Molbio Prelim Topics

MOLECULAR BIOLOGY AND DIAGNOSTICS PREPARED BY:
INSTRUCTOR: Dyan Jumamoy, MsBio RALPH JAYSON GARCIA

TOPIC 1: INTRO TO MOLECULAR BIOLOGY BMLS 2E
INTRODUCTION Replication: Faithful reproduction of the DNA

from parent to daughter cell during cell division.
Enzyme and proteins involved are:
Molecular Biology:  Helicase
 The study of biological phenomenon at  DNA – Binding Protein
the molecular level.  Primase
 The study of molecular structure of  DNA Polymerase
DNA and the information it encodes,
and the biochemical basis of Gene Polymerase Chain Reaction (PCR): In
Expression. vitro method in amplifying DNA.
 Gene Expression: Conversion of
information from RNA to Protein or Genes: Unit of DNA that specifies production
Gene to Protein. of proteins and RNA molecules required for
cellular function.
Molecular Diagnostic:  Exons: Coding region, translated to
 Application of molecular biology proteins.
techniques for the purpose of  Introns: Noncoding region, not
prevention, diagnosis and follow – up translated to proteins
or prognosis for disease, and selection,
and optimization and monitoring Alleles: Alternative version of gene at a given
therapies. location (locus) along a chromosome. An
 Prognosis: likely outcome or course individual inherits 2 alleles, 1 from each parent.
of the disease. It is also the chance of  Homozygous: 2 identical allele
reoccurrence or recovery of the  Heterozygous: 2 different allele
disease.  Genotype: describes the organism’s
allele or genetic information.
DNA: molecule that carries genetic
 Phenotype: observable
information.
characteristics or the outward
: Histones: associated proteins.
projection.
Nucleosomes: Unit of chromosomes
consisting of nucleosome core particles.
Gene Expression: The process responsible
Chromatin: Nuclear DNA and its
for the flow of genetic information from gene to
associated proteins.
protein.
Chromosome: Highly ordered structure of
 Transcription: DNA to mRNA
single double stranded DNA Molecule.
 Translation: mRNA to Protein
Nucleic Acid: polymer made up of
Nucleotide monomer
Chromosomes
: Nucleotide: unit of nucleic acid
 Autosome: non-sex chromosome, 22
consisting of one chemical base plus sugar
pairs in the human genome. This
molecule and at least one phosphate group.
determines the somatic
Base Pair: Purine and pyrimidine nucleotide
characteristic of an individual
bounded by hydrogen bond.
 Sex Chromosome: 1 pair, frequency with which recombination
Determines the sex and sex related occurs between them.
hormonal trait.
Epigenetics: Process that alters gene
Essential parts of the chromosome function or its interpretation by mechanism
 Telomere: DNA seqeuences at the end other than those that rely on changes in
of the chromosome DNA sequence.
 Centromere: Primary constriction in a  DNA Methylation: Addition of
chromosome. methyl group typically to the 5th
 Origin of Replication parts carbon position of cytosine ring.
p- arm – short arm  Genomic Imprinting: process by
q arm- long arm which only one copy of gene is
expressed while the other is
Genomic Regions suppressed.
 Euchromatin: Rich gene, compact and  Histone Modification: Key
organized during interphase epigenetic regulator that control
 Heterochromatin: Gene poor or span chromatin structure and gene.
transcriptionally short gene and more  Chromatin Rearrangement:
densely pack during interphase. Rearrangement of chromatin from a
condensed state to a
Genome: Complete set of chromosome. transcriptionally accessible state.
 Haploid genome: Half set
 Diploid genome: Complete set Human Genome Project: A project
undertaken by the International Human Genome
Cells: Basic unit of life Sequencing Consortium to decipher 3 billion
 Prokaryotic Cell: Membrane bound base pair in the human genome. It is completed
organelles is absent and DNA is not in 2003. It identified the following:
organized into chromosome. Organisms  Full set human genes and sequenced
are called prokaryotes. them all.
 Eukaryotic Cell: Cells with true  Some of alleles
nucleus bounded by nuclear membrane.
Recombination: process of exchange of genes

or segments of DNA between chromosomes.
This produces gametes with gametes that are
different from its parents.
 CentiMorgan (cM): Unit of measure
that refers to the distance between 2
gene loci determined by the
MOLECULAR BIOLOGY AND DIAGNOSTICS

TOPIC 1: INTRO TO MOLECULAR BIOLOGY
PREPARED BY RALPH JAYSON GARCIA
BRIEF HISTORY AND  1902: Archibald Garrod: Explains
DISCOVERY OF the concept of human inborn errors of
metabolism, linking inheritance to
Three lines of Research that leads to the proteins.
discovery that DNA is hereditary material: Each  1902: Walter S. Sutton: Genes for
chromosome is a single molecule of DNA and Mendels “character”
genes are sequence of DNA.  1905: Reginald Punnett: Punnett
 Heredity and Genes Square
 DNA  1906: William Bateson: Coined the
 Chromosome term “Genetics”
 1910: Thomas Morgan and Alfred
HEREDITY AND GENES Sturtevant: Announces the Gene
theory and chart the first linear map of
genes
 384 – 322 B.C. : Aristotle: Theory  1941: George W. Beadle and
of Pangenesis. Eduard L. Tatum: One – gene, One
- Pangenesis: process by which heredity – enzyme hypothesis.
transmission is by parts of the
parents. CHROMOSOME
 1886: Gregor Mendel: Father of
Modern Genetics. Inheritance of trait  1875: E. Strasburger: described what
in peas. will be called “chromosome”
 1884: E. Strasburger, Oscar  1882: Walther Fleming: described
Hertwig, R.A. Von Kollikv: the behavior of the chromosome during
Independently identify the cell nucleus mitosis.
as the basis of inheritance.  1883: W. Waldeyer: named
 1893: August Weismann: “Germ chromosome (colored bodies)
plasm theory” – Theory of heredity by  1902: Walter S. Sutton and
which the germ plasm is the hereditary Theodere Bovern: Observed that
material in which the parent can only chromosomes in cells behave in ways
transmit if it is present in the germ cells parallel to mendel’s character during
 1889: Hugo de Vries: hypothesize meiosis.
the existence of Pangenes  1905: Nettie M. Stevens and
o Pangenes: heritable Edmund B. Wilson: sex
characteristics are determination by chromosome.
transmitted during cell division.
 1900: Hugo de Vries, Karl Correns DNA
and Erich Von Tschermark:
Rediscovered and verified Mendel’s  1869: Friedrich (Fritz) Mieshcer:
law. “Nuclein”
 1928: Fredrick Griffith:
“Transforming Principle”

 1929: Phoebus Ann Levene:
Characterizes and named the compunds
RNA, DNA and tetranucleotide
structure of DNA in which four bases of
DNA are arranged after another in a set
of 4
 1938: Rudolf Signer, Torbjorn
Cassperson and Einer
Hammersten: Found the molecular
weight for DNA between 500,000 and
1,000,000 daltons. Proteins and DNA
are studied by many scientists using X-
ray crystallography
 1944: Oswald Avery, Collin
Macleod and Maclyn McCarthy:
demonstrated that Griffith’s bacterial
transforming principle is not protein but
DNA and suggest that it may function as
Genetic material.
 1938: Warren Weaver: Molecular
Biology
 1949: Roger and Collete
Mendreley and Andre Boivin:
Showed a constant amount of DNA in
all tissues of the same animal and found
half as much DNA in the nuclei of
sperm cells as they are in body cells.
 1950: Erwin Chargaff: Shows
amounts of the base A & T and G & C
are equeal.
 1952: Alfred Hershey and Martha
Chase: Bacteriophage to confirm that
DNA is the hereditary material.
 1952: Maurice Wilkins and
Rosalind Franklin: Use X-Ray
crystallography to reveal the repeating
structure of B – form DNA
 1953: James Watson and Francis
Crick: DNA Double Helix
conformation.

