Compre Molbio

RNA: Transcription, Types, Polymerases, TRANSCRIPTION
Enzymes and Regulation of

Transcription o Copying of 1 strand of DNA into RNA
o mRNA carries the information in DNA to
RNA Structure the ribosomes, where it is translated into
protein
Is a macromolecule of: Carbon, Nitrogen,
Oxygen, Phosphorus and Hydrogen DNA can only store information. How will
this information be used?
Is a polymer nucleotides similar to DNA:
Phosphate Group, Ribose Sugar and GENE EXPRESSION: It is the production of
Nitrogen base RNA and protein using a DNA template
Nitrogen bases: o Catalyzed by RNA polymerase occurs

mostly in interphase
 Pyrimidines: uracil (instead of - In most prokaryotes: single type
thymine) & cytosine - Eukaryotes: RNA polymerase pol I, pol
 Purines: adenine & guanine II, & pol III
o Synthesized as single strand rather

than as a double helix
o Some fold & loop upon themselves to
take on a double-stranded character
that is important for their function
o Can pair with complementary single
strands of DNA or another RNA & form
a double helix
 Complementary base pairs: A:U
& C:G
Types:
1. Ribosomal RNA (rRNA)

2. Transfer RNA (tRNA)
3. Messenger RNA (mRNA)
4. Small nuclear RNAs
o Copied, or transcribed from DNA

 Exception: 1 family of RNA
viruses (retroviruses) → RNA to
DNA
Steps:  Transcription Elongation
- Both prokaryotes & eukaryotes: RNA
1. Initiation - Beginning of transcription, polymerases synthesize RNA using
when RNA polymerase & supporting the base sequence of 1 strand of the
proteins bind to the promoter. double helix as a guide
2. Elongation - Addition of nucleotides to - RNA polymerases (50-100 bases/sec)
the mRNA strand work more slowly than DNA
3. Termination - Ending of transcription, polymerases (1,000 bases/sec) & with
occurs when RNA polymerase crosses less fidelity
a stop (termination) sequence in the - RNA synthesis does not require
gene priming
Setting the stage for transcription to - 1st ribonucleoside triphosphate retains
begin: all of its phosphate groups
- Subsequent ribonucleoside
DNA must be released locally from histones triphosphates retain only the alpha
& the helix unwound, involve the phosphate & the other 2 (beta &
participation of: gamma phosphates) are released as
orthophosphates during the
 DNA-binding proteins synthesis reaction
 Transcription factors  Transcription Termination
 Histone-modification enzymes
 RNA polymerase Prokaryotes:
 Transcription Initiation - Some are responsive to protein

- RNA polymerase & its supporting products; high levels of a gene
accessory proteins assemble on DNA product induce termination of its own
at a specific site (promoter) synthesis
 Prokaryotes: Basal - In some genes, by interactions
transcription complex = between RNA polymerase &
assembly of large & small nucleotide signals in the DNA
subunits of RNA polymerase & template
additional sigma factors at the - In other genes, additional factor (rho)
promoter is required
 Eukaryotes: Transcription - Rho-independent termination occurs
complex = assembly of RNA at G:C-rich regions in the DNA & the
polymerase & up to 20 additional G:C bases are transcribed into RNA &
factors for accurate initiation fold into a short double-stranded
hairpin; elongation complex
dissociates as it reaches A:T-rich area
Eukaryotes: Eukaryotes
- Transcription catalyzed by poll: - Synthesized from highly repeated

terminates transcription just prior to gene clusters
Sal box (site in the DNA) with the - Copied from DNA as a single 45S
cooperation of a termination factor precursor RNA (pre-ribosomal
(TTF1) RNA)→18S species of the ribosome
- Transcription catalyzed by pol II: small subunit & 5.8S & 28S species of
termination will be activated once the the large subunit 5S species: found in
polyadenylation signal (polyA site) the large ribosome subunit,
along the DNA template will be synthesized separately
encountered
- Transcription catalyzes by pol III:
termination signal is a run of adenine
residues in the template, requires a
Rho termination factor
TYPES/STRUCTURES OF RNA
Types of RNAs found in the cell:

3. Messenger RNA (mRNA) 2. Messenger RNA (mRNA)
4. Small nuclear RNAs - Initial connection between the
information stored in DNA & the
translation apparatus that will
ultimately produce the protein
- Largest component of cellular RNA
products responsible for the
(80%-90% of the total cellular RNA)
phenotype
- Various types are named for their
sedimentation coefficient (S) in Prokaryotes
density-gradient centrifugation
- Important in structural & functional part - Synthesized & simultaneously
of the ribosomes translated into protein
- Sometimes polycistronic (1 mRNA
Prokaryotes codes for more than 1 protein)
 16S rRNA: found in the ribosome
small subunit
 23S rRNA & 5S rRNA: found in the
ribosome large subunit
All are synthesized from the same gene.

Eukaryotes Messenger RNA (mRNA) Processing
- Monocistronic (having only 1 protein In eukaryotes, the newly made RNA

per mRNA) (primary transcript/ pre-mRNA is further
- Can produce different proteins from processed before it is functional:
the same DNA sequences by:
 starting the RNA synthesis in 1. Polyadenylation
different places or - Addition of adenosines to the 3' end of
 by processing the mRNA mRNA
differently - Most mRNAs carry a sequence of
polyadenylic acid at the 3' terminus
(polyA tail), which will be added to the
RNA after synthesis of the pre-mRNA
- Polyadenylate polymerase is the
enzyme responsible for adding the
adenines to the end of the subscript
- Copying of RNA from DNA & protein
- mRNA mammalian cells: a run of up to
synthesis from RNA are separated by
200 nucleotides of polyA
the nuclear membrane barrier
- Undergoes a series of post-
transcriptional processing events
before it is translated into protein 2. Capping
(mRNA processing)  Cap
- A structure (5'-5' pyrophosphate
linkage of 7-methyl guanosine to either
2' O-methyl adenine of the mRNA)
that blocks the eukaryotic mRNA at
the 5' terminus
- Confers a protective function & serves
as a recognition signal for the
translational apparatus
Amount of a particular mRNA in a cell is
3. Splicing
related to the requirements for its final
- Removal of intron sequences from
product
mRNA
 Constitutive transcription: - Introns: noncoding (intervening)
messages are transcribed constantly sequences, does not code for amino
& are relatively abundant in the cell acids
 Inducible/regulatory transcription: - Exons: remaining sequences that
messages are transcribed only at code for the protein product
certain times during the cell cycle/ - Heteronuclear RNA (hnRNA)
under particular conditions  newly transcribed mRNA,
much larger than mature RNA
because it contains introns
 Capped & tailed, transitioning Some of the diseases resulting from
from hnRNA to mRNA, abnormalities in splicing process:
removing introns from hnRNA
 Some ẞ-thalassemias: mutations
Splicing Mechanism: in the splice recognition
sequences of the ẞ-globin genes
1. Self-Splicing RNAs - Special  Certain autoimmune conditions:
sequences of RNA that are able to production of antibodies to RNA-
splice themselves protein complexes
2. Spliceosome - Nuclear
macromolecule complex within
which splicing reactions occur to
3. Small Nuclear RNA (snRNA)
remove introns from pre-mRNAs - Functions in splicing in eukaryotes,
allowing for precise alignment and
correct excision of introns Stays in the
nucleus after its transcription by RNA
polymerase I or III

