Molecular Biology

& Diagnostics

GROUP
1
PRESENTATION
 TABLE OF CONTENTS

I II III

Review of DNA, RNA, Techniques for DNA

and DNA and RNA Hybridization
Protein Structure and Isolation Techniques
Function

IV V

Fundamental of PCR Molecular Techniques

and other in Protein Analysis
Amplification
Techniques
 REVIEW OF DNA, RNA,
AND PROTEIN STRUCTURE
AND FUNCTION

Bea Angeles
Tricia Kaye Sausa
1. DNA: Hereditary
Molecule
DNA: Hereditary Molecule
• Deoxyribonucleic acid
• A macromolecule of carbon, nitrogen, oxygen,
phosphorus, and hydrogen atoms.
• Assembled in units of nucleotides that are composed
of phosphorylated ribose sugar and a nitrogen base.

Nitrogen Bases that make up the majority of DNA:

1. Adenine
2. Cytosine
3. Guanine
4. Thymine
 DNA: Hereditary Molecule
• Nitrogen bases are attached to deoxyribose sugar,
which forms a polymer with the deoxyribose sugars of
the nucleotides through a phosphodiester bond.

• The information in the DNA storage system is based on

the order or sequence of nucleotides in the nucleic acid
polymer.
A. STRUCTURE
OF DNA
• The double helical structure of DNA
was first described by James Watson
and Francis Crick.

• The helical structure of DNA results

from the physicochemical demands of
the linear array of nucleotides.

• Specific sequence (order) of

nucleotides can affect DNA.
 B. HUMAN
GENOME PROJECT
 Human Genome Project
• A large, well-organized, and highly
collaborative international effort that
generated the first sequence of the
human genome.
• 1990 – 2003
• A special committee of the US National
Academy of Sciences outlined goals
that lead to discovering list of
organisms.
• Organisms discovered: Bacterium E.
coli, baker’s yeast, fruit fly, nematode,
and mouse.
 Francis Collins
(Led the Human Genome Project and NIH)
 C. GENES,
GENOMES, AND
GENETIC MATERIAL
 GENE
• Defined as the ordered sequence of
nucleotides on a chromosome that
encodes a specific functional product.

• Is the fundamental physical and

functional unit of inheritance.
 GENOME
• Entire set of DNA instruction found in a
cell
• Consists of 23 pairs of chromosomes
located in the cell’s nucleus.
• A genome contains all the information
needed for an individual to develop
and function.
 GENETIC MATERIAL
• Also known as hereditary material
• Is the medium by which instruction are
transmitted from one generation to the
next.
• DNA is the main hereditary material,
but some contain RNA too.
 D. COMPOSITION
OF DNA STRUCTURE
• The phosphodiester backbones of the
two nucleic acid chains from the helix.
• Nitrogen bases are oriented toward the
center, where they hydrogen bond with
homologous bases to stabilize the
structure.
• Two hydrogen bonds form between
adenine and thymine.
• Three hydrogen bonds form between
cytosine and guanine.
2. Structure and
Types of RNA
Types of Ribonucleic Acid

• Messenger RNA (mRNA)

• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
 Messenger RNA (mRNA)

• Carries genetic information to be translated

into protein.

• It is the initial connection between the

information stored in DNA and the translation
apparatus that will ultimately produce the
protein products responsible for the phenotype.
Messenger RNA (mRNA)
 Ribosomal RNA (rRNA)

• Protein-synthesizing organelle
• The largest component of cellular RNA
comprising 80% to 90% of the total cellular
RNA.
• Is an important structural and functional part of
the ribosomes, cellular organelles where
proteins are synthesized.
 Transfer RNA (tRNA)

• Translation of the information from nucleic acid

to proteins requires reading of the mRNA by
ribosomes, using adaptor molecules or transfer
RNA.