MOLECULAR BIOLOGY AND DIAGNOSTICS PREPARED BY:
INSTRUCTOR: Dyan Jumamoy, MsBio RALPH JAYSON GARCIA
TOPIC 2: DNA: STRUCTURE, REPLICATION, ENZYMES, RECOMBINATION AND PLASMIDS BMLS 2E
CENTRAL DOGMA OF LIFE Also, is assemblage of units of

nucleotides that are composed of:
 Replication: DNA to DNA
 Phosphate group
 Transcription: DNA to RNA
 Deoxyribose sugar
 Translation: RNA to Protein
 Nitrogen Base
James Watson: When he coined the term

Nucleoside:
“Molecular Biology”, he was referring to the
 Nitrogen bound to a unphosporylated
biology of DNA.
sugar.
 A (Adenosine), G (Guanosine),
Johan Friedrich Mischer: Discovered
C(Cytonidine), T(Thymidine)
DNA in 1869 and 1871, he published a
paper on nuclein, the viscous substance
Nucleotide:
extracted in nucleus.
 A nitrogen base bound to a
phosphorylated sugar.
DNA
 Nucleoside mono-, di-, or
triphosphate
 Functions to store information.
 Examples: Adenosine
STRUCTURE Monophosphate (AMP),
Adenosine Triphosphate
James Watson and Francis Crick:  Free nucleotides are
 They first described the double deoxyribonucleoside triphosphate
helical structure of DNA.  Can be converted to nucleoside by
 Their molecular model was found on hydrolysis.
previous observation of the chemical
nature of DNA and physical NITROGEN BASES
evidence including diffraction  Planar C-N ring structures.
analysis performed by Rosalind Pyrimidines (single ring): Thymine
Franklin. and cytosine
 Helical Structures results from Purines (double ring): Adenine and
psychochemical demands of the Guanine
linear array of nucleotides. Complementary Base Pairs: A-T ( 2 H
DNA is a macromolecule of bonds), C-G ( 3 H bonds)
 CARBON  Attached to deoxyribose sugar,
 NITROGEN which forms a polymer with the
 OXYGEN deoxyribose sugar of other
nucleotides through a
 PHOSPHOROUS
phosphodiester bonds.
 HYDROGEN
Erwin Chargaff: Numbering of positions in the
 In DNA, the amount of Adenine nucleotide molecule:
corresponds to the amount of  Starts with the ring position of the
Thymine and the amount of Cytosine nitrogen base. (C or N1,2,3 and so
to the amount of Guanine. on)
 Watson and Crick suggested that this
arrangement was the basis of a Modified Nucleotides
coping mechanism.  Often formed in nature
 Complementary strands could  Base modifications have significant
separate and serve as guides or effects on phenotype
templates for producing  Some results from DNA damage,
complementary strand. naturally modified or specific
function or to affect gene expression
DEOXYRIBOSE SUGAR  Used by bacteria and viruses as a
primitive immune system.
 5 Carbon sugar of DNA.
 Ribose with number the number 2 Nucleic Acid
carbons of deoxyribose linked to
 Macromolecules made up of
Hydrogen.
nucleotide bonded together by the
 OH on the 3rd carbon is important in phosphate and OH group.
forming a phosphodiester bond.
st
 Chain grows by attachment of th 5’
1 carbon: nitrogenous bases
phosphate group to the 3’ hydroxyl
2nd carbon: hydrogen
group of the last nucleotide on the
3rd carbon: OH group
growing chain ( Polarity)
4th carbon: 5th carbon
 DNA orientation: 5’ to 3’
5th carbon: Phosphate group.
DNA
SUGAR PHOSPHATE BACKBONES
 Mostly double stranded
 Arranged at specific distance from
 Sugar phosphate backbone is
one another in the double helix.
oriented in spiral/helix around the
 2 regions in the helix by the
nitrogen bases
backbones: major groove and minor
 Antiparallel orientation: 2
groove.
strands exist in opposite 5’ to 3’
orientation, held together by the H
bond between their respective bases.

TOPIC 2: DNA: STRUCTURE, REPLICATION, ENZYMES, RECOMBINATION AND PLASMIDS
 Hybridization: Formation of the  Discontinuous synthesis
H bonds between 2 produces Okazaki fragments on a 5’
complementary strands of DNA to 3’ template.
Helicase: Untangles DNA by cutting and

DNA REPLICATION ligating one or both strands of the double
helix.
 Process by which DNA makes a
copy of itself during cell division. Primase: Produces short
complementary RNAs to prime DNA
Semiconservative: synthesis
 Commonly found replication in
DNA. DNA polymerase: Builds a new duplex
 1 complete old and 1 complete new DNA strand by adding nucleotides in the 5’
 Key to maintaining the sequence of to 3’ direction; performs proof - reading
the nucleotide in DNA through new and error correction; many types each of
generation. which performs different functions in
different types of cells.
Matthew Meselson and Franklin Stahl
 Demonstrated the mechanism Ligase: Catalyzes the formation of a
semiconservative replication using phosphodiester bond between nucleotides on
the technique of equilibrium density 1 strand of a double stranded DNA
centrifugation on cesium gradient. molecule.
Process of DNA replication Prokaryotes

 Helicase binds to origin and  It is continuous process
separate strands  Circular, double stranded DNa
 Binding proteins keep the  Occurs in cytoplasm
strands apart  Single origin of replication
 Primase makes a short stretch of  Small amount of DNa
RNA on the DNA template
 DNA polymerase I and III are
 DNA polymerase adds DNA involved
nucleotides to the RNA primer
 Large okazaki fragment
 DNA polymerase proofreading
 The process is rapid, 200 base
activity checks and replace incorrect
pair per second
base.
 Continuous Strands
Eukaryotes
synthesizes continuous in a 5’ to 3’
 This process occurs in the S-
direction.
phase of cell cycle

 Linear, double stranded with end Catalytic domain of E. coli
 Occurs in the nucleus
 Multiple origins of replication DNA pol I
 DNA is 50 times more than  Large fragment (Klenow
prokaryotic DNA fragm,ent)
 DNA polymerase alpha, delta  Polymerizing activity
and epsilon  Small fragment: exonuclease
 Small okazaki fragment function
 The process is slow, 100 base
pair per second Nick Translation
 Addition of labeled nucleotides at
nick (single stranded breaks) in DNa
POLYMERASE  During DNA replication, E. coli
DNA Pol III can synthesize and
degrade DNA simultaneously
DNA Polymerase I (Pol I)  Often used In vitro as a method to
 1st purified enzyme shown to introduce labeled nucleotides into
catalyze DNA replication in DNA molecules, which will be used
prokaryotes. for DNA detection in hybridization
 Followed by DNA Polymerase II and and analysis.
III.
 Polymerization: addition of Pol alpha
nucleotides.  Loc: Nucleus
 Function: Most active, identified
DNA Polymerase III (Pol III) with chromosome replication
 Main polymerizing enzyme during Pol beta
bacterial replication.  Loc: Nucleus
 Functions as a multi subunit  Function: Associated with DNA
Holoenzyme repair
Function Pol gamma
 Polymerization  Loc: Mitochondria
 Pyrophospholysis and  Function: Replication of
pyrophosphate exchange Mitochondrial DNA
 Proof reading Pol delta
 Exonuclease function  Loc: Bone marrow
 3’to5’ exonuclease Activity;
associated with DNA repair

Terminal Transferase ENZYMES THAT METABOLIZES DNA
 Type of DNA polymerase that can
synthesize polynucleotide chains DNA Metabolism: The process by
without template which cellular DNA is maintained.
 By adding nucleotide to the end of a
DNA strand in the absence of H Restriction Enzyme
base pairing with a template.  Endonuclease hat recognize specific
base sequence at the sugar phosphate
Polymerase backbone
 Play a central role in modern  Originally isolated from bacteria, as
biotechnology a part of a primitive immune system
 Cloning and some amplification and  Named from organism in which they
sequencing technique were isolated, for example:
 Classified into Families based on o BamHI (Bacillus
sequence structure amyloliquefaciens H)
 A and B are most useful for
biotechnological engineering Type I Restriction Enzyme
 Classified also based on  Nuclease and methylase activity in
structural similarities single enzyme.
 Bind to host specific DNA sites 4-
6 bp separated by 6-8 bp and
Polymerases are also produced containing methylated adenines.
through chemical manipulation of the
amino acid structure with Type II Restriction Enzyme
characteristics that are useful in the  Used most frequently in the
laboratory: laboratory.
 Processivity: staying with the  Do not have inherent methylation
template longer to make longer activity.
product.  Bind as simple dimers to
 Fidelity: Faithful copying of the symmetrical 4 – 8 bp DNA
template recognition sites (palindromic)
 Substrate Specificity: Affinity  Cleave DNA directly at the binding
for altered nucleotide. site, producing fragments of
predictable site.
 Subclassified by their
characteristic activity.
 Type II S Restriction
endonucleases: recognizes 5- to
7- bp nonpalindromic sequences

and alter DNA w/in 20 bases of Nucleases
the recognition site. Exonuclease
 Type II T enzymes: composed of  Degrade DNA from 3’ hydroxyl or
2 subunits, each of which 5’ phosphodiester
contains 1 catalytic site.  Used under controlled conditions to
 Type II M restriction enzymes: manipulate DNA invitro
cut methylated DNA  Different substitute requirements and
will therefore degrade specific type
Type III restriction enzyme of DNA ends.
 Resemble type I enzymes in their  Exonuclease I E. Coli: degrades
ability to both methylate and restrict single stranded DNA (ssDNA) from
DNA. the 3’ hydroxyl end into
 Have multiple subunits, including mononucleotides.
helicase activity.  Exonuclease III from E.Coli:
 Recognition sites are removes 5’ mononucleotides from
asymmetrical and the cleavage of the the 3’ end of double strands DNA in
substrate DNA occurs 24-36 bp from the presence of Mg2+ and Mn2+
the site to the 3’ side.
 Exonucleate VII from E.Coli:
digests ssDNA from 5’ phosphate to
Type IV Restriction Enzymes
3’ hydroxyl end.
 Similar subunit structures and
 recBC nuclease (Exonuclease
enzymes requirements
V) from E. Coli:
 Cutting and methyltransferase
o ATP dependent ssDNa &
functions.
dsDNA nuclease
o Digest ssDNa from either 3’
Restriction Enzymes
hydroxyl or the 5’ phosphate
 Can be used for mapping a DNA
end
fragment.
o Endonuclease activity
 Basis for forensic identification
o Unwinds DNA double helix
using restriction fragment analysis
at high levels of ATP
of Human DNA.
 Micrococcal nuclease: Digest
ssDNA and dsDNA and RNa at AT
DNA Ligase
OR AU rich regions
 Catalyzes the formation of
 Deoxyribonuclease I (DNAse I)
phosphodiester bond between
from bovine pancreas: Digest ssDNa
adjacent 3’ hydrogen and 5’
and dsDNA and RNa at pyrimidines
phosphodiester nucleotide ends.
to oligonucleotides
 Discovered in 5 different laboratories
in 1967.