- Adaptor molecules during the
translation process
- Relatively short, single-stranded
polynucleotides of 73-93 bases in
Why are eukaryotic genes interrupted by
length, MW 24,000-31,000
introns?
- At least 1 tRNA for each amino acid
1. Theory: Introns evolved as a means of - CCA at the 3' end: amino acid will be
increasing recombination frequency covalently attached to the tRNA
within genes & between genes without - Acceptor arm: the end of tRNA to
breaking coding sequences. which an amino acid becomes bound
2. May protect the coding regions from - tRNA loops:
genetic damage  T( )C loop
 Anticodon loop
Alternative splicing  Variable loop
 D loop
- Removal of introns from RNA using
 T( )C loop
different breakpoints
- ( stands for the modified
- Exons from the same gene are joined
pseudouridine), seven-base loop,
in different combinations, leading to
contains the sequence 5'-TTCG-3'
different, but related, mRNA
- Special recognition site for the
transcripts
ribosome to allow a tRNA-ribosome
- Modifies products of gene by alternate
complex to form during the process of
insertion of different exons
protein synthesis
- Has been found in over 40 different
 Variable loop
genes
- Larger in longer tRNAs, helps in
recognition of the tRNA molecule
 Anticodon loop - RNA Polymerases 3 multisubunit RNA
- Seven-base loop, contains the polymerases (all are DNA-dependent
anticodon polymerases)
1. RNA polymerase I
 D loop
2. RNA polymerase II
- 8- to 12-base loop, relatively rich in 3. RNA polymerase III
dihydrouridine (modified nucleotide),
plays an important role in stabilizing - Single-subunit mitochondrial RNA
RNA structure polymerase imported to organelles
HISTORICAL HIGHLIGHT
Robert Holley & colleagues at Cornell

University
In 1964, solved the 1st tRNA sequence

(alanine tRNA of yeast = 76 bases long &
10 of these are modified)
5. Other RNAs
 RNA viruses
- Late 1900s, increasing varieties of - RNA-dependent RNA polymerases
 Hepatitis C virus, Zika virus, &
RNA species have been described
Dengue Virus: to replicate their
- Functions: RNA genomes
 RNA synthesis & processing  Also found in plants & lower
 Influence numerous cellular eukaryotes: associated with gene
processes: silencing
1. plasmid replication  PolyA polymerase
2. bacteriophage - Template-independen t RNA
polymerase
development
- Adds adenine nucleotides to the 3'
3. chromosome structure and end of mRNA
development - Result: polyA tail, important for
mRNA stability & translation into
RNA POLYMERASES protein
- Catalyze the RNA synthesis
Prokaryotes
- 1 multisubunit enzyme for all types of

RNA 
- Bacterial RNA polymerase: 5 subunits
(2 α & 1 of each β, β’, & σ)
OTHER RNA-METABOLIZING ENZYMES Regulation of mRNA Synthesis at
Initiation
What is RNA metabolism?
2 factors responsible:
- It refers to any events in the life cycle
of RNA molecules: 1. Cis factors: DNA sequences that
 Transcription mark places on DNA involved in the
 Folding/unfolding initiation & control of RNA synthesis
 Modification 2. Trans factors: Proteins that bind to
 Processing the cis sequences & direct the
 Degradation assembly of transcription complexes
at the proper gene
1. Ribonucleases
- Ubiquitous, stable enzymes that
degrade all types of RNA
2. RNA helicases
- Required in RNA synthesis &
processing to catalyze the unwinding
of dsRNA
- Have been characterized in
prokaryotes & eukaryotes
- Some work exclusively on RNA,
others on DNA (RNA heteroduplexes
& DNA substrates
- Involved in removal of proteins from  Operon
RNA-protein complexes - Series of structural genes transcribed
together on 1 mRNA & subsequently
RNA Helicase: separated into individual proteins
- Example: lactose (lac) operon in
 Ribosome biogenesis Escherichia coli (metabolism of
 Translation initiation lactose)
 RNA processing
 RNA decay
REGULATION OF TRANSCRIPTION
Gene Expression
- Production of RNA and protein using a

DNA template
- Key determinant of phenotype
- Some genes (products are in continual
use by the cell), gene expression is
constant (constitutive)
- Other genes, gene expression is
tightly regulated throughout the life of
the cell
- Most immediate & well-studied level of
control of gene expression:
transcription initiation
 Lac operon in the absence of lactose - H bonding of complementary bases
- Repressor protein binds to the (allow/prevent transcription)
operator sequence & prevents
transcription of the operon General arrangement of cis factors:
- Usually 4-20 base pairs in length

- Some are inverted repeats with the
capacity to form a cruciform structure
in the DNA duplex recognizable by
specific proteins
 Lac operon in the presence of lactose

- Lactose binds to repressor protein &
changes its conformation & lowers its
affinity to bind the operator sequence,
resulting in the expression of the
operon
Post-Transcriptional Regulation
Several factors affecting the stability of

the RNA transcript:
 RNA sequence structure

 Presence of exonucleases &
endonucleases that digest the RNA
 Attenuation
- Another mechanism of control in
bacteria
- Formation of stems & loops in the
RNA transcript by intrastrand
[MIDTERM] MOLECULAR BIOLOGY AND DIAGNOSTICS
LEC TOPIC 1: DNA AND RNA ISOLATION
NUCLEIC ACID EXTRACTION LATER ROUTINE LABORATORY PROCEDURES OF DNA