• Serves as adaptor or link

3. Central Dogma of
Molecular Biology
 Central Dogma of
Molecular Biology

Is the theory stating that genetic

information flows only in one direction, from
DNA, to RNA, to protein, or RNA directly to
protein.
 • The two strands of a double helix have an
A. DNA Replication antiparallel orientation because of the way DNA is
replicated.
• As DNA synthesis proceeds in the 5’ to 3’ direction,
DNA polymerase, the enzyme responsible for
polymerizing the nucleotide chain, uses a guide, or
template, to determine which nucleotides to add
to the chain.
• The enzyme reads the template in the 3’ to 5’
direction. The resulting double strand, then, will
have a parent strand in one orientation and a
newly synthesized strand arranged in the opposite
orientation.
 STEPS IN DNA REPLICATION

1. Helicase unwind the parental double helix.

2. Single-strand binding proteins stabilize the
unwound parental DNA.
3. The leading strand is synthesized continuously in
the 5’ to 3’ direction by DNA polymerase.
4. The lagging strand is synthesized discontinuously.
Primase synthesizes a short RNA primer, which is
extended by DNA polymerase to form an Okazaki
Fragments.
5. After the RNA primer is replaced by DNA (by
another DNA polymerase), DNA ligase joins the
Okazaki fragment to the growing strand.
 B. RNA
TRANSCRIPTION

• Transcription is the copying of one strand of DNA into

RNA by a process similar to that DNA replication.

• This activity, catalyzed by RNA polymerase, occurs

mostly in interphase.

Steps in RNA Transcription

• Initiation
• Elongation
• Termination
 INITIATION
• In the nucleus, RNA polymerase
recognizes the recognition sites
causing it to bind to the promoter; (the
start of a gene).
• The RNA polymerase then separates
the DNA into single strands to the
template strand can be read in the 3’ to
5’ direction.
 ELONGATION
• Pre-mRNA nucleotides are quickly
paired with their complementary bases
which correspond with the template
strand of DNA.
• The pre-mRNA moves in the 5’ to 3’
direction while the template strand of
DNA moves oppositely from the 3’ to 5’
direction.
• Pre-mRNA does not contain thymine,
instead uracil is used as the
complementary base for adenine.
 TERMINATION
• When the RNA polymerase reaches the
terminator, it signals the RNA
polymerase to stop and release from
the DNA.
• Once separated the two DNA strands
come back together and reform the
double helix.
• The newly formed pre-mRNA molecule
is then released.
C. RNA PROCESSING

• Capping and Polyadenylation

Processes
• RNA Splicing
• Alternative Splicing
 Capping and Polyadenylation Processes
• The 5’ capping occurs soon after the growing RNA emerges
from RNA polymerase II, and it is accomplished in three
successive steps.
• First, and RNA 5’ triphosphate catalyzes the removal of one
phosphate from the triphosphate at the 5’ end of the
mRNA. A guanyl transferase then attaches a molecule of
guanosine monophosphate (GMP) to this end. Finally, the
transferred guanine is methylated by a guanine-7-
methyltransferase.
 Capping and Polyadenylation Processes
• Polyadenylation of the 3’ end of eukaryotic mRNAs begins
with an initial cleavage of the mRNA. The cleavage is usually
occurred after a CA nucleotide pair that lies somewhere
between a conserved AAUAAA hexamer sequence and a U-
or GU- rich region further downstream.
• After cleavage, a tail of approximately 200 adenosines is
added by poly(A) polymerase. This process is more complex
than 5’ capping because it involves recognition and
processing of different polyadenylation sites in different
mRNAs.
 RNA Splicing
• an RNA processing event where specific parts of pre-mRNA,
called introns are recognized and removed by a protein-and-
RNA complex called spliceosome.
• Intron ca be viewed as “junk” sequences that must be cut
out so the “good parts version” of the RNA molecule can be
assembled.
• The pieces of the RNA that are not chopped out are called
exons. The exons are pasted together by the spliceosome to
make the final, mature mRNA that is shipped out of the
nucleus
 Alternative Splicing
• It is a process in which more than one mRNA can be made
from the same gene. Throughout alternative splicing, we
(and other eukaryotes) can sneakily encode more different
proteins than we have genes in our DNA.
• In alternative splicing, one pre-mRNA may be spliced in
either of two (or sometime many more than two) different
ways. For example, in the diagram, the same pre-mRNA can
be spliced in three different ways, depending on which
exons are kept. This results in three different mature
mRNAs, each of which translates into a protein with a
different structure.
 D. PROTEIN
TRANSLATION
• In protein translation, the small ribosomal subunit first
binds to initiation factor 3 (IF-3) and then to specific
sequences near the 5’ end of the mRNA, the ribosomal
binding site. This guides the AUG codon (the “start”
codon) to the proper place in the ribosomal subunit.

