA fragment from a
dead, degraded bacterium enters a competent recipient bacterium

As part of his experiments, Griffith tried injecting mouse with heat-killed S bacteria (S
bacteria heated to high temperatures, causing the cells to die).
the heat-killed S bacteria did not cause disease in mice.

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University
Harmless R bacteria were combined with harmless heat-killed S bacteria and injected into a
mouse.
the mouse develop pneumonia and die
contained living S bacteria (because of Transformation principle)
• Oswald Avery, Colin MacLeod, and Maclyn McCarty
o Demonstrated that the transforming principle can be destroyed by DNase
thus, DNA is the genetic material

Purified heat killed S cells were used in the study

Purified heat killed S cells were treated with Deoxyribonuclease, Ribonuclease, and a
protease and cultured to a petridish with R strain.
o Deoxyribonuclease or DNAse for short is an enzyme that degrades DNA
o Ribonuclease is an enzyme that degrades RNA
o Protease is an enzyme that degrades protein.
1st Petridish
o Treated with DEOXYRIBONUCLEASE
o RNA is not present
o NO transformation occur with live R cells.
nd
2 Petridish
o Treated with RIBONUCLEASE
o DNA is PRESENT
o transformation occur with live R cells.
rd
3 Petridish
o Treated with PROTEASE
o Transfomation occurs between live R cells and the heat killed S strain.
o Protein is not present
Control Group
o no enzyme present
C.P.Tolenada, RMT, MSMT
ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University
o Contains live R cells and heat killed strain without any enzymes
o transformation occur.

DNA is the genetic material for bacteriophage

• A. D. Hershey and Martha Chase

o Used bacteriophage (labelled with phosphorus-32 in DNA and sulfur-35 in
protein) and E.coli to confirm that DNA is the genetic material
Bacteriophage: Virus that attack bacteria
o composed of protein and DNA (outer structures made of protein and
the inner core consisting of DNA)
o Attached to the surface of bacteria and inject the genetic material

2. 1.

Hershey and Chase prepared two different batches of phage.

o first batch was labeled with phosphorus, which is incorporated into the DNA .
o second batch of phage was labeed with S or sulfur, which is incorporated into the
protein coat.
Both batches showed that the bacteriophage replicates and lysed the cell.
Lysis release the phage so they can infect new cells.

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University

Both bacteriophages infect bacteria

Infected cells were subjected to blender agitation in order to remove the bacteriophage.
(Genetic information and protein remain inside the cell)

1. 2.

Infected cells were again invaded by a new bacteriophage.

The bacteriophage acquired the DNA present inside the cell.
1st bacteria: the phage reproduces a progeny containing the labeled DNA from the
parent/ old bacteriophage
2nd bacteria: the bacteriophage reproduces but no offspring or progeny containing S
labeled proteins.
Hershey and Chase concluded that DNA is made up the genetic material of the
bacteriophage that can easily pass to the offspring
TRANSDUCTION: Process of genetic recombination in bacteria in which genes from a
host cells are incorporated into the genome of a bacteriophage and then carried to
another host cells when the bacteriophage initiates another cycle of infection.

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University

RECOMBINANT DNA TECHNOLOGY

Making a new DNA sequence from the original DNA segments.

Plasmid vector is isolated

o Plasmid: genetic structure in a cell that can replicate independently of the
chromosomes
Cut the plasmid and insert a desired gene that was cleaved into a fragment by an
enzyme.
The plasmid containing the desired gene is taken up by a new bacterium for replication

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University

The cloned gene may undergo replication or expression

o Replication meaning it obtain many copies of cloned gene
o Expression is a process of obtaining many copies of gene’s product which is the
protein. (DNA-RNA-Protein)

RNA is the genetic material for viruses

Virus contains only 1 genetic material (either RNA or DNA)
• Tobacco mosaic virus
• purified RNA caused infection
• Retroviruses (HIV)
• reverse transcription is used to replicate genetic material using RNA to create
complimentary DNA.
• Enzyme: Reverse transcriptase
• Process: Reverse Transcription

Nucleic acids: Macromolecules made out of units called nucleotides,

The nucleotides is divided in two naturally occurring varieties: deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA).
Each nucleotide is composed of a phosphate group, a ribose sugar and nitrogenous base

PENTOSE SUGAR
FIVE-CARBON SUGAR in DNA is called deoxyribose, while in RNA, the sugar is ribose.
Difference: the second carbon of ribose bears a hydroxyl group (meaning it contains
oxygen bonded with hydrogen), while the equivalent carbon of deoxyribose has a
hydrogen instead.
Sugar occupies a central position, with the base attached to its 1′ carbon and the
phosphate group (or groupS) attached to its 5’ carbon

HETEROCYCLIC BASES
Four possible nitrogenous bases: adenine (A), guanine (G) cytosine (C), and thymine (T).
Pyrimidine – C,U,T
Purine - A, G
C.P.Tolenada, RMT, MSMT
ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University