Helicase:
Recombination in
 Series of enzymes that cut and re-
Sexually Reproducing
close the DNA sugar – phosphate
backbone. Mendel’s Law
 2 types: Topoisomerase and Gyrase  Each generation of sexually
 Topoisomerase: interconvert reproducing organisms is a new
topological isomers or relax combination of this parental
supertwisted DNA genomes – generates genetic
 Gyrase: untangles DNA through diversity
double – strand breaks and separate  Analysis of peas (Pisum sativum)
linked rings of DNA – traits are inherited in a particular
manner.
Methyltransferases
 Catalyze the addition of methyl 3 ways:
groups to nitrogen bases, usually  Beginning with meiosis: duplicated
adenines and cytosine in DNA chromosomes line up and recombine
strands. by crossing over or breakage and
 Most prokaryotic DNA is methylated reunion of the 4 DNA duplexes
or hemimethylated  Recombined duplexes are randomly
 Eukaryotic DNa is methylated in assorted into gametes
specific regions.  Gametes will merge with the gamete
from other parent carrying its own
RECOMBINATION set of recombined chromosomes.
 Resulting offspring will contain a
 It is the mixture and assembly of new set or recombination of the
new genetic combinations. genes of both parents.
 Occurs through the molecular
process of crossing over or physical  Movement and inmanipulation of
Recombination
exchange between molecules. genes inAsexual
the laboratory began with
 The one that holds a new the study of natural recombination in
combination of DNA sequences is asexually reproducing bacteria.
called the recombinant molecule
or organism.

 3 ways:Conjugation, Transduction
Transduction and Transformation  Transfer of genetic information from
1 cell to another through a viral
Conjugation: intermediate
 Transfer of genetic information  1960s, studied by Francis Jacob and
by physical association of cells Elie Wollman. Viral intermediate:
 2 types of participating bacteria: Bacteriophage (Bacterial Viruses)
F+ and F-  1952: Alfred Hershey and
 Conjugating cells must be in Martha Chase: confirmed that the
physical contact with each other DNA of the bacterial virus was the
for successful transfer of the F+ carrier of its genetic determination in
phenotype the transduction process.
 J. Lederberg and William
Hayes: demonstrated polarity in Transformation:
the process of conjugation  Transfer of genetic information
– genetic information could among cells without physical
move from F+ to F- bacteria association, such that a new
 F+ bacteria had a fertility phenotype is produced in the
factor: Carried information recipient cells.
from 1 cell to another. They are  A bacterium takes up piece of DNA
responsible for establishing floating in its environment (often
physical connection between the DNA that has been shed by other
mating bacteria. bacteria)
 F factor: Extrachromosomal  1st observed by Frederick Griffith:
circle dsDNA or plasmid o Investigative virulence in
carrying the genes coding for Diplococcus
construction of the mating o 2 strains of bacteria: rough
bridge. After mating: both type ( a virulent) and smooth
bacteria are F+ type (virulent) – to develop a
 High Frequency vaccine against pneumonia.
recombination (Hfr) o Griffiths conclusion:
bacteria: something from the dead
o Strains with smooth type bacteria had
chromosomally transformed the rough type
embedded F factors. bacteria into virulent smooth
o Used in first mapping type bacteria
studies.

o Griffith observation: o Occur in small numbers (1 or
Transfer of DNA from 1 2 copies per chromosome
organism to another without equivalent)
the protection of a
conjugative bridge or viral  Small Plasmid
coat. o more numerous in the cell
 1994: Oswald T. Avery, Collin (20 copies per
MacLeod, & M.J. MacCarthy: chromosome)
o Identified the transforming o Do not carry genes directing
material as DNA their
o Prepared boild virulent maintainance
bacterial cell lysates and o Rely on high numbers for
sequentially treated with distribution into daughter
them with enzymes. cells at cell division or uptake
o Conclusion: transforming by host cells in
factor was DNA transformation.
PLASMIDS Resistance Transfer Factors (RTF)

 Class of plasmids carrying genes for
 These are small, usually
inactivation/ circumvention of
circular,dsDNAs, often carrying antibiotic action
genetic information, that replicates
 Promote resistance to common
independently or in synchrony with
antibiotics: chloramphenicol,
the host cell replication.
tetracycline, ampicillin and
 Most are 2,000 to 100,000 (2-100 streptomycin
kilobase pairing) in size
 Acquisition of the resistance genes
 Can carry limited amount of genetic
from the host to unknown bacteria
information
 DNA Duplex is compacted, or Colicinogenic Factor
supercoiled and can be relaxed by
 Class of plasmids carrying resistance
nicking or by local unwinding of the
to bacteriocins (toxic proteins
double helix.
manufactured by bacteria)
Initial Classification:
 Large plasmids
o Include F factor and some of
the R plasmids
o Carry genes for their own
transfer and propagation and
self – transmissible

PRELIM LEC TOPIC 3-RNA: TRANSCRIPTION, TYPES, POLYMERASES, ENZYMES AND REGULATION OF
RIBONUCLEIC ACID (RNA): STRUCTURE  Catalyzed by RNA polymerase, occurs
 is a macromolecule of: mostly in interphase
1. Carbon  In most prokaryotes: single type
2. Nitrogen  Eukaryotes: RNA polymerase pol I,
3. Oxygen pol II, and pol III
4. Phosphorus
5. Hydrogen
 Is a polymer of nucleotides similar to DNA
1. Phosphate group
2. Ribose sugar
3. Nitrogen base
 Nitrogen bases:
 Pyrimidines: uracil (instead of
thymine) and cytosine
 Purines: adenine and guanine
 Synthesized as single strand rather than as
a double helix
 Some fold and loop upon themselves to
take on a double-stranded character that
is important for their function
 Can pair with complementary single
strands of DNA or another RNA and
form a double helix
 Complementary base pairs: A:U &
C:G
 Types:
 Ribosomal RNA (rRNA)
 Transfer RNA (tRNA)
 Messenger RNA (mRNA)
 Small nuclear RNAs
 Copied, or transcribed from DNA
 Exception: 1 family of RNA viruses
(retroviruses) -> RNA to DNA
TRANSCRIPTION
Process:
 Copying of 1 strand of DNA into RNA
1. Initiation: Beginning of transcription, when
 mRNA carries the information in DNA to
RNA polymerase and supporting proteins
the ribosomes, where it is translated
bind to the promoter.
DNA can only store information. How will this 2. Elongation: Addition of nucleotides to
information be used? the mRNA strand
3. Termination: Ending of transcription,
GENE EXPRESSION: It is the production of RNA occurs when RNA polymerase crosses a
and protein using a DNA template. stop (termination) sequence gene
Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-
23
Setting the stage for the transcription to begin: Transcription Termination
DNA must be released locally from histones Prokaryotes:
and the helix unwound, involve the participation
of:  Some are responsive to protein
products; high levels of a gene product
 DNA-binding proteins induce termination of its own synthesis
 Transcription factors  In some genes, by interactions between
 Histone-modification RNA polymerase & nucleotide signals in
 RNA polymerase the DNA template
 In other genes, additional factor (rho) is
Transcription Initiation required
 RNA polymerase and its supporting  Rho-independent termination occurs at
accessory proteins assemble on DNA at a G:C-rich regions in the DNA & the G:C
specific site (promoter) bases are transcribed into RNA & fold into
 Prokaryotes: Basal transcription a short double-stranded hairpin;
complex = assembly of large and elongation complex dissociates as it
small subunits of RNA polymerase reached A:T-rich area
and additional sigma factors at Eukaryotes
the promoter
 Eukaryotes: Transcription complex  Transcription catalyzed by pol I:
= assembly of RNA polymerase terminated transcription just prior to Sal
and up to 20 additional factors for box (site in the DNA) with the cooperation
accurate initiation of a termination factor (TTF1)
 Transcription catalyzed by pol II:
Transcription Elongation
termination will be activated once the
 Both prokaryotes and eukaryotes: RNA polyadenylation signal (polyA site) along
polymerases synthesize RNA using the the DNA template will be encountered
base sequence of 1 strand of the double  Transcription catalyzes by pol III:
helix as a guide termination signal is a run of adenine
 RNA polymerases (50-100 bases/sec) residues in the template, requires a
work more slowly than DNA polymerases termination factor
(1,000 bases/sec) and with less fidelity Types of RNAs Found in the Cell
 RNA synthesis does not require priming
 1st ribonucleoside triphosphate retains all 1. Ribosomal RNA (rRNA)
of its phosphate groups  Largest component of cellular RNA (80%-
 Subsequent ribonucleoside triphosphates 90%)
retain only the alpha phosphate and the  Various types are named for their
other 2 (beta & gamma phosphates) are sedimentation coefficient (S) in density
released as orthophosphates during the gradient centrifugation
synthesis reaction
 Important in structural and functional
part of the ribosomes