• Translation, as related to genomics, is the process

through which information encoded in mRNA directs the
addition of amino acids during protein synthesis.
Translation takes pace on ribosomes in the cell cytoplasm,
where mRNA is read and translated into the string of
amino acid chains that make up the synthesized protein
 4. Enzymes that
Metabolize DNA and
RNA
a. Enzymes Necessary to
Metabolize DNA
 *DNA Polymerases:
These enzymes are responsible for
synthesizing new DNA strands during
replication and repair. They add
complementary nucleotides to the existing
DNA template, ensuring accurate copying of
genetic information.
 *DNA Ligases**:
DNA ligases seal the breaks in the sugar-
phosphate backbone of DNA strands. They
play a crucial role in the final stages of
DNA replication, repair, and recombination
by joining the Okazaki fragments on the
lagging strand and sealing nicks in the DNA
backbone.
 *DNA Helicases:
These enzymes unwind the double-
stranded DNA during replication and
repair by breaking the hydrogen bonds
between complementary bases. This
unwinding is essential to provide access
to the DNA template for various
enzymatic processes.
 *Topoisomerases:
DNA strands can become tangled or
overwound during replication and
transcription. Topoisomerases help by
cutting and rejoining DNA strands,
relieving the tension and allowing the
strands to unwind or rewind as needed.
 *DNA Primase:
Primase is responsible for
synthesizing short RNA sequences
called primers, which provide a
starting point for DNA polymerases
during replication. These primers
are essential for initiating DNA
synthesis.
 Exonucleases and Endonucleases:
These enzymes are involved in DNA
repair mechanisms. Exonucleases
remove nucleotides from the ends of
DNA strands, while endonucleases cut
DNA at specific points to excise
damaged or mismatched bases.
 DNA Methyltransferases:
These enzymes add methyl groups
to specific cytosine residues in
DNA, a process known as DNA
methylation. DNA methylation
plays a role in gene expression
regulation and other cellular
processes.
 Telomerase:
Telomerase is an enzyme that adds
repetitive DNA sequences called
telomeres to the ends of linear
chromosomes. This prevents
chromosome shortening during
replication and helps maintain
genomic stability.
a. Enzymes Necessary to
Metabolize RNA
 **RNA Polymerases:
These enzymes synthesize RNA
strands by using DNA templates
during transcription. They catalyze the
formation of phosphodiester bonds
between nucleotides to create RNA
molecules.
 Ribonucleases:
Ribonucleases are responsible for
degrading RNA molecules. They can
be endonucleases, which cleave RNA
within the molecule, or exonucleases,
which remove nucleotides from the
ends of RNA strands.
5. Recombination in
Sexually Producing
Organisms
 1. Crossing Over:
During meiosis, the process by which sex cells
(gametes) are produced, homologous chromosomes pair
up. In a step called "crossing over," sections of
chromatids from one chromosome are exchanged with
the corresponding sections on the other chromosome.
This results in the mixing of genetic information
between the parents and generates new combinations
of alleles (gene variants) on the chromosomes.
 2. Genetic Diversity:
Recombination increases genetic diversity within
a population. It leads to the shuffling of alleles,
creating offspring with unique combinations of
traits. This diversity is advantageous for
populations to adapt to changing environments
and provides the raw material for natural
selection.
 3. Independent Assortment:
Another aspect of genetic recombination occurs
during the segregation of homologous
chromosomes in meiosis. The independent
assortment of chromosomes ensures that
different combinations of alleles are inherited
independently of each other, further increasing
genetic variability.
 4. Linkage and Recombination Frequency:
Genes located closely on the same chromosome
are said to be linked. However, due to
recombination, linked genes can still undergo
crossing over, leading to the formation of
recombinant offspring with new allele
combinations. The frequency of recombination
between two genes is used to map their positions
on a chromosome.
 5. Evolutionary Advantage:
Recombination promotes evolutionary
adaptability by introducing novel genetic
combinations into a population. This allows
organisms to respond to selective pressures and
increases the chances of advantageous traits
arising.
 5. Evolutionary Advantage:
Recombination promotes evolutionary
adaptability by introducing novel genetic
combinations into a population. This allows
organisms to respond to selective pressures and
increases the chances of advantageous traits
arising.
6. Recombination in
Asexually Producing
Organisms
Conjugation -