ISOLATION
 Removal of nucleic acids (DNA and/or RNA from  Solubility differences among chromosomal DNA,
the cells in which they normally resides plasmids, and proteins in alkaline buffers
PURPOSE IS TO RELEASE NUCLEIC ACID FROM THE CELL

FOR
1. Detecting a specific pathogen (bacteria and

viruses)
2. Diagnosing disease and genetic disorders
3. Other applications:
a. Forensics
b. Paternity tests
c. Ancestry tracking
d. Genetic engineering
e. Vaccines
f. Hormones I. PREPARING THE SAMPLE
TARGET NUCLEIC ACID  Yield of DNA from Different Specimen Sources
 Free from contamination with macromolecules

(proteins, carbohydrates, lipids, or other nucleic
acids)
OVERVIEW OF HOW THE NUCLEIC ACID IS REMOVED

FROM THE CELL
ISOLATION OF DNA
FIRST EXTRACTION OF NUCLEIC ACID
Friedrich Miescher in 1869

 Isolated the nuclein (DNA) from the WBCs he
obtained from the pus on collected surgical
bandages in a nearby hospital
o Precipitation
EARLY ROUTINE LABORATORY PROCEDURES OF DNA

ISOLATION
Developed from density-gradient centrifugation strategies

 Meselson & Stahl (1958): demonstrate the semi-
conservative replication of DNA
:) I BMLS 2D | MOLBIO MIDTERM 1

A. Bacteria and Fungi Fixed, embedded tissue
 May be deparaffinized by soaking in xylene and
Gram-negative bacteria tissue is rehydrated by soaking it in decreasing
 Can be lysed by high pH and detergents concentrations of ethanol
 May be used directly without dewaxing
Some bacteria & fungi with tough cell walls
 Can be lysed by enzyme products or mechanically
by grinding or by vigorously mixing with glass Tissue Fixatives Influencing Nucleic Acid Quality
beads
 Detergent (1% sodium dodecyl sulfate) a strong
base (0.2 M NaOH) in the presence of Tris base,
ethylene-diaminetetraacetic acid (EDTA) &
glucose
Yeast, filamentous fungi, &gram-positive bacteria
 Commercial reagents designed for isolation of
DNA in amplification procedures (PCR)
B.Viruses
Viral DNA
 Held within free viruses or integrated into the
host genome along with host DNA
 Cell-free specimens (plasma) will be used for viral
detection
 May require concentration of viroids by
centrifugation or other methods
C. Nucleated Cells in Suspension (Blood & Bone Marrow

Aspirates)
White blood cells (WBCs) II. DNA ISOLATION CHEMISTRIES

 Anticoagulants will be added to prevent clotting
and will also trap WBCS
A. Organic Isolation Methods
 Obtained from the blood plasma
 Can be accomplished using an organic mixture
 Purified of red blood cells (RBCs) & other
(phenol & chloroform)
components by either differential density-gradient
 Use a combination of high salt, low pH & an
centrifugation or differential lysis
organic mixture of phenol & chloroform
 Differential osmotic fragility of RBCs & WBCs
o Readily dissolves hydrophobic
contaminants (lipids & lipoproteins)
o Collect cell debris & strips away most
DNA-associated proteins
D.Plasma
Exosomes
 Small vesicles (30-100 nm in diameter), which B. Inorganic Isolation Methods
form by invagination & budding from the inside of  Does not include organic extraction step
cellular endosome vesicles & are secreted by  Sometimes called “salting out”, makes use of low-
living cells pH & high-salt conditions to selectively precipitate
 Contain nucleic acid & can be collected by proteins, leaving the DNA in solution
centrifugation
 Diagnostic & prognostic analyses purposes (liquid
biopsy)
 Solid-phase collection of nucleic acid
E.Tissue Samples
Fresh tissue
 Dissociated by mincing the tissue
Frozen tissue
 Dissociated by grinding and homogenizing the
tissue

C. Solid-Phase Isolation 2. Genomic DNA Purification from Saliva
 Uses solid matrices (silica-based products) to bind  Cells found in the saliva: exfoliated buccal
& hold DNA for washing epithelial cells & other cells
a. Buccal swabs (cotton swabs/

cytobrushes)
 Suspended in lysis buffer which
includes Tris, EDTA, SDS, &
proteinase K
 Incubated
III. PROTEOLYTIC LYSIS OF FIXED MATERIAL
 Paraffin-embedded specimens
o Dewaxed with xylene or other agents
o Rehydrated before nucleic acid isolation b. Mouthwash method
 Some tests (somatic mutation analyses)  Samples from saline rinses need to
o Microscopic examination of adjacent be processed/ frozen immediately
stained serial section after collection
 Alcohol-containing moutwash must
be used
3. Genomic DNA Extraction from Urine Sample

a. Urine specimen
 Inverted & swirled in a specimen
cup to create a homogenous
suspension of cells followed by
the centrifugation
 Supernatant is removed & a dry
pellet containing cells is chilled
(-20 C) for 15mins followed by
addition of lysis buffer (Tris,
EDTA, SDS, & proteinase K)
 Sample is incubated (56 C) for 2h
& then DNA is extracted from the
solution
IV. NONINVASIVE HUMAN DNA ISOLATION
1. Genomic DNA Purification from Hair

a. Simple alkaline lysis method
 Hair with root is incubated (95C)
for 10 mins in NAOH buffer & the V. PROTEOLYTIC LYSIS OF FIXED MATERIAL
supernatant is subjected to DNA
purification after centrifugation  Before lysis
b. Smooth chemical digestion method using o Cells may be washed by suspension &
Dithiothreitol (DTT) centrifugation in saline/ other isotonic
 Hair sample can be incubated buffers
(56C) for 2h with buffer containing  Reagents for cell lysis
Tris-HCl, EDTA, NaCl, SDS, DTT, & o Simple screens: detergents (SDS or
proteinase K, followed by gentle mixing Triton)
& incubation (60C) for 2h or until the o PCR amplification: mixture of Tris buffer
& proteinase K
hair has dissolved completely