23
Prokaryotes  Undergoes a series of post-transcriptional
processing events before it is translated
 16S rRNA: found in the ribosome small
into protein (mRNA processing)
subunit
 23S rRNA & 5S rRNA: found in the Amount of a particular mRNA in a cell is related
ribosome large subunit to the requirements for its final product
All are synthesized from the same gene.  Constitutive transcription: messages are
transcribed constantly and are relatively
Eukaryotes
abundant in the cell
 Synthesized from highly repeated gene  Inducible/regulatory transcription:
clusters messages are transcribes only at certain
 Copied from DNA as a single 45S times during the cell cycle/under particular
precursor RNA (pre-ribosomal RNA) -> conditions
18S species of the ribosome small
Messenger RNA (mRNA) Processing
subunit & 5.8S & 28S species of the large
subunit In eukaryotes, the newly made RNA (primary
 5S species: found in the large ribosome transcript/ pre-mRNA) is further processed before
subunit, synthesized separately it is functional:
2. Messenger RNA (mRNA) 1. Polyadenylation
2. Capping
 Initial connection between the information 3. Splicing
stored in DNA and the translation
apparatus that will ultimately produce Polyadenylation
the protein products responsible for the
 Addition of adenosines to the 3’ end of
phenotype
mRNA
Prokaryotes  Most mRNAs carry a sequence of
polyadenylic acid at the 3’ terminus (polyA
 Synthesized and simultaneously translated tail), which will be added to the RNA
into protein after synthesis of the pre-mRNA
 Sometimes polycistronic (1 mRNA codes  Polyadenylate polymerase is the enzyme
for more than 1 protein) responsible for adding the adenines to the
Eukaryotes end of the subscript
 mRNA mammalian cells: a run of up to
 Monocistronic (having only 1 protein per 200 nucleotides of polyA
mRNA)
 Can produce different proteins from the Capping
same DNA sequences by:  Cap
 starting the RNA synthesis in  A structure (5’-5’ pyrophosphate
different places or linkage of 7-methyl guanosine to
 by processing the mRNA differently either 2’ O-methyl adenine of the
 Copying of RNA from DNA & protein mRNA) that blocks the eukaryotic
synthesis from RNA are separated by the mRNA at the 5’ terminus
nuclear membrane barrier

23
 Confers a protective function & Why are eukaryotic genes interrupted by introns?
serves as a recognition signal for
1. Theory: Introns evolved as a means of
the translational apparatus
increasing recombination frequency within genes
Splicing and between genes without breaking coding
 Removal of intron sequences from Mrna sequences.

 Introns: noncoding (intervening) 2. May protect the coding regions from genetic
sequences, does not code for amino acids damage.
 Exons: remaining sequences that code for
the protein product  Alternative splicing
 Heteronuclear RNA (hnRNA)  Removal of introns from RNA using
 Newly transcribed mRNA, much different breakpoints
larger than mature RNA because it  Exons from the same gene are
contains introns joined in different combinations,
 Capped and tailed, transitioning leading to different, but related,
from hnRNA to mRNA, removing mRNA transcripts
introns from hnRNA  Different forms of mRNA
(transcript variants/splice
Splicing Mechanism: variants/isoforms)
1. Self-Splicing RNAs  Modifies products of gene by
 Special sequences of RNA that are alternate insertion of different
able to splice themselves exons
2. Spliceosome  Has been found in over 40 different
 Nuclear macromolecule complex genes
within which splicing reactions  Some of the diseases resulting from
occur to remove introns from pre- abnormalities in splicing process:
mRNAs  Some ß-thalassemias: mutations in
the splice recognition sequences of
the ß-globin genes
 Certain autoimmune conditions:
MECHANISM OF production of antibodies to RNA-
TYPES OF
LOCATION REMOVAL
INTRONS protein complexes
FROM hnRNA
Nuclear, 3. Small Nuclear RNA (snRNA)
mitochondrial,
GROUP 1 Self-splicing
& chloroplast  Functions in splicing eukaryotes, allowing
genes for precise alignment and correct excision
Mitochondrial of introns
GROUP 2 & chloroplast Self-splicing  Stays in the nucleus after its
genes transcription by RNA polymerase I or III
NUCLEAR Nuclear genes Spliceosome
tRNA splicing
tRNA Nuclear tRNA
endonuclease

23
PRELIM LEC TOPIC 3-RNA: TRANSCRIPTION, TYPES, POLYMERASES, ENZYMES AND REGULATION OF TRANCRIPTION
 T𝛙C loop
 (𝛙 stands for the modified
pseudouridine), seven-base loop,
contains the sequence 5’-TWCG-3’
 Special recognition site for the
ribosome to allow a tRNA-ribosome
complex to form during the process
of protein synthesis
 Variable loop
 Larger in longer tRNAs, helps in
recognition of the tRNA molecule
 Anticodon loop
 Seven-base loop, contains the
anticodon
 D loop
4. Transfer RNA (tRNA)  8- to 12-base loop, relatively rich in
dihydrouridine (modified
 Adaptor molecules during the translation nucleotide), plays an important role
process
in stabilizing RNA structure
 Relatively short, single-stranded
polynucleotides of 73-93 bases in HISTORICAL HIGHLIGHT
length, MW 24,000-31,000
Robert Holley & colleagues at Cornell University
 At least 1 tRNA for each amino acid
 CCA at the 3’ end: amino acid will be In 1964, solved the 1st tRNA sequence (alanine
covalently attached to tRNA tRNA of yeast = 76 bases long & 10 of these are
 Acceptor arm: the end of tRNA to which an modified)
amino acid becomes bound 6. Other RNAs
 tRNA loops:
 T𝛙C loop  Late 1900s, increasing varieties of RNA
 Anticodon loop species have been describes
 Variable loop  Functions:
 D loop  RNA synthesis & processing

23
 Influence numerous cellular RNA viruses
processes:
1. Plasmid replication  RNA-dependent RNA polymerases
2. Bacteriophage development  Hepatitis C virus, Zika virus, &
3. Chromosome structure and Dengue Virus: to replicate their
development RNA genomes
 Also found in plants & lower
RNA POLYMERASES eukaryotes: associated with gene
 Catalyze the RNA synthesis silencing
 Prokaryotes
PolyA polymerase
 1 multisubunit enzyme for all
types of RNA  Template-independent RNA polymerase
 Bacterial RNA polymerase: 5  Adds adenine nucleotides to the 3’ end of mRNA
subunits (2 α & 1 of each ß, ß’, &  Result: polyA tail, important for mRNA stability &
𝜎) translation into protein
What is RNA metabolism?
It refers to any events in the life cycle of RNA
molecules
 Transcription
 Folding/unfolding
 Modification
 Processing
 Degradation
OTHER RNA-METABOLIZING ENZYME
1. Ribonucleases
 Eukaryotes
 3 multisubunit RNA polymerases  Ubiquitous, stable enzymes that degrade
(all are DNA-dependent all types of RNA
polymerases)
1. RNA polymerase I
2. RNA polymerase II
3. RNA polymerase III
 Single-subunit mitochondrial RNA
polymerase imported to organelles

23
2. RNA helicases Structural Activity of the
Product
genes gene product
 Required in RNA synthesis and processing
Hydrolyzes
to catalyze the unwinding of dsRNA
lactose into
 Have been characterized in prokaryotes & lacZ B-galactosidase
glucose &
eukaryotes galactose
 Some work exclusively on RNA, others on Transports
Lactose
DNA (RNA heteroduplexes & DNA lacY lactose into
permease
substrates) the cell
 Involved in removal of proteins from RNA- Thiogalactosidase Transacetylates
lacA transacetylase galactosides
protein complexes
GENE EXPRESSION
Lac operon in the absence of lactose
 Production of RNA and protein using a
DNA template  Repressor protein binds to the operator
sequence & prevents transcription of the
 Key determinant of phenotype
operon
 Some genes (products are in continual use
 Lactose binds to repressor protein &
by the cell), gene expression is constant
changes its conformation & lowers its
(constitutive)
affinity to bind the operator sequence,
 Other genes, gene expression is tightly
resulting in the expression of the operon
regulated throughout the life of the cell
 Most immediate & well-studied level of
Modes of
control gene expression: transcription
regulation in Description Example
initiation prokaryotes
REGULATION OF mRNA SYNTHESIS AT INITIATION Inducer
prevents the
2 factors responsible repressor
Enzyme Found in lac
from
1. Cis factors: DNA sequences that mark induction operon
binding the
places on DNA involved in the initiation operator to
& control of RNA synthesis turn on
2. Trans factors: Proteins that bind to the cis expression
sequences & direct the assembly of Corepressor
transcription complexes at the proper must bind to
gene Enzyme a repressor in Found in
repression order to turn arg
Operon off operon
transcription
 Series of structural genes transcribed
Activator
together on 1 mRNA & subsequently
binds
separated into individual proteins with RNA Found in mal
 Example: lactose (lac) operon in Activation
polymerase to operon
Escherichia coli (metabolism of lactose) turn on