Conjugation:
Conjugation is a process of genetic exchange
observed in asexually reproducing organisms,
particularly bacteria. It involves direct physical
contact between two bacterial cells, where one
acts as a donor and transfers genetic material,
often in the form of a plasmid, to the recipient.
This mechanism allows for the rapid spread of
advantageous traits, such as antibiotic resistance,
throughout bacterial populations, contributing to
their adaptation and survival. Conjugation is a form
of horizontal gene transfer that enhances genetic
diversity in asexual organisms.
Conjugation -

Transduction:
Transduction is a process of genetic
transfer in asexually reproducing organisms,
commonly observed in bacteria. It involves
the transfer of genetic material between
bacteria through viruses called
bacteriophages. These viruses can carry
bacterial DNA from one host cell to another,
introducing new genes and potentially leading
to genetic diversity and adaptation in
bacterial populations.
Conjugation -

Transformation:
Transformation is a mechanism of genetic
transfer in asexually reproducing organisms,
particularly bacteria. It involves the uptake
and incorporation of external genetic
material, such as DNA fragments, by a
bacterial cell. This introduced DNA can
become integrated into the recipient cell's
genome, potentially leading to the acquisition
of new traits and genetic diversity in bacterial
populations.
7. Types of Mutation
 Frameshift mutation:

This type of mutation occurs when the addition or loss

of DNA bases changes a gene’s reading frame. A
reading frame consists of groups of 3 bases that each
code for one amino acid. A frameshift mutation shifts
the grouping of these bases and changes the code for
amino acids. The resulting protein is usually
nonfunctional. Insertions, deletions, and duplications
can all be frameshift mutations.
 Point mutation:

• A point mutation is one type of mutation caused by a

change in a single nucleotide.

• This mutation results when a DNA nucleotide is

added, removed or replaced by a different
nucleotide.
 Nonsense mutation:

A nonsense mutation is also a change in one

DNA base pair. Instead of substituting one
amino acid for another, however, the altered
DNA sequence prematurely signals the cell to
stop building a protein. This type of mutation
results in a shortened protein that may
function improperly or not at all.
 Missense mutation:

This type of mutation is a change

in one DNA base pair that results
in the substitution of one amino
acid for another in the protein
made by a gene.
 Silent mutation:

Some mutations that change DNA bases

do not have any effect on the sequence
of amino acids in the protein. These
mutations are called silent mutations and
they do not affect the structure or
function of the protein because there is
no effect on the amino acid sequence.
 Conservative Mutation:

Where the new amino acid is of

the same type as the original.
 Non-conservative Mutation:

Results in the replacement of an

amino acid with a biochemically
different amino acid, which
changes the biochemical nature of
the protein.