Topic 3: Nucleic Acids: DNA & RNA [MT 203]

SY 2024-2025
[MT 203] : Cytogenetics 1ST SEMESTER
[Ms. Dyan Jumamoy] prepared by Lance, Kezziah Rapha D.

Adenine = Thymine & Guanine =

NUCLEIC ACIDS Cytosine (Chargaff’s Rule)
Maurice Wilkins & Rosalind Franklin
● Molecular repositories for genetic information
- Bombarded DNA with X-rays (X-ray
and are jointly referred to as the “molecules of
1950 diffraction), then deduced the overall
heredity” structure of the molecule
● They are the main information of carrying - Photo 51
molecules of the cells and by directing the
Linus Pauling
process of protein synthesis - Suggested a triple helix structure of
● Determine the inherited characteristics of DNA
every living thing
1953 James Watson & Francis Crick
DEOXYRIBONUCLEIC ACID (DNA) - Constructed the double-helical model
for DNA & published their findings in
● Serves as the genetic material in all living April 25, 1953 issue of Nature Magazine
organisms as well as in most viruses - Received Nobel Prize

RIBONUCLEIC ACID (RNA) NUCLEIC ACID: STRUCTURE

● Involved in protein synthesis and sometimes DNA STRUCTURE

in the transmission of genetic information
● A double-stranded molecule with long chain
of nucleotides
DISCOVERY OF NUCLEIC ACIDS Nucleotides

Freidrich Miescher ● Single building block of DNA

- Isolated nuclei from pus cells (WBCs) ● Deoxyribose sugar, phosphate group &
1869 and found that they contained “nuclein” nitrogenous base
- First term named but it is changed into Nitrogenous Bases
‘nucleic acids’
Emil Fischer ● Information-containing parts of DNA
- Discovered purine (nucleic acid-base, ● DNA sequences are measured in numbers of
1880s base pairs
two-ring structure) and pyrimidines
(nucleic acid-base, single-ring structure) ● Purines: Adenine (A) & Guanine (G)
Geheimrat Albrecht Kossel - Double ring
- Recognizes that nucleins are associated ● Pyrimidines: Cytosine (C) & Thymine (T)
1894
with histones (a type of protein that coils - Single ring
the DNA in a regular pattern)
Richard Altmann
1899
- Introduced the term nucleic acid
P.A. Levene
- Recognized the 5-carbon ribose sugar
1909
and later discovered deoxyribose un
nucleic acids
Robert Feulgen
1914 - Demonstrated a color test known as
Feulgen test for the DNA
P.A. Levene Sugar Phosphate Backbone
1929 - Stressed that there are 2 types of
nucleic acids ● Formed when the nucleotides are joined into
long chains when strong attachments called
Erwin Chargaff
1950s - Discovered that several species contain
phosphodiester bonds form between the
equal amounts of the bases: deoxyribose sugars & the phosphate

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 Antiparallelism DNA Packaging

● Opposing orientation of the 2 nucleotide ● Process by which the long, linear DNA
chains in DNA molecules are tightly compacted & organized
into a more condensed structure within the
cell

RNA STRUCTURE

● Single stranded molecule in most of its

biological roles and has a shorter chain of
nucleotides
Nucleotide

● Ribose sugar, phosphate group and

Complementary Base Pairs nitrogenous bases
Nitrogenous bases
● Pairs of DNA bases that hydrogen bonds
● Adenine is for Thymine; Guanine is for ● Purines: Adenine (A) & Guanine (G)
Cytosine ● Pyrimidines: Cytosine (C) & Uracil (U)
● Purine-pyrimidine couple

DIFFERENCES OF DNA & RNA

DNA RNA
1. Usually double stranded 1. Usually single stranded
2. Thymine as base 2. Uracil as Base
3. Deoxyribose as the sugar 3. Ribose as the sugar
4. Carries protein encoding
4. Maintains protein
information and controls
encoding information
how information is used
5. Cannot function as an 5. Can function as an
enzyme enzyme
6. Persists 6. Transient