ISOLATION OF DNA
Before isolation of RNA

 Strict precautions to avoid sample RNA
degradation by ribonucleases (RNAses)
 RNAses must be eliminated or inactivated
o RNAses inhibitors: diethyl pyrocarbonate
(DEPC) added to water & buffers;
vanadyl-ribonucleoside complexes; and
macaloid clays
 Allocate a separate RNAse-free (RNF) area of the
laboratory
TOTAL RNA
VI. RAPID EXTRACTION METHODS
 rRNA
 PCR o most abundant (80% - 90%) RNA in all
 Rapid lysis methods & DNA extraction/ storage cells
cards  mRNA
 Chelex o Next most abundant RNA fraction (2.5%
o Cation-chelating resin that can be used for to 5%)
simple extraction of DNA from minimal  tRNA & snRNA
samples o also present
o 10% Chelex resin beads is mixed with the
specimen & the cells are lysed by boiling
VII. ISOLATION OF MITOCHONDRIAL DNA

I. SPECIMEN COLLECTION
Approaches:
1. 1st isolate the mitochondria by centrifugation  RNA tests (array analysis or quantitative reverse
2. Isolate total DNA by hybridization or PCR transcriptase PCR)
o RNA is stabilized from further
metabolism or degradation after
collection
 Specialized collection tubes
o To stabilized RNA immediately upon
draw
 Tissue or cell pellets can be stored and/or
transported in preservative reagents
II. PREPARATION OF SPECIMEN MATERIAL

FOR RNA EXTRACTION
 Reticulocytes in blood & bone marrow samples

o Lysed by osmosis or separated from
WBCs by centrifugation
 Tissue
o Kept frozen in liquid nitrogen/immersed
in buffer that will inactivate intracellular
RNAses
 Bacterial & fungal RNA
o Isolated by chemical lysis/ by grinding in
liquid nitrogen
 Viral DNA
o Isolated directly from the serum, plasma,
culture medium, or other cell-free fluids
by means of specially formulated spin
columns or beads

III. RNA ISOLATION CHEMISTRIES
1. Organic Isolation
 Cell lysis V. PROTEOLYTIC LYSIS OF FIXED MATERIAL

o Detergent or phenol in the presence of
high salt (0.2 to 0.5 M NaCl) or RNase  The quality of RNA from fixed, paraffin-embedded
inhibitors tissue will depend on the fixation process and
o Guanidine isothiocyanate (GITC) can also handling of the specimen before fixation
be used  Commercial reagent sets are available for
o Strong reducing agents (2- extraction of RNA from fixed-tissue specimens
mercaptoethanol) may also be added  Specialized reagents or spin columns for removal
 Acid phenol: chloroform: isoamyl alcohol of genomic DNA contamination are included in
(25:24:1) solution efficiently extracts RNA some systems
 Automated isolation systems have reagent kits
and cartridges
2. Solid-phase Isolation
 Begins with similar steps as described for organic

extraction
 The strong denaturing buffer conditions must be
adjusted before application of the lysate to the
column
VI. ISOLATION OF polyA (MESSENGER) RNA
 RNA often required for analysis: mRNA (2.5%-5%

of the total RNA yield)
 Single-stranded oligomers of thymine or uracil
immobilized on a matrix resin column or beads
are used
o Approximately 30-40mg of mRNA can be
IV. YIELD OF RNA FROM VARIOUS SPECIMEN obtained from 1µg of total RNA
SOURCES
 1 million eukaryotic cells or 10-50 mg of tissue

= 10 µg of RNA
 Yield of RNA from biological fluids will depend on
the concentration of microorganisms or other
target molecules present in the specimen

MEASUREMENT OF NUCLEIC ACID QUALITY & QUANTITY
MEASUREMENT OF THE QUALITY & QUANTITY OF

DNA & RNA
I. ELECTROPHORESIS
 Separation of particles through a solution or

matrix under the force of an electric current
 Nucleic acids absorb light at 260 nm through the

adenine residues
 Beer-Lambert Law: A=𝜖bc
 Absorbance is directly proportional to the
concentration of the nucleic acid in the sample
 DNA and RNA can be analyzed for quality

(detection & size analysis) by resolving an aliquot
of the isolated sample on an agarose gel
 Uses an electric current to propel charged
biomolecules through a porous gel matrix at a rate
that is the function of the charge, size, & shape of
the molecules
 Fluorescent dyes used: ethidium bromide,
SybrGreen I, silver stain
o Less frequently, silver stain has been used
to detect small amounts of DNA by visual
inspection.
 Appearance of DNA on agarose gels depends on
the type of DNA isolated
 Beer-Lambert Law: A=𝜖bc

o 𝜖 = molar absorptivity (L/mol-cm) 50 for
dsDNA, 40 for RNA
Using absorptivity, as a conversion factor from optical

density to concentration:
 At 260 nm,
 1 optical density unit (or absorbance unit)
 = 50 mg/L (or 50µg/ mL) of dsDNA & 40 µg/ mL
of RNA
To determine concentration:
 Spectrophotometer reading in absorbance units x
appropriate conversion factor
If the DNA/ RNA preparation require dilution before
spectrophotometry, to determine concentration:
 Absorbance reading x conversion factor x dilution
factor
II. SPECTROPHOTOMETRY
 Technique that uses light absorption to measure

the concentration of an analyte in solution

 May also be used to estimate the purity of nucleic

acid
 Detection of contaminants: reading the 2. NanoDrop Spectrophotometry
concentration over a range of wavelength  Similar in principle with the previous, but
 Indication of contamination: absorbance over has many additional capabilities:
background at any wavelength other than the o Functions by combining fiber
maxima of the nucleic acid A200 optic technology & natural
o Absorbance of DNA at 260nm = 1.6 to surface tension properties
2.00 times more than the absorbance at o Accompanied by special software
280 nm to enable analysis of signal from
o RNA = 2.0 to 2.3 small quantities of sample
o If the 260-nm / 280-nm ratio is <1.6, the o Displays the entire absorbance
nucleic acid preparation may be spectrum of the sample in
contaminated with unacceptable amounts graphical form→ allows detection
of protein & not of sufficient purity for of contaminants
use o Capable of determining a wide
 Most likely contaminant protein (absorbs light at range of sample concentrations
280 nm through the aromatic tryptophan & w/o requiring serial dilutions
tyrosine residues)
In practice, nucleic acid solutions are read at two or three

distinct wave lengths ( Table 3.4 ).
1. UV Spectrometry
 Standard nucleic acid quantitation III. FLUOROMETRY
 Nucleic acid sample is placed into quartz (FLUOROSCENT SPECTROSCOPY)
cuvette, which is then placed inside the
UV spectrometer  Measurement of emitted fluorescent light
 UV light passed through the sample at a  Measures fluorescence related to DNA
specified path length, & the absorbance of concentration in association with DNA-specific
the sample at specific wavelengths is fluorescent dyes
measured  Early methods: 3,5-diaminobenzoic acid 2HCI
 Does not require additional (DABA), combined with alpha-methylene
reagents/incubation time aldehydes (deoxyribose) to yield a fluorescent
product
 Modern methods: DNA-specific dye Hoechst
33258, combined with adenine-thymine base
pairs in the minor groove of the DNA double helix

o Fluorometric determination of DNA
concentration: down to 200 ng DNA/mL
 Other DNA-specific dyes
o PicoGreen = detection down to 25
pg/mLconcentrations
o OliGreen = detection down to 100 pg/mL
of ssDNA
 RNA: SybrGreen II RNA gel stain is used
Absorption and fluorometry readings may not always agree