Differences: DNA (A with T, G with C); RNA (A with U, G with C)

o A with T and A with U : 2 hydrogen bond
o C with G: 3 hydrogen bonds

PHOSPHATE GROUP
Derived from phosphoric acid
Attached to the 5’ carbon of the sugar
The phosphate group on one nucleotide links to the 3' carbon atom of the next pentose
sugar

NUCLEOTIDES
FORMED IN 2-STEP PROCESS
pentose sugar + nitrogenous base = nucleoside
nucleoside + phosphate group=nucleotide

NUCLEOSIDE
Base is attached to C1' of sugar (beta configuration)
o BETA CONFIGURATION: hydroxyl group is projecting on the opposite side as that
of oxygen ring
NUCLEOTIDE
PO4- is attached to C5' position
consists of a base plus a molecule of sugar and the phosphate group

ESTER BOND: bond that connect the 3rd carbon of the pentose sugar to the phosphate
group of the next nucleotide

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University
Phosphodiester bond: the linkage between the 3rd carbon of one pentose sugar
molecule and the 5’ carbon atom of another pentose sugar. (linkage between 2
nucleotide)

STRUCTURE OF DNA MOLECULES

• DOUBLE HELIX
• X-ray diffraction by Rosalind Franklin and Maurice Wilkins
• Proposed by James Watson and Francis Crick
• CHARGAFF'S RULES
• Discovered by Erwin Chargaff
• DNA should have a 1:1 stoichiometric ratio (base pair rule) of pyrimidine and
purine bases
• The amount of guanine should be equal to cytosine and the amount of
adenine should be equal to thymine.
Two long polynucleotide chains are coiled around a central axis, forming a right-
handed double helix.
The two DNA strand are antiparallel, that is, their 5' → 3' orientation runs in opposite
directions.
The base of both chains lie perpendicular to the axis, and they are stacked on one
another.
The nitrogenous bases of opposite chains are paired as the result of the formation of a
hydrogen bond in DNA.
Each complete turn of helix is 34 A˚ long.
The double helix has a diameter of 20 A˚ .
A=T;G=C
Purines = Pyrimidine.
The percentage of (G+C) is not necessarily equal the percentage of (A+T).

Alternative Forms of DNA Exist

• B-DNA
• Most common
• right-handed structure
• identified at 92% relative humidity
• helical turn in 10bp
• A-DNA
• same with B-DNA but shorter and thicker
• observed when DNA is dehydrated
• originally identified at 75% relative humidity
• helical turn in 11 bp
• Z-DNA
• longer and narrower than B- DNA
• left-handed structure
• zigzag pattern
• helical turn in 12 bp

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University

Various Forms of RNA: almost always single-stranded

DNA Denaturation
o Ability to separate the two strands by disruption of H bonds by high temperature
(95 degrees Celsius), low salt concentration, or high pH in vitro
o DNA unwinds and the hydrogen bonds that hold the two strands together
weaken and finally break.

Secondary Structure of DNA

• slipped
• tandem repeats
• Formed by sequences containing stretches of direct repeats and is involved in
frame shift mutagenesis during replication.
• cruciform
• paired-stem loop
• cross-links structures
• They are likely to form when a DNA sequence contains a palindrome or inverted
repeats.
• triple-helix
• Involve single stands interacting with duplexes, resulting in the formation of
triple strand structures.

Tertiary Structure of DNA

Nucleic acid tertiary structures reflect interactions which contribute to overall three
dimensional shape.
This includes interactions between different secondary structure elements, single
strands, and topological properties of nucleic acids.

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University

Secondary and Tertiary Structure of RNA

DNA Topoisomerase Catalytic Mechanisms

• Topoisomer
• DNA with same sequences yet differ in linkage number
• visualized using gel electrophoresis
• Topoisomerase
• enzymes that modulate DNA supercoiling; can convert one DNA isomer to another
• Type I - does not use ATP
• responsible for relaxing positively and/or negatively supercoiled DNA.

TYPES FUNCTION

IA form a 5'- phosphotyrosyl intermediate; relaxes negatively supercoiled DNA

and topoisomerase III; clamp-like structure.

IB forms a 3'-phosphotyrosyl intermediate and relaxes positive and negative

supercoils; C-shaped protein clamp

IC same mechanism of action as IB

• Type II - use ATP

• transiently cleave both strands of a DNA duplex to allow the
unidirectional passage of another DNA duplex through the protein-
linked DNA gate
• ALSO KNOWN AS DNA GYRASE

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph
Molecular Biology
Department of Medical Technology
Far Eastern University

DNA RNA
• Usually double-stranded • Usually single-stranded
• Thymine as a base • Uracil as a base

• Deoxyribose as the sugar • Ribose as the sugar

• Carries protein-encoding information and

• Maintains protein-encoding information
controls how information is used

• Cannot function as an enzyme • Can function as an enzyme

• Persists • Short-lived

C.P.Tolenada, RMT, MSMT

ctolenada@feu.edu.ph