DNA REPLICATION
● A process by which DNA makes a copy of
itself during cell division (S Phase)
Example 1:
Given the DNA strand - ACGTCG, What is its
complementary strand? TGCAGC

Example 2:
Given the DNA strand = CATCATT, What is its
complementary strand? GTACGTAA

DNA CONFIGURATION IN THE NUCLEUS

DNA Molecules are extremely long

● DNA of smallest chromosome: 14 mm long if

stretched out, but is packaged into a
chromosome (2um long)

KRL | BMLS 2E 2
 Central Dogma

● This illustrates the flow of genetic informations

in the cells and the DNA REPLICATION
holding the RNA through the
TRANSCRIPTION process and further RNA
codes for the proteins by the process of
TRANSLATION
Alfred Hershey & Martha Chase
Genetic Material
- Confirmed that the DNA of the
bacteriophage was the carrier of its genetic
● "A genetic material must carry out two jobs: determination
duplicate itself and control the development of
the rest of the cell in a specific way." - Francis
Crick

Discovery of Genetic Material

1953
Freidrich Miescher
- Isolated the genetic material from WBC
1869 nuclei; he noted it had an acidic nature
containing nitrogen (N) & phosphorus (P), &
called it nucleins

Archibald Garrod
- 1st linked inherited disease & proteins
1902
- People who have certain inborn errors of
metabolism doesn't have certain enzymes
Fredrick Griffith
- Investigated virulence in Diplococcus (now Why must DNA be replicated?
known as Streptococcus pneumoniae)
- 2 strains: Rough type (avirulent) & smooth
● DNA must be replicated so that the
type (virulent)
information it holds can be maintained and
- Transforming principle
passed to future cell generations.

Mode of DNA Replication

1928
Semiconservative Replication

● Replicated DNA would consist of one old and

one new strand (used template)
Conservative Replication

● 2 newly created strands are brought together

& the parental strands reassociate
Dispersive Replication
Oswald T. Avery, Colin MacLeod & M.J. MacCarty
- Identified the transforming material as DNA ● Each strand would consist of both old and
by preparing boiled virulent bacterial cell new DNA
lysates & sequentially treated them with
1944
enzymes
- Conclusion: “transforming principle” in
Griffith’s experiment was DNA”

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 5. DNA polymerase proofreading activity checks
and replaces incorrect bases

OVERVIEW OF DNA REPLICATION

6. Continuous strand synthesis continuous in a
● DNA replication during S phase (Interphase) ‘5 to 3’ direction
7. Discontinuous synthesis produces Okazaki
fragments on the ‘5 to 3’ template

8. Enzymes remove RNA primers. Ligase seals

sugar phosphate backbone (Human replicates
STEP BY STEP PROCESS OF DNA REPLICATION at a rate about 50 bases per second)

1. Helicase binds to origin and separate strands

DNA AMPLIFICATION
2. Binding proteins keep strands apart ● Laboratory technique used to produce
3. Primase makes a short stretch of RNA on the multiple copies of DNA sequence
DNA template ● DNA replication conducted outside cell

Polymerase Chain Reaction (PCR)

● 1st and the best known DNA amplification

technique
4. DNA polymerase adds DNA nucleotides to the ● Uses DNA polymerase to rapidly replicate a
RNA primer specific DNA sequence in a test tube

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 2. Forensics: ID of suspects/victims & provide
evidence for criminal investigations
3. Disease diagnosis: infectious diseases
caused by microorganisms.
4. Biomedical research: study of gene
expression patterns, genetic variations, DNA
sequencing, etc.
5. Environmental monitoring: detection & ID of
microorganisms in soil, water, & air samples
6. Paternity & relationship testing: establish
biological relationships
7. Ancient DNA studies: extraction &
amplification of DNA from ancient specimens

DNA SEQUENCING
● Determines the order of the 4 chemical
building blocks called ‘bases’ that make up
the DNA molecule
● Basis for DNA SEQUENCING:
Complementary Base Pairing
Adenine (A) <~> Thymine (T)
Cytosine (C) <~> Guanine (G)