 These methods recognize different targets:
o Single nucleotides do not bind to
fluorescent dyes, but they can absorb
ultraviolet light
 Absorption measurements do not distinguish
between DNA & RNA
 Deciding which instrument to use is at the
discretion of the laboratory:
o Most use spectrophotometry because the
samples can be read directly without
staining or mixing with dye
o Methods requiring accurate
measurements of low amounts of
DNA/RNA (in the range of 10 to 100
ng/mL), fluorometry may be preferred
IV. MICROFLUIDICS
 Science & technology of systems that process or

manipulate small amounts of fluid (10-9 to 10 to
18L), using channels measuring from tens to
hundreds of micrometers
 Sample is applied to a multiwell chip & then
moves through microchannels across a detector
 Instrument software generates images in
electropherogram (peak) or gel (band)
configurations
 RNA integrity number: quantification estimate for
RNA, determined as a standard measure of RNA
integrity
 Uses a minimal volume of sample (as low as 1 μl)
& can test multiple samples simultaneously
 Useful for analysis of studies on small RNAS
(microRNAs) in eukaryotes & gene expression in
bacteria

PREFI LEC TOPIC 2 – GENE MUTATIONS
MOLECULAR BIOLOGY I PROF EARN IAN CARO, RMT I MARCH 20, 2023
GENE MUTATIONS 2 Immunohistochemistry

 Affect single genes and are often, but not always,  Longstanding method that allows detection of
small changes in the DNA sequence. protein abnormalities on situ.
 Deletions, insertions, inversions and  Formalin-fixed paraffin-embedded tissue: <5
translocations microns slices (microtome)
 Alterations of a single and few base pairs (point  Fixation can affect tissue antigens  altering/
mutations). covering some epitopes  can be solved by
antigen retrieval:
a. Enzyme digestion (proteinase K,
chymotrypsin, pepsin, pronase)
b. Heating tissue sections in water/ buffer
 Snap frozen tissue (in isopentane, at -160C): 5
to 15-micron slices (cryostat inside of a chamber
held at 20C)
 Fixation: acetone
 Sections are dried and stored frozen
 Rehydration of the dried sections in phosphate-
buffered saline
Transition – purine replaces a purine or pyrimidine with a
pyrimidine.
Transversion – purine replaces a pyrimidine or
pyrimidine replaces a purine.
DETECTION OF GENE MUTATION

A. Biochemical methods
B. Nucleic acid analyses
Biochemical methods
 Used to directly analyze the change in protein
structure of function.
 Other uses:
o Metabolic defects where several genes are
involved in the disease phenotype.
o Detection of the actual protein/ amino acid
alterations.
 Include:
1. Enzyme Immunoassays
2. Immunohistochemistry  To minimize nonspecific binding: blocking
3. High-performance liquid chromatography solution with serum protein (albumin),
(HPLC) detergent (Tween 20), and unlabeled
4. Gas chromatography antibodies.
5. Mass spectrometry  Positive and negative controls: included
with samples to ensure the adequacy and
1 Enzyme Immunoassays specifity of staining.
 Detects the presence of metabolites in the blood,
urine, or other biological fluids.  Imaging/ microscopic observation of antibody
 Involve the use of specific antibodies or other requires a signal from the antibody:
ligands to detect the presence of the target a. Fluorescent signal: fluorescent molecules
molecules. (fluorescein, Cy5, phycoerythrin)
 Widely used EIA: enzyme-linked b. Colorimetric signal: substrate solution is
immunosorbent assay (ELISA) added, oxidized by the enzymes
(horseradish peroxidase/ alkaline
phosphatase)
o Most frequently used: red/ brown IHC
staining
3 High-performance liquid chromatography

(HPLC)
 Separation of molecules (nucleic acids and
proteins) in solution through interaction with a
solid support in the column.
MATEO, JENDY ROMAE EHILLA I BMLS-2A I SY. 2022-2023 I DAVAO DOCTORS COLLEGE, Inc.
 2 phases:
o Mobile phase (solvent)
o Stationary phase (solid support)
 Ultra-HPLC (UHPLC): increase resolution and
lower separation time while using less solvent;
faster flow rates (5mL/ min).
 Types of detectors: light scattering,

fluorescence, refractive index, UV light
absorption, and mass spectrometry (MS)
4 Gas chromatography
 Mobile phase: inert gas
 Stationary phase: high-boiling point liquid that
absorbed to an inert solid support in the column
 Detector: flame ionization detector  2 ionization methos for large biomolecules
 Used for detection of drugs and poisons and their (proteins):
metabolites in biological samples. a. Electrospray ionization (ESI)
 May be coupled with MS to detect biomarkers o
disease.
b. Matrix-assisted laser desorption/

ionization (MALDI)
5 Mass spectrometry
 Converts molecules to ions that can be moved in
a magnetic field based on their charge and mass.
 Readout: spectrum with mass/ charge value on
the x-axis and abundance of the ion on the y-axis
 ID of molecule: by their characteristic spectrum/
set of peaks
MALDI-TOF (time-of-flight) Spectrometry  Dilute concentrations of short, ds-PCR

 High MW molecules products (100-400 bp) are denatured
 Ionized molecules are accelerated at a. 10-20 mM NaOH, 80% formamide for 5
a fixed point and allows to drift minutes at 90C
through the flight tube to the detector. b. 10-20 mM NaOH, 0.004 mM EDTA, 10%
formamide for 5 minutes at 55C-60C
Surface-Enhanced Laser Desorption/  Brand/ peak patterns
Ionization (SELDI) o Different from the normal sequence
 Combined with TOF offers flexibility in (control) conformers prepared
the ID and quantification of peptides. simultaneously with the test conformers 
presence of gene mutation
Nucleic acid analyses o Detected by silver stain, radioactivity, or
 Performed on a variety of specimen types: blood fluorescence
or buccal cells.
 DNA mutations from single-base pair changes to
large chromosomal rearrangements can be
detected.
 PCR amplification: simplified mutation detection
(limiting specimens).
 DNA sequencing: most definitive method for
detecting mutations.
B Allele-Specific Oligomer Hybridization