23
PRELIM LEC TOPIC 3-RNA: TRANSCRIPTION, TYPES, POLYMERASES, ENZYMES AND REGULATION OF TRANCRIPTION
Attenuation  RNA transcription &
translation are
 Another mechanism of control in bacteria concurrent, protecting the
 Formation of stems & loops in the RNA RNA from the processing
transcript by intrastrand H bonding of & exogenous factors
complementary bases (allow/prevent  RNA stability is affected
transcription) by secondary structure
Prokaryotes
(folding of the RNA
General arrangement of cis factors: molecule) &
 Usually 4-20 base pairs in length polyadenylation of 3’
end of the transcripts
 Some are inverted repeats with the
 Codon usage & cofactor
capacity to form a cruciform structure in
availability may alter
the DNA duplex recognizable by specific translation speed
proteins
Location of cis regulatory
Organisms elements
Close to the gene (proximal
Prokaryotes
elements only)
Thousands of base pairs
away from the genes they
control (distal elements,
Eukaryotes examples include enhancers
& silencers);
In or around the genes
they control (proximal
elements)
POST-TRANSCRIPTIONAL REGULATION
Several factors affecting the stability of the RNA
transcript:
 RNA sequence structure

 Presence of exonucleases &
endonucleases that digest the RNA
Organisms Post-Transcriptional Regulation

23
 Enzymatic & structural
alterations in RNA
interfere with its
processing
 Alternate splicing in
Eukaryotes combination with protein
factors responsible for
many tissue- &
development-specific
gene-expression patterns
(hematopoiesis)

23
PRELIM LEC TOPIC 4-PROTEINS: AMINO ACIDS, GENES, GENETIC CODE AND TRANSLATION
PROTEINS
 Products of transcription & translation of

nucleic acids
 Polymers of amino acid
 Manifest the phenotype directed by the
nucleic acids
AMINO ACIDS
 Monomers of proteins
 Structure:
a. Carboxyl group (COO-)
b. Amino group (NH2 group bonded to
C atom)
c. R (radical) group/side chains
d. Central C atom
 Side chains (R groups) of the 20 amino
acids
 Charged R groups
 Lysine
 Arginine
 Aspartic acid
 Glutamic acid
 Histidine
 Polar R groups
 Serine
 Threonine
 Cysteine
 Their properties that make up a protein
 Asparagine
determine the shape & biochemical nature
 Glutamine
of the protein
 Proline
 Single protein -> Separate domains with
 Nonpolar R groups
different properties
 Glycine
 Example: Transmembrane proteins
 Alanine
 Valine
 Methionine
 Leucine
 Isoleucine
 Aromatic R groups
 Phenylalanine
 Tyrosine
 Tryptophan
Transcribed by: JANROSE R. TERRAZULA, BMLS

2F
PRELIM LEC TOPIC 4-PROTEINS: AMINO ACIDS, GENES, GENETIC CODE AND
 Peptide bonds: covalent C-N bonds that
connect amino acids in proteins
 Peptide: polymer of few amino acids
 Peptides with additional units are
di-, tri-, tetra-, pentapeptides, &
so forth (depending on how many
units are attached to each other)
 1 end: amino group (amino-
terminal / NH2 end)
 Opposite terminus: carboxyl group
(carboxyl-terminal / COOH end)
 Peptide chain grows from the
amino to the carboxy terminus
PROTEINS
 Polypeptides of up to more than

a thousand of amino acids in length
 Information in the DNA -> transcribed ->
translated = genetically coded
phenotype
 Constitute the most abundant
macromolecules in cells
 Proteome: collection of proteins encoded
in all of an organism’s DNA
 Larger than genome (10x)
Take a look at this different kind of wedding Structure of Proteins

proposal: 1. Primary Structure
The couple are licensed chemists working as  Sequence of amino acids in proteins which
science researchers at UP Diliman.
determines the nature and activity of that
The boyfriend made the proposal last July 22, protein
2021 by making her girlfriend run a synthetic
peptide sample at work which later on resulted to
as synthesized “MARRYME” peptide.
 pH 7: most of the carboxyl groups are

ionized & amino groups are not
 ionization can switch between amino &
carboxyl groups -> amino acids
zwitterions at physiological pH
 pK values: amino acids become completely
+/- charged at certain pH levels
 pI values: pH where amino acids are
neutral

2F
2. Secondary Structure CLASSIFICATION OF PROTEINS BASED ON
COMPOSITION
 Local interactions between amino acid
side chains PROTEINS COMPOSITION EXAMPLES
 Ordered beta / beta-pleated sheets and Albumins,
Simple
less-ordered alpha helices, or random Only amino acids globulins,
proteins
coils histones
Lipoproteins
 First described by Linus Pauling and
(low density
Robert Corey Protein and
lipoproteins),
nonprotein
Conjugated glycoproteins
component
proteins (mucin),
(prosthetic
metalloproteins
groups)
(ferritin &
hemoglobin)
Simple/conjugated
proteins having
Derived been partially Proteases,
proteins hydrolyzed by fibrin
acids, enzymes, or
alkalis
3. Tertiary Structure
CLASSIFICATION OF PROTEINS BASED ON
 Further folding of secondary structures STRUCTURE
of proteins
4. QUATERNARY STRUCTURE
 Protein structure consisting of more than

one polypeptide

2F
PROTEINS STRUCTURE FUNCTIONS EXAMPLE
Enzyme,
messenger,
Globular transporters,
Spherical Hemoglobin
proteins regulators, &
sometimes
structural
Protection,
Fibrous Sheet-like
structural Collagen
proteins filamentous
role
Intermediate
Intermediate Blood
to fibrous to Fibrinogen
proteins clotting
globular

2F
CLASSIFICATION OF PROTEINS BASED ON The Genetic Code
FUNCTIONS
 Gene’s nature was further clarified
PROTEINS FUNCTION EXAMPLE  Deciphered by Francis Crick, Marshall
Act as Nirenberg, Philip Leder, Gobind Khorana,
Enzymes Nucleases
catalysts & Sydney Brenner
Transport
Channel  A dictionary to translate the 4-nucleotide
substances
Transport proteins sequence information in DNA to the 20-
across
Proteins (sodium ion amino acid sequence information in
biological
channels) proteins
membranes
Reserves of  1965:
Storage metal ions  3-nucleotide code: 43 = 64 triplets
Ferritin
Proteins and amino were assigned to amino acids
acids  Redundant (all, except 2 methionine &
Motility Generate tryptophan, have more than 1 codon)
Myosin
Proteins movement
 Wobble in the 3rd position
Structural Maintain cell
Collagen  Triplets coding for the same
Proteins shape
Protection amino acid often differing in the
Defensive against 3rd base of the triplet
Antibodies  Nonsense codons
Proteins harmful
agents  Terminate protein synthesis: UAG,
Regulate UAA, UGA
Regulatory different
Enzymes TRANSLATION
Proteins processes
and activities Amino Acid Charging
 Transcription -> Translation -> Phenotype
GENES & THE GENETIC CODE
 Adaptor hypothesis: “There must be a
Genes molecular factor that can recognize
components of both nucleic acid & protein
 Ordered sequence of nucleotides on a
sequences.” -> tRNA (over 50 in humans
chromosome that encodes a specific
& 40 in bacteria)
functional protein
 Fundamental physical & functional unit Start of Protein Synthesis
of inheritance
 tRNA charging: activation of the amino
 1st studied by tracking mutations acids by covalent attachment to tRNA &
 Composition: catalyzed by 20 aminoacyl tRNA
 Structural sequences synthetases
 Regulatory sequences (Ex:  Classes of aminoacyl tRNA synthetases:
promoter) 1. Class I: act on the 2’ OH of the tRNA
acceptor stem
2. Class II act on the 3’ OH

2F
Ribosomes Initiation
 Site of protein synthesis  Beginning of translation, when the small

1. Prokaryotes: 70S ribosomes ribosome subunit assembles with mRNA &
 50S large subunit (1.8 million then the large ribosomal subunit
1. Small ribosomal subunit first binds to initiation
daltons) = 5S rRNA + 23S rRNA
factor 3 (IF-3) & then to specific sequences
+ 34 ribosomal proteins near the 5’ end of mRNA (ribosomal binding
 30S small subunit (1 million site)
daltons) = 16S rRNA + 21 2. Initiation factor 2 (IF-2) bound to GTP &
ribosomal proteins initiating tRNAMet (eukaryotes) / tRNAfMet
 mRNA & initiating factors (bacteria, mitochondria, & chloroplasts), then
joins the complex
3. Large ribosomal subunit associated with
hydrolysis of GTP resulting to formation of the
initial complex
 tRNAMet (eukaryotes) / tRNAfMet (bacteria,
mitochondria, & chloroplasts) is situated & can
only bind to the peptidyl site (P site)
 All other tRNAs bind to aminoacyl site (A site)
of the ribosome
Elongation
 Binding of charged tRNAs & formation of the

peptide bond producing growing polypeptide
1. tRNA carrying the next amino acid binds to the
A site in a complex
2. 1st formation of peptide bond between amino
2. Eukaryotes: 80S ribosomes
acids in A& P sites by transfer of the 1st
 60S large subunit (2.7 million amino acid to the next, generating a
daltons) = 5S rRNA + 5.8S rRNA dipeptidyl-tRNA in A site (catalyzed by
+ 28S rRNA + 40 ribosomal peptidyl transferase)
proteins 3. Dipeptidyl-tRNA shifts from A site to P site
 40S small subunit (1.3 million (translocation) with the release of the “empty
daltons) = 18S rRNA + 30 tRNA" from the E site
ribosomal proteins  During translation, the growing polypeptide
begins to fold into its mature conformation
 Assisted by molecular chaperones
Termination
 Ending of translation occurs when the complex

encounters a nonsense codon
 Signaled by 1 of the 3
stop/nonsense/termination codons: UAA, UAG,
or UGA
 Termination/release factors = hydrolysis of the
finished polypeptide from the final tRNA ->
release of the final tRNA from the ribosome ->
dissociation of large & small ribosomal

2F
subunits

2F
MOLECULAR BIOLOGY AND
DIAGNOSTICS PRELIM LEC TOPIC 5-
What is EPIGENETICS?  Histones:
 Structural proteins, also regulate
 1942 – Conrad Weddington: It is a
access of trans factors & RNA
developmental phenomenon that allowed
polymerase to the DNA helix
cells to take on different phenotypes
without changes in the genetic structure Histone Modification
 1958 – David Nanney: It is used to
emphasize the reliance of the auxiliary  Affects the activity of chromatin-associated
system on the genetic information proteins & transcription factors that
 Subsequent years – Defined as mitotically increase / decrease gene expression
& meiotically heritable changes in  Types of histone modification
phenotype not encoded in genotype 1. Methylation
 Current study – It involves chemical & 2. Phosphorylation
structural modifications in chromatin & the 3. Ubiquitination
activity of particular noncoding RNAs 4. Acetylation
Examples of Epigenetic Phenomena Where does modifications occur?
1. Histone modification Specific amino acids on amino terminal &

carboxyterminal ends
 Key epigenetic regulators that control
chromatin structure & gene transcription, “Histone Code”
impacting on various important cellular Correlating specific histone modifications with
phenotypes biological effects
2. Nucleic acid methylation
 Regulates gene expression by recruiting

proteins involved in gene repression or by
inhibiting the binding of transcription
factor(s) to DNA
3. Noncoding RNA
 Can regulate expression at the level of the

gene and the level of chromosome to
control cell differentiation
1. HISTONE MODIFICATION
Chromatin
 Nuclear DNA & its associated proteins

 Eukaryotes: chromosomal DNA
is compacted into nucleosomes
 Nucleosomes:
1. 150 bases of DNA
2. Complex of 8 histones proteins (2
histone 2A, 2 histone 2B, 2 histone 3,
& 2 histone 4)
23
Nomenclature for Core Histone (H2A, H2B, H3, 2. NUCLEIC ACID METHYLATION
H4) Modifications
a. DNA Methylation
 Name of histone followed by amino acid &
 Involves the attachment of small chemical
its position in the protein using single-
groups (methyl groups -CH3) to DNA
letter code, followed by the modification
building blocks
 Examples:
1. Acetylation of lysine at  Another type of epigenetic regulation of
position 27 of histone H3 = gene expression (eukaryotes &
H3K27Ac prokaryotes)
2. Methylation of lysine at position  Vertebrates: occurs in CG-rich sequences
20 of histone H2B = in the DNA (CpG islands)
H2BK20Me  Human genomes: over 45,000 CpG
islands
 Aberrant DNA methylation at the CpG
islands is a source of dysregulation of
genes in disease states
 Methylation of cytosine residues
in the promoter regions of tumor-
suppressor genes is a mechanism
of inactivation of these genes in
cancer
ENZYMES THAT METHYLATE DNA IN

MAMMALS
Maintenance enzyme
& mainly methylates
DNA
hemimethylated DNA
methyltransferase 1
(cytosines that are
(DNMT1)
methylated on
1 strand only)
DNA De novo methylase &
methyltransferase methylate
3a/3b (DNMT3) unmethylated DNA
Likely nonfunctional
as a methylase
Involvement of Histone Modification with Gene because it lacks a
Expression functional catalytic
DNA
site; may regulate
methyltransferase 3L
 Can induce/repress gene expression DNMT3A & 3B
(DNMT3L)
 Has led to the study of aberrations in these activities by
occupying DNMT3A &
modifications in disease states (viral
3B protein / DNA-
infections & neoplastic cells) binding sites
 Therapeutic agents (histone deacetylase May be involved in
inhibitors) are currently in use for some DNA
modifying bases in
hematological malignancies methyltransferase 2
tRNA or other

23
Genomic Imprinting CLASSIFICATION OF EPIGENETIC FACTORS
 Only one copy of a gene in an individual Writers Methyltransferases, phosphorylases,

(either from mother or father) is expressed, acetylases, & other enzymes that
while the other copy is suppressed modify
 Regulated by DNA methylation: Maintains histones, RNA, & DNA
Erasers Deacetylases, TET enzymes, & enzymes
the balanced expression of genes in
that reverse the modification of
growth & embryonic development by histones, RNA, & DNA
selective methylation of homologous Readers Transcription factors, methyl-group-
genes binding proteins & others that
 Examples: Mules (progeny of female recognize the state of chromatin & act
horse & male donkey) & hinnies accordingly
(progeny of male horse & female
donkey)
CLASSIFICATION OF EPIGENETIC FACTORS (IN
 Genetic diseases in humans:
CANCER CELLS)
 Angelman syndrome: both copies
of a section of chromosome 15 is Modulators Activities that activate the epigenetic
inherited from the father process; includes genetic factors
 Prader-Willi syndrome: from the (IDH1/2 & the KRAS & TP53 cancer
mother genes)
Modifiers Writers & erasers of the previous
b. DNA Demethylation system that change / maintain the
chromatin structure
 Process of removal of methyl group Mediators Those genes whose products are the
 Passive DNA methylation: takes place in targets of the modifiers
the absence of methylation of newly
synthesized DNA strands by DNMT1
during several replication rounds Non-Coding RNAs
 Active DNA methylation: occurs via direct  Carry out their function as RNA & are
removal of a methyl group independently transcribed but not translated into
of DNA replication & requires action of protein
enzymes (TET enzymes)  Roles in gene regulation & even in
c. RNA Methylation information transfer across generations
 Found in oocytes & spermatozoa
 Attachment of methyl groups in RNA  Piwi RNA (piRNA): affects transposon
 More than 100 post-transcriptional transcription in germ cells
modifications in RNA  Classification based on their length:
 Methylated bases: N6- methyladenosine 1. Small non-coding RNAs (microRNAs,
(m6A) in a 3-base recognition site, GAC small interfering RNAs, etc.)
(most frequently), & AAC (less commonly) 2. Intermediate non-coding RNAs
 Enzymes involved: methyltransferase 3. Long non-coding RNAs
enzymes encoded by METTL14 &
METTL3
 Affects RNA stability, splicing, &
translation