Methods used for DNA Sequencing

● There are 3 billion base pairs in a human body

1. SANGER SEQUENCING
● Invented by Frederick Sanger in 1977 a way
to determine the base sequence of a small
piece of DNA
● The PCR, a standard laboratory procedure ● Generates a series of DNA fragments (about
applied in molecular biology for the 900 base pairs in length) of identical sequence
amplification of specific segments of that are complementary to the DNA sequence
double-stranded DNA. PCR is based on the
of interest
mechanisms of DNA replication, though it's
performed in vitro. ● AKA chain termination method:
● First, the double-stranded DNA, which serves dideoxynucleotides (ddNTPs) are added,
as the template in the reaction, is separated terminating the DNA synthesis Uses ddNTPs
by heat. Next, a DNA polymerase synthesizes to determine the order/sequence of
a new daughter strand on each single strand nucleotides in a nucleic acid
in one direction. By repeating these reactions
continuously, a defined segment of DNA, the
target, is amplified.
● This and the high specificity of the reaction
enable the amplification of target DNA
segments

Uses/Application of PCR

1. Genetic testing & diagnostics: identify

genetic markers associated with disease,
detection of mutation, ID of pathogens, &
screening of genetic disorders

KRL | BMLS 2E 5
 ● Requirements: ● Features:
1. Template to be sequenced - Highly parallel
2. Primers - Micro scale
3. DNA polymerase - Fast
4. 4 DNA nucleotides (dNTPs = dATP, - Low cost
dTTP, dCTP, dGTP) {dNTPs - - Shorter length
Deoxynucleotide triphosphate}
5. Different ddNTPs (ddATP, ddTTP,
ddCTP, ddGTP), each labeled with a
different color of dye {ddNTPs -
dideoxyribonucleotides}
6. Sequencing reaction (thermal cycler)

STEPS IN SANGER SEQUENCING

1. DNA amplification
2. DNA denaturation
3. Dispersion of primed DNA
4. Addition of DNA, polymerase, dNTPs and
Nanopore Sequencing
ddNTPs
5. Attachment of DNA polymerase
● NGS sequencing platform
6. Chain Termination
● Each nucleotide can be identified by a
7. Denaturing Polyacrylamide Gel
disruption in current as it passes through the
Electrophoresis
pores at about 1,000 bases per second
8. Sequencing Ladder
● Uses Grapheme: 1-atom-thick sheet of
- Ladder is read from the bottom to top
carbon
- No. of bases per sequence: 300- 400
(depending on the reagents & gel
used)

USES/APPLICATION OF DNA SEQUENCING

1. Genome Sequencing
2. Next Generation Sequencing
- Provides comprehensive information
about an organism’s genetic makeup
2. Medical diagnostics & personalized
● Collectively referred to the most recent set of
medicine
DNA sequencing technologies
- Diagnosing genetic diseases &
● Ability to sequence millions of small pieces at identifying diseases causing
once that can handle much larger DNA mutations
molecules much faster 3. Cancer genomics
- ID of genetic mutations associated
with the development & progression
of tumors

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4. Forensics & Human ID
- Analysis of DNA for crime scene
samples or unidentified human
remains & establishing genetic
relationships
5. Evolutionary studies & phylogenetics
- Reconstructing evolutionary
relationships between organisms &
studying their genetic diversity
6. Metagenomics & microbial ecology
- Analysis of complex microbial
communities present in various
environments
7. Pharmacogenomics
- ID of genetic variations affecting an
individual’s response to medications
8. Ancient DNA studies
- Providing insights into the genetic
history and evolution of extinct
species, including human ancestors.

Enzymes in DNA REPLICATION

1. Helicase - unwinds parental double helix

2. Binding proteins - stabilize separate strands,
keep the strands apart.
3. Primase - adds short primer to template
strand, makes and creates RNA.
4. DNA polymerase - binds nucleotides to form
new strands, proofreads the arrangement of
bases.
5. Ligase - joins Okazaki fragments and seals
other nicks in the sugar phosphate backbone

KRL | BMLS 2E 7