(ASO)
 Utilizes differences in the Tm of short sequences
(2o bases) with 1 or 2 mismatches and those
with no mismatches.
 Synthetic ss-probes (labeled) with normal/
mutant target DNA sequence (immobilized) in
1 Hybridization-based Methods a solution.
a. Single-Strand Conformation Polymorphism  At specific annealing temperatures and
(SSCP) conditions (stringency)
b. Allele-Specific Oligomer Hybridization (ASO) a. Probe will not bind to a near complementary
c. Melt-Curve Analysis (MCA) target sequences with 1 or 2 mismatched
d. Heteroduplex Analysis bases
e. Array Technology b. Probe with perfect complementary sequence
will bind
A Single-Strand Conformation Polymorphism
(SSCP)
 Based on the preference of DNA to exist in a
double-stranded state.
 Absence of the complementary strand: nucleic
acids form intrastrand duplexes, 3D structure
(conformer).
o Shape: kinks, loops, bubbles, tail
 Bound probes will be detected with a

conjugated horseradish peroxidase-
antibiotin Fab fragment and will be
exposed to a chromogenic/
chemiluminescent substrate,
generating a color/ light signal

indicating the binding of the test DNA D Heteroduplex Analysis
to the probe.  Formed when single strands that are not
completely complementary hybridize to one
C Melt-Curve Analysis (MCA) another.
 Method of analyzing the dissociation of double  Can be resolved through polyacrylamide/
stranded DNA during the heating cycles. agarose gel electrophoresis: presence of
 PCR amplicons in the presence of a DNA- bands different from a homozygous reference
specific fluorescent dye (EtBr, SYBR green, LC control is indicative of mutation.
green) are heated (0.3C/ sec).  Can also be resolved in denaturing high-
 Rise in the temperature, DNA duplexes begin to performance liquid chromatography (DHPLC).
separate into single strands, losing the dye.
 Black line  targets with different mismatches to
the hybridization probe.
 Sequence differences result in different melting
characteristics and Tm for each sequences.
 Interpretation of results by the temperature peak
placement with respect to the temperature on the
x-axis
a. Specimen with identical sequences –
overlaying peaks at expected Tm
b. Specimen with different sequences – 2 or
more peaks at different temperatures
E Array Technology
 High-density oligonucleotide arrays: test DNA is
fragmented by treatment with DNase before
binding to the complementary probes on the
array.
High-resolution melt-curve analysis (HR-MCA)
 Hybridization formats:
 High specifity
a. Standard tiling – base substitution is always
 Uses fluorescent resonance energy transfer in the 12th position from the 3’ end of the
(FRET) probes
probe.
 Raise in b. Redundant tiling – same mutation id placed
temperature, at different positions in the probe.
probes  After hybridization: fluorescent signal is read on a
dissociate at
scanner with appropriate software and mutations
specific Tm are identifies as indicated by which probes are
 donor no
bound.
longer close
 Bead array technology: uses sets of solor0-
to the
coded polystyrene beads in suspension as the
acceptor 
solid matrix.
fluorescence
 Combination of bead color and test label =
drops
presence/ absence of a mutation/ polymorphism
Indication of
mutation: 2 Sequence (polymerization)- Based Methods
Tm lower than that of
A Sequence-Specific (Primer) PCR (SSP-
the probe and its
perfect complement, PCR)
or sequence  Detect point mutations and other SNPs
difference between
probe reference Primer 3’ end falls on the nucleotide to be analyzed
sequence and the o Must match the template perfectly to be
test sequence. extended by Taq polymerase.
o Prescence/ absence of the product = presence/ B Nonisotopic RNase Cleavage Assay

absence of the mutation. (NIRCA)
 Heteroduplex analysis using duplex RNA
 T7 or SP6 phage RNA polymerase
 Detection mutation: heteroduplexes form
between normal and mutant transcripts 
targets for cleavage by RNase enzymes (E.
coli RNaw and Aspergillus RNase T1)
 Remaining dsRNA fragments can then be
separated by aragose gel electrophoresis.
B Allelic Discrimination with Fluorogenic

Probes
 Thermal cyclers with fluorescent detection.
 RT-PCT, using 2 probes labeled 3’ quencher
molecules and different fluors on the 5’ ends
(complementary to either normal/ mutant
sequence).
 Prescence of corresponding fluorescent signal
indicates whether the test sequences is normal/
mutant.
3 Enzymatic/ chemical Cleavage Methods
A Restriction Fragment Length

Polymorphisms (RFLPs)
 Can detect sequence alterations C Cleavage Assay
 Mutand changes the structure of a restriction  Bases on the characteristic enzymatic activity of
enzyme target site/ changes the size of a cleavase.
fragment.  Premixed reagents (including cleavase) +
 PCR-RFLR is used standard 96 well-plate + test specimens +
controls
 Clevase: recognizes the structure formed by
hybridization of the normal/ mutant probes to the
test sequences
 If the probe and test reactions occur 
fluorescent signal (standard fluorometer)
4 Other Methods
 Combination of methods to increased
sensitivity and detection (RFLP with modified
primers).
 Array-based methods and massive parallel
sequencing methods provide specific multiplex
detection and sensitivity required for clinical
applications.
 Method selected will depend on the available
instrumentation, genetic target, and the
nature of mutation.
GENE VARIANT NOMENCLATURE

 Descriptive and consistent system of expressing
mutations/ polymorphisms.
A DNA AND cDNA

 1st nucleotide of the 1st amino acid in the B ALLELES
sequence: A for ATG (methionine) = position +1 1. If both alleles are affected:
 Preceding nucleotide +position -1 o Designation of each mutation
o Separated by ;
If there is nucleotide change:
 Position/ nucleotide interval
 Type of nucleotide change
 Changed nucleotide
 Symbol >
 New nucleotide
2. If 2 mutations in the same allele:
3. If both alleles are affected, one loses the

entire reference coding sequence
C INTRONS OF GENOMIC DNA 1. If there is amino acid change:

 Indicated by: o Amino acid changed
o Position of the mutation in the genomic o Position
sequence of the DNA/ position from the end o New amino acid
of the coding sequence (exons) + position in
the intron
2. If there is nonsense mutation in the 4th codon:
3. If there is a deletion of Arg and His:
D PROTEIN LEVEL 4. If there is insertion of Gly (G), Ala (A), and Thr
 Numbering begins with the initial amino acid (T):
(Met) in the protein sequence, designated +1 Amino acid interval, “ins”, inserted amino acids
 Single-letter code/3- letter denotations of amino
acids
 Stop codons are designated as X
5. If there is frameshift mutations

Amino acid, its position, “fs”
REMEMBER!!!
 Prefixes used to distinguish between mutation
nomenclature
o g. = genomic DNA
o c. = coding (complementary/ copy)
o m. = mitochondrial DNA
o r. = RNA
o p. = protein sequences
 RNA sequences are written in lowercase letter
o Coding DNA: c.89T>C
o RNA sequence: r.89u>c
GENE NAMES
 Set by the Human Genome Organization
(HUGO) gene nomenclature committee
 Gene names should be capitalized and set in
italics with no hyphens
 Protein names are not italicized nor completely

capitalized
Thus, the report will contain the official gene name and
the appropriately named change in the DNA sequence, if
present.
PREFI LEC TOPIC 3 – DNA SEQUENCING
MOLECULAR BIOLOGY I PROF EARL IAN CARO, RMT I MARCH 27, 2023
DNA SEQUENCE
 Refers to the order of the nucleotides in the
DNA molecule.
 Applications of DNA sequencing in medical
laboratory:
o Detection of mutation
o Typing microorganisms
o Identifying human haplotypes
o Designating polymorphism
o Treatment strategies
SEQUENCING METHODS
1. Direct sequencing: manual and automated
2. Pyrosequencing
3. Bisulfite DNA sequencing
4. RNA sequencing
5. Next-generation sequencing
I DIRECT SEQUENCING
 Direct determination of the order, or sequence of
nucleotides in a DNA polymer. Addition of a strong reducing agent (10% piperidine) =
 Most specific and direct method for identifying ssDNA would break at specific nucleotides
genetic lesions (mutations)/ polymorphisms.
 Types:  After reactions: fragments will be separated by size
1. Manual sequencing (chemical (Maxam- on a denaturing polyacrylamide gel (6-20%)
Gilbert & Sangers sequencing) o Sequence = bands
2. Automated fluorescent sequencing (dye o Lane in which the band appeared = ID of
primer & dye terminator sequencing) the nucleotide
o Sequence is read from the bottom (5’ end)
Manual Sequencing: to the top (3’ end) of the gel
Chemical (Maxam-Gilbert) Sequencing  Run times:
 Allan M. Maxam & Walter Gilbert o Short fragments (up to 50 bp) = 1-2 hours
 Requires a ds/ss version of the DNA region to be o Long fragments (>150 bp) = 7-8 hours
sequenced with 1 end radioactively labeled (32P)
 Sequencing proceeds in 4 separate reactions Manual Sequencing:
 Template: labeled fragment Dideoxy Chain Termination (Sanger) Sequencing
 Frederick Sanger
 Uses dideoxynucleotides (ddNTPs) to determine
the order/sequence of nucleotides in a nucleic acid
 Primer complementary to DNA to be sequenced
 Product detection of sequencing:
o Primer is attached at 5’ end to a 32P-
/fluorescent dye-labeled nucleotide
o Incorporate 32p/35S-labeled dNTPs in the
nucleotide sequencing reaction mix
(internal labeling)
JACOB, K. A. and MATEO, J. R. I BMLS-2A I SY. 2022-2023 I DAVAO DOCTORS COLLEGE, Inc.
 ddNTPs are added, terminating the DNA synthesis

(chain termination)
o Lack OH = 5’-3’ phosphodiester bond
cannot be established to incorporate a
subsequent nucleotide.
Automated Fluorescent Sequencing

 Automated reading of DNA sequence ladder
requires fluorescent dyes (4 distinct colors) to label
primers/ sequencing events
1. Fluorescein
2. Rhodamine
 Components: Mixed in 4 reaction tubes 3. Bodipy (4,4-difluoro-4-bora-3a-diaza-s-
1. DNA template (PCR product) indacene)
2. Radioactivity-labeled primer  Fluorescent dyes can be distinguished by
3. Enzyme (DNA polymerase) automated sequencers
4. dNTPs (all 4)
 Approaches (to label fragments according to their
5. Buffer (20mM EDTA, formamide, gel tracking/
terminal ddNTP): dye primer and dye terminator
loading dyes)
sequencing
6. Different ddNTPs in each of the 4 tubes
 Sequencing reaction: thermal cycler (cycler
sequencing
Dye Primer Sequencing o If not clear, N will replace A, C, T, or G.

 4 different fluorescent dyes are attached to 4
separate aliquots of the sample.
 Dye molecules are attached to the 5’ end of the
primer = 4 versions of the same primer with
different dye labels.
 Products are labeled at the 5’ end using the dye
color associated with the ddNTP at the end of the
fragment.
Dye Terminator Sequencing

 1 of the 4 fluorescent dyes attached to each of the
ddNTPs.
 All 4 sequencing reactions are performed in the
same tube.
 Products fragments are labeled at the 3’ end.
II PYROSEQUENCING
 Determines a DNA sequence without having to
make a sequencing ladder.
 Relies on the generation of light (luminescence)
when nucleotides are added to a growing DNA
strand.
 No gels, fluorescent dyes, ddNTPs
Automated Electrophoresis  Reaction mix components:
 4 sets of sequencing products in each reaction are 1. ssDNA template
loaded onto a single gel lane/ capillary. 2. Sequencing prime
 Fluorescent dye colors distinguish which nucleotide 3. Sulfurylase
is at the end of each fragment. 4. Luciferase
 Fluorescent detection equipment yields results as 5. Substrates: adenosine-5’-phosphosulfate
electropherogram. (APS) and luciferin
 Base calling: process of bases ID in a sequence by 6. 1 of the 4 dNTPs is added to predetermined
sequencing software. order of the reaction
IV RNA SEQUENCING
 Early approaches: used RNase to cut end-labeled
RNA at specific nucleotides
 Other approaches:
o Based on amino acid sequence
o Based on sequencing of its complementary
DNA
 Direct RNA sequencing
o Based on single-molecule sequencing
technology and virtual terminator nucleotides
 mRNA is captured by immobilized polydT
oligomers (through their polyA tails).
o RNA without polyA tails: initial treatment with
polyA polymerase
o 4 reversible dye-labeled nucleotides are
sequentially added.
III BISULFITE DNA SEQUENCING

 AKA methylation-specific sequencing
 Chain termination sequencing designed to detect
methylated sequencing cytosine nucleotides.
 2-4 ug of genomic DNA is cut with restriction
enzymes to facilitate denaturation.
 DNA is denatured (97C for 5 mins) and exposed to
bisulfate solution (sodium bisulfite, NaOH,
hydroquinone) for 16-20 hours.
o Cytosines are deaminated  uracil
o 5-methylcytosines are unchanged
o Can be detected by Sanger sequencing/
pyrosequencing
V NEXT-GENERATION SEQUENING (NGS)

 AKA massive parallel sequencing
 Designed to sequence large numbers of templates
carrying millions of bases.
 Powerful computer data assembly systems
(bioinformatics, computer software and support)
are required.
 Require the preparation of a sequencing library
(sets of DNA fragments representing the regions to
be sequenced).
Gene Panels o Selected by multiplex PCR with gene-

 Collection of genes that have been grouped for specific primers tailed with binding sites for
testing, enabling simultaneous sequencing of all a secondary primer sets.
genes (2 to >1000 genes).  Allele dropout: loss of library fragments from the
 Types: sequenced regions.
1. “Hot-spot” panels – target regions of specific
genes known to affect treatment response, Sequencing Platforms
disease state, or clinical condition. 1. Ion-conductance
2. Targeted panels – critical genes in particular o Indexed libraries (gene panels) are
diseases (hematological-cancer specific, solid- amplified using primers immobilized on
tumor specific). microparticles (beads) in aqueous oil
3. Very large panels (≥3000 genes) – emulsion using adapters on the library
diagnostic, prognostic, discovery purposes. fragments complementary to the
immobilized primers.
NGS Library Preparations
 Sequencing library: collection of DNA library
fragments (100-1000 bp) to be sequenced.
1. Shearing of high-frequency acoustic energy

2. Sonication
3. Nebulization (forcing DNA molecules through a
small opening)
4. Enzymatic treatments
 Adapters: synthetic short dsDNA carrying

sequences complementary to a single primer pair,
which may contain short sequences that will ID the
sample (indexing/ bar coding).
2. Reversible dye terminator sequencing

o Captured/ amplified fragments are
hybridized to immobilized primers on a
solid surface (flow cell).
o Labeled nucleotides are applied to the flow
cell and incorporated into growing chains
by DNA polymerase at each polony
location.
Targeted Libraries
3. Sequencing by ligation
 The regions to be sequenced are enriched by: o Uses a pool of labeled oligonucleotides DNA
1. Probe hybridization ligase to identify the template sequence
o Probes: biotinylated oligonucleotides through the known probe sequences.
complementary to specific gene regions.
2. Amplification with region-specific primers
(amplicon-based targeted libraries)
4. Nanopore sequencing  Variants that remain after filtering may be

o Does not require fragmentation and annotated by searching in disease-specific
amplification of the template DNA. databases:
o Each nucleotide can be identified by a 1. Cancer Genome Atlas (TCGA)
disruption in current as it passes through the 2. Catalogue of Somatic Mutations in Cancer
pore. (COSMIC)
o Also used for direct RNA sequencing. 3. My Cancer Genome
4. Leiden Open (source) Variation Database
(LOVD)
5. Human Genome Mutation Database (HGMD)
 Final reports of variants: information, such as
effects on therapeutic treatments (targeted
therapies, clinical trials, and prognosis).
Data Analysis
 Optical signals are translated to a nucleotide
sequence (base calling), which is measured by
the Phred score, acceptable = 2-3 (100-1000-fold
certainty of a correct call).
 Each sequence is compared to a reference
sequence (“normal”) through read alignment.
 Variant ID based on camparison with the reference
sequence (SNVs, indels, rearrangement
sequences, CNVs).
 Sequence variations from the reference are
arranged in a variant call file (VCF).
BIOINFORMATICS
 Involves using computer technology (in silico) to
collect, store, analyze, and disseminate biological
data and information (computational biology).
BIOINFORMATICS TERMINOLOGY
 Annotations: performed for critical variants ID TERM DEFINITION
 Confidence in the variant call is determined by Identity The extent to which two sequences are
sequence quality and coverage = at least 500x the same.
(recommended). Alignment Lining up two or more sequences to
 Chromosomal and sequence location of the search for the maximal regions of
variants is identified along with the type of variant identity in order to assess the extent of
(subject to filtering). biological relatedness or homology.
Local Alignment of some portion of two  Matches/ hits = diagram, showing alignments and
alignment sequences. color code
Multiple Alignment of three or more sequences
sequence arranged with gaps so that common
alignment residues are aligned together.
Optimal The alignment of two sequences with
alignment the best degree of identity.
Specific sequence changes (usually
Conservation protein sequence) that maintain the
properties of the original sequence.
Similarity The relatedness of sequences, the
percent identity or conservation.
Algorithm A fixed set of commands in a computer
program.
Domain A discreet portion of a protein or DNA
sequence.
Motif A highly conserved short region in International Union of Pure and Applied
protein domains. Chemistry and the International Union of
A space introduced in alignment to Biochemistry and Molecular Biology (IUB)
Gap compensate for insertions or deletions  Assigned a universal nomenclature for mixed,
in one of the sequences being degenerate, or wobble bases.
compared.
Homology Similarity attributed to descent from a
common ancestor.
Orthology Homology in different species due to a
common ancestral gene.
Paralogy Homology within the same species
resulting from gene duplication.
The sequence presented for
Query comparison with all other sequences in
a selected database.
Description of functional structures,
Annotation such as introns or exons in DNA or
secondary structure or functional
regions to protein sequences.
The point of meeting between a
Interface computer and an external entity, such
as an operator, a peripheral device, or a
communications medium.
The genetic sequence database
GenBank sponsored by the National Institutes of
Health.
Search service sponsored by the
PubMed National Library of Medicine that
provides access to literature citations in
Medline and related databases.
Protein database sponsored by the
SwissProt Medical Research Council (United
Kingdom).
Basic Local Alignment Search Tool (BLAST)

 System for homology searches