23
1. Small Non-Coding RNAs  Ribonuclease III enzyme is responsible for its
generation as well as the microRNA, from
Bacteria – small RNAs dsRNA precursors
(sRNAs)  Not normally found in animal cells
 Frequently used in laboratory to artificially
Eukaryotes – noncoding RNAs (ncRNAs) turn off the expression of specific genes
 Regulation of Gene Expression by RNA
A. MicroRNAs (miRNAs)
Interference
 Regulatory RNAs, 17-27 nucleotides (nt) in RNA interference (RNAi)
length, derived from endogenous RNA  1st discovered in C. elegans in 1993
hairpin structures  Not present in humans
 Useful tool in the research laboratory
 Discovered in the worm Caenorhabditis to selectively eliminate expression of
elegans specific genes in vitro
 Control gene expression by pairing with  Mediated through siRNAs
imperfectly complementary sequences in  Uses small dsRNA as triggers to direct
mRNAs to inhibit translation homology-dependent control of gene
 Humans: over 5,600 miRNAs have been activity
identified C. Other small RNAs
 Eukaryotes: affects gene expression, cell
development, & defense  Late 1990s: increasing varieties of small RNAs
 Regulation of Gene Expression by (both prokaryotes & eukaryotes):
1. Tiny coding RNAs (tncRNAs, 20-22 b)
MicroRNAs
2. Small modulatory RNAs (smRNAs, 21-23
 Regulate gene expression post- b)
transcriptionally by binding to the 3. Small nucleolar RNAs (smRNAs)
3’ end of mRNA & preventing its 4. Transfer-messenger RNAs (tmRNAs)
translation 5. Guide RNAs (gRNAs)
 Hybridization between 6. Others
microRNA & mRNA
Influence the following cellular process:
prevents translation of the
target RNA & eventually 1. RNA synthesis & processing
results in degradation of 2. Plasmid replication
the mRNA 3. Bacteriophage development
4. Chromosome structure & development
 Their dysregulation is associated with a
variety of diseases, such as cancer, where 2. Long Non-Coding RNAs
they promote oncomirs (tumor-cell
phenotype)  Length: 200-100,000 bases
 Important regulators of chromatin structure,
 Molecular mimics of miRNA & molecules
affecting gene expression & disease
targeted at miRNAs (antimirs) are being processes
tested as therapeutic agents  Human genome: 15,000-57,648 lncRNAs
B. Small interfering RNAs (siRNAs)  Acts as:
1. Decoy
 Functional intermediates of RNA 2. Scaffold
interference (RNAi) 3. Guide
 Act inside the cell to reduce gene 4. Enhancer
expression
23
MOLECULAR BIOLOGY LAB NOTES Specimen: RNA
QUALITY ASSURANCE AND QUALITY CONTROL IN THE A. Blood, bone marrow, fluids
MOLECULAR LABORATORY
● <2hrs, 23C or 4C
● 5 D, 23C; 7 D, 4C in denaturant
Specimen ● 1-2 weeks, -70C in denaturant
● Check your specimen
● WBC:2-4 weeks, -20C; >6mos, -70C
● Pre- analytical (prior analysis)
B. Tissue
● Specimen:
● <2hrs, 4C
● With requisition form/ electronic test order ● Snap frozen, -70C, > 2yrs
○ SPX material (is it blood? Bone marrow? ● Nitrogen vapor -140C to -150C, > 2 yrs
Etc.)
C. Isolated RNA
○ Test ordered, date and time collected
● 2-25C (not recommended)
○ Reason and physician contact info
● <30D, -70C in depc-treated water
● Not tampered, hemolyzed, degraded or clotted
● >6mo, -70C in ethanol
● Labels identification
Solid tissue samples
Additional requirements
● Can be fresh or frozen- best
● For molecular genetics, forensics, or parentage testing
○ For southern blot or long-range PCR
○ Patient consent form
methods
○ Ethnicity
○ Can be snap frozen by liquid nitrogen
○ Photo id of individuals tested
● Fixed, paraffin embedded
○ Pxt label verification
○ Lower quality DNA and RNA
○ Transfusion history/ pedigree
○ RT-PCR and PCR
○ Chain of custody- forensics
Blood samples
Bar coding system - quicker accessions ● Acid citrate dextrose tubes- most routine
● Heparin may inhibit enzymes (RT and DP) - may still
Procedures
depend on level of resistance
● Written procedures: for handling and proper accession ● PPT (plasma preparation tube)- WBC separation
● Books and electronics records: “log books”
○ HIV and HCV
○ Date of receipt, laboratory identifier, PXT
○ Genetics, flow cytometry
info
○ Engraftment testing
○ If SPX is unacceptable, disposal/ retention
is recorded
All specimens are potentially infectious
● Handle with standard precautions
Delayed processes, aliquoting
○ PPE
● Stored in a secure, limited access area + appropriate
○ Gloves
conditions.
○ Part of SP
Aliquoting - for buccal cell suspension and CFS
○ Protects nucleic acids from nuclease
● Centrifuged
○ Required RNA
● Avoid pipetting carryover
● Transmission based precautions
● Aliquots are not returned from the original vessel.
○ Prevents airborne/contact transmissible
○ Agents
Storage
○ Respirations
Specimen: DNA
● Contact precautions
A. Blood, bone marrow, fluids
○ For direct PXT care
● Less than a day, 23C; 3 days, 4C
● WBC: more than 1 year; -20C or -70C
PRECAUTIONS
B. Tissue
Hazard
● 23C (not recommended)
● Biological hazards
● b< a day, 4C
○ Ingestion, inoculation, contaminated,
● > 2 weeks, -20C
needles aerosol inhalation
● Less than 2 years; -70C
○ Body fluids, tissues, supplies, or materials
C. Isolated DNA
■ HBV, HCV, HIV, tuberculosis
● < 26 weeks, 2-25C
Avoid
● 1-3 years. 4C (1 year for southern blot)
● Mouth pipetting
● < 7 years, -20C (not frost free)
● Consumptions of food
● Smoking
Storage
● Applying cosmetics
● Potential needlestick situation
● Leaving unprotected skin, eyes, etc.
Hazard - work area should be decontaminated on the

regular basis Reproducibility Consistency of A qualitative
test results method yields
Common decontamination agents produced from 100 positive
● Heat the same results when
● Ethylene oxide procedure performed in 10
● 2% glutaraldehyde independent
● 10% hydrogen peroxide laboratories, a
● 10% formalin reproducibility of
● 5.25% hypo chloride 100%
● Formaldehyde
● Detergents Analyte The range within A qPCR HSV
● Phenols measurement which a array yields
● Ultraviolet radiation range specimen may reproductive
● Ionizing radiation be measured reproducible
● Photo- oxidation directly (without linear results
or with dilution) from 10 to 107
Chemical hazard copies of HSV
● Stored in explosion proof cabinet/ ref units per 10 ul of CSF.
○ Xylene Specimens
○ Methanol within this range
○ Ethanol are measured
○ Isopropanol directly
● Secondary/ reinforced containers
○ Phenol Reference range Expected analyte The reference
○ Concentrated acids frequency or range for
● Radioactive chemicals are handled with absorbent levels from a prostatic specific
paper, drip trays or other protective containers population of antigen is 0-4
○ Labeled individuals. ng/Ml.
Hazards Analytic Production of Of 100

● The NFPA/ National Fire Protection ASSN has accuracy correct results specimens with
developed a series of warning labels for universal use mutations in the
on all chemical containers. HCM gene, 99
● MSDS/ Material safety data sheets are detected by
○ Gives detailed info about the material + sequencing, with
hazards associated no mutations
● Check labels detected in
● Acid to water normal
● Do not touch, taste or smell any reagents specimen.
● Use hoods, clean spills properly and promptly
● Chemical tracking system Quantitative Quantitative A graph of test
correlation correlation procedure
Commercial reagents/kits between test between test results input
result and result and actual analyte yields a
● FDA require validation of the performance of clinical
test methods and reagents in accurately detecting/ amount of straight line.
measuring analytes prior to use in human testing analyte
The following should be established by manufactured and

approved by FDA A. True positive (TP)- outcome where the model correctly
● Calibration predicts the positive class
● Validation B. True negative (TN)- outcome where the model
● QA measures correctly predicts negative class
C. False positive (FP)- outcome where the model
Test performance measurements incorrectly predicts the positive class
D. False negative (FN)- outcome where the model
Analytical test performance is assessed by several incorrectly predicts negative class
criteria:
TP and TN are determined using a gold standard/method b) Random error
reference
Instrument maintenance
The analytical sensitivity of an assay equals:
● Manufacturers supply recommendations for
TP/TP+FN(100) routine maintenance
● Laboratory maintains a schedule and instructors for
The analytical sensitivity of an assay equals: all routine maintenance
TN/TN+FN(100) ● Technologists should be aware of the limits of user-
recommended repairs/maintenance
The analytical accuracy of an assay equals: ○ Check temp settings, timers, signals bg
TN+TP/TN+TP+FN+FP(100) levels
○ Capillary, bulb, battery replacements,
Test validation cleaning
● Commercially developed and FDA approved molecular ○ And when service calls are indicated
methods are verified by using the purchase reagent sets ■ Repair/replacement
to test validation SPX in the lab ○ Document maintenance,
● If the commercial test is modified, validation is repairs/replacement, service calls,
required to show equal superior performance of the calibrations
modified procedure Instruments
● Once a procedure is established, the method is ● Thermal cyclers - thermometers with flexible probes
documented in the laboratory according to CLSI ● Centrifuge - check speed using tachometer
guidelines. (annually/6 mos)
● Adjustable and fixed-volume automatic pipettes - 6
Proficiency testing mos
● Refers to external SPX from reference source supplied ● Fridge/freezer - check temps daily, established
to independent laboratories tolerance limits
● Reference laboratories supply SPX for molecular ● Incubators and water bath - same with fridge
analysis ● Autoclaves - heat sensitive tape/biological
indicator (b. stearothermophilus)
Controls ● Analytical balance: standard (defined) weights -
● Samples of known type or amount that are treated like avoid vibrating areas
and run with PXT SPX ● Biosafety cabinets/ laminar air flow hoods -; verify air
○ Qualitative tests: positive, negative, and intake grills are not obstructed (daily), weekly
sensitivity controls cleaning.
○ Quantitative: high positive, low positive
and negative controls Calibrations
Amplification procedures: Can be:
■ Amplification control ● Previously tested measurements (delta checking)
■ Prevent false negs ● Reference standards
● Commercial calibrators
Quality assurance ● Prof testing material
● Periodic review and documentation of test results is Should cover 3 levels: low, medium, high points
required
● Molecular quantitative methods should have a defined Reagents
dynamic range, sensitivity level and accuracy Instructions for prep and quantities for each assay should be in the
● Assay levels that distinguish positive from negative written lab protocol
results (cut off values) must also be well defined and ● Sequences of primers and probes
verified regular intervals ● Primer binding sites and sizes of expected amplicons
Quality controls Analyte specific reagents (ASR)

Positive, sensitivity and negative controls should be included each ● Detects a specific target (cell surface protein or
run DNA mutation)
1) Tolerance and acceptability levels of controls Class 1- for molecular labs; Class 2 and 3- blood banks,
a) Levey Jennings plot/modified levey screening for infectious diseases.
Jennings
b) Cusum plots In- vitro diagnostic devices/ reagents (IVD)
2) Tolerance limits will be expressed by westgard rules ● For use in the DX of disease
a) Systematic errors
● Products that may use to collect, prepare, and examine
SPX
Class 1- lowest risk; Class 2- moderate risk; Class 3- higher
risk
Test results
● In form of electropherograms, gel images,
autoradiograms should be sufficiently high quality that
results are unequivocal
● Documentation of assay conditions, rgt lot, no, and
quality of the isolated DNA//RNA is required
● Fish are correlated with tissue findings (stained slides)
● Raw data is retained with final report and interpretation
of results
Test results must

● Convey the method/ manufactured kit used
● Locus
● Mutation/organism tested
● Analytical interpretation of raw data
● Clinical interpretation of the analytical result
● Likelihood of false+/false-
DIAGNOSTICS PRELIM LAB TOPIC 2-
OPTICAL DEVICES Light source
 SPECTROPHOTOMETRY
 Yields high output of polychromatic light
 FLUOROMETRY
over a wide range of spectrum
 LUMINESCENCE
 Most common: incandescent
 TURBIDIMETRY AND NEPHELOMETRY tungsten/tungsten-iodide lamp
 Types:
SPECTOPHOTOMETRY  Xenon Flash
 A process which measure the light  For visible and UV
transmitted by a solution to determine  Do not heat up the
concentration of the light-absorbing instrument
substance in the solution.  Reduce warm up time
PRINCIPLE: BEER-LAMBERT LAW Monochromators
“Absorbance is directly proportional to the
 Isolation of individual wavelength of light
concentration of the solution”
 Consist of 3 parts
 Entrance slit
 Exit slit
 Dispersion device
Dispersion Devices
 Causes different wavelength of light to be

dispersed in different angles
 Types:
 Prism
 If parallel beam of radiation
falls on the prism, the radiation
of 2 wavelengths will be bent
through different angles.
 The prism can be rotated,
allowing only the desired
wavelength to pass through an
exit slit.
 Glass = Visible range; 3x
dispersion than quarts
 Quartz = Visible and UV
23
 Filter SPECTROPHOTOMETER CLASSES
 Absorbs/reflects certain  Single beam – measures blank and the
wavelengths and sample not the same time
transmitting other
 Double beam – measures blank and the
wavelengths. sample simultaneously
 Absorption filters
o Absorb short  DOUBLE BEAM IN TIME: 1
wavelengths, Photodetectors, with
transmit long chopper/splitter
wavelengths  DOUBLE BEAM IN SPACE: 2
 Interference filters Photodetectors (FOR SAMPLE AND
o Selectively transmit REFERENCE)
or reflect a certain FLUOROMETRY
range of
wavelengths; with  Determines the concentration via
dielectric films excitation of the substance via
 Diffraction Gratings electromagnetic radiation. Light emission
 Splits/diffracts light into will be measured over a zero
several beams travelling in background and is proportional to the
different directions. concentration.
 Consists of many parallel  More specific and sensitive (1000x) than
grooves (15,000 or 30,000 conventional spectrophotometer
per inch) etched onto a  Very SENSITIVE to ENVIRONMENTAL
polished surface. CHANGES
 Wavelengths bend as they
Light sources:
pass a sharp corner
Sample Cell or Cuvette  Xenon
 Mercury Arc
 Holds the sample
QUENCHING = reduces fluorescent/light emitted
 Sold in matched sets
by:
 Scratches may scatter light
 Ph
Photodetectors
 Temperature
 Convert the transmitted radiant energy  Contamination
into an equivalent amount of electrical  UV light changes
energy.
 Types LUMINESCENCE
 Barrier layer/Photocell  Excitation of the substance in the saple
 Phototube is cause by CHEMICAL, BIOCHEMICAL, or
 Photomultiplier ELECTROCHEMICAL reaction. Emission is
measured by LUMINOMETERS.
 Differs from spectrophotometry and
fluorometry:
 No monochromator
 No light source
23
TYPES OF LUMINESCENCE LIGHT SCATTERING DEVICES
Chemiluminescence Light Scattering depends on wavelength and
particular size.
 Chemical reactions (oxidation)
 Emission of light when an electron returns - Immunoassays for Proteins and Haptens
from an excited or higher energy level to a TURBIDIMETRY
lower energy level
 Chemicals: Luminol, isoluminol, acridinium  Determines the amount of light blocked
esters, luciferin, hydrogen peroxide, (light reduction) by a particulate matter
hypochlorite, oxygen in a turbid solution
 Catalysts:  Application: Protein Measurements (CSF
1. Enzymes (Horseradish peroxidase, and Urine); Bacterial growth detection in
alkaline phosphatase, broths, AST (antimicrobial susceptibility) in
microperoxidase) broths, Detection of clots.
2. Metal ions/Metal complexes (Copper
NEPHELOMETRY
and Iron phthalocyanine complex)
3. Hemin  Determines of amount of scattered light by
 Chemiluminescence in Molecular Biology a particular matter suspended in a turbid
Chemiluminescence assays are solution
ULTRASENSITIVE with WID DYNAMIC  Physical phenomenon that results from
RANGES light particle interactions in a solution.
Utilized in the Molecular Biology  Application: For antigen-antibody
laboratory in form of: complexes (proteins)
- Automated immunoassays
- DNA probes assay NEPHELOMETRY VS TURBIDIMETRY
systems Bioluminescence
 Chemiluminescence found in Biological
systems.
 Luciferin and aequorin
Electrochemiluminescence
 Reactive species from chemiluminescence

reaction are electrochemically generated
 Ruthenium and tris(bipyridyl)
chelate

23
OTHER OPTICAL DEVICES ATOMIC ABSORPTION SPECTOPHOTOMETRY
 Measures the light absorbed by atoms

dissociated by heat
 Principle: an element is not excited by
merely dissociated from its chemical bond and
placed in unionized, unexcited ground state.
 Hollow-Cathode Lamp as Light source
 Flame as cuvette
 VERY PRECISE
 FOR DETECTION OF METALS AND HEAVY
METALS
LASER SPECTROMETRY
 Based on interaction of radiant energy with

suitably excited atoms/molecules
FLAME EMISSION PHOTOMETERS  FOR DETERMINATION OF STRUCTURE, IF OF
SAMPLES, AND QUANTIFICATION
 Measures the light emitted by a single  Employed in HEMATOLOGY as FLOW
atom burned in a flame CYTOMETERS for WBCs
 Principle: Excitation of electrons from  Side scatter –
lower to higher energy state. complexity/granularity of cell
 Flame is the Cuvette and Light source  Forward scatter – size of cell
 FOR THE DETERMINATION OF SODIUM, ELECTROCHEMICAL DEVICES
POTASSIUM, LITHIUM, AND OTHER
Electrochemistry – the study of electron movement
METALS/IONS
 ION SELECTIVE ELECTRODES
- used alternative for flame test
(photometer)
- sensitive toward individual ions
 pH ELECTRODES
- used to measure the potential Hydrogen
 GAS SENSING ELECTRODES
- Almost the same with pH electrodes but
this gas sensing electrodes are designed
to detect specific gases that is on the
solution
- Separated from the solution by a thin
gas permeable membrane
 ELECTROPHORESIS
Element Wavelength Flame Color - Used for us to determine the migration or
Sodium 589 nm Yellow movement of the charge solutes of
Potassium 766 nm Violet particles particularly in molecules such as
Lithium 670 nm Red proteins and nucleic acids
 OSMOMETRY
- In need device called osmometer used to
measure the concentration of the solute
particles that is in the solution
23

Molbio Prelim Topics

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MOLECULAR BIOLOGY AND DIAGNOSTICS PREPARED BY:

INSTRUCTOR: Dyan Jumamoy, MsBio RALPH JAYSON GARCIA

INTRODUCTION Replication: Faithful reproduction of the DNA

Recombination: process of exchange of genes

MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY AND DIAGNOSTICS

CENTRAL DOGMA OF LIFE Also, is assemblage of units of

James Watson: When he coined the term

MOLECULAR BIOLOGY AND DIAGNOSTICS

Helicase: Untangles DNA by cutting and

Process of DNA replication Prokaryotes

MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY AND DIAGNOSTICS

MOLECULAR BIOLOGY AND DIAGNOSTICS

PLASMIDS Resistance Transfer Factors (RTF)

MOLECULAR BIOLOGY AND DIAGNOSTICS

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

 Removal of intron sequences from Mrna sequences.

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

 RNA sequence structure

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

 Products of transcription & translation of

Transcribed by: JANROSE R. TERRAZULA, BMLS

 Polypeptides of up to more than

Take a look at this different kind of wedding Structure of Proteins

 pH 7: most of the carboxyl groups are

Transcribed by: JANROSE R. TERRAZULA, BMLS

 Protein structure consisting of more than

Transcribed by: JANROSE R. TERRAZULA, BMLS

Transcribed by: JANROSE R. TERRAZULA, BMLS

Transcribed by: JANROSE R. TERRAZULA, BMLS

 Site of protein synthesis  Beginning of translation, when the small

 Binding of charged tRNAs & formation of the

 Ending of translation occurs when the complex

Transcribed by: JANROSE R. TERRAZULA, BMLS

Transcribed by: JANROSE R. TERRAZULA, BMLS

Examples of Epigenetic Phenomena Where does modifications occur?

1. Histone modification Specific amino acids on amino terminal &

 Regulates gene expression by recruiting

 Can regulate expression at the level of the

 Nuclear DNA & its associated proteins

ENZYMES THAT METHYLATE DNA IN

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

 Only one copy of a gene in an individual Writers Methyltransferases, phosphorylases,

Transcribed by: JANROSE R. TERRAZULA, BMLS 2F 22-

Hazard - work area should be decontaminated on the

Hazards Analytic Production of Of 100

The following should be established by manufactured and

Quality controls Analyte specific reagents (ASR)

Test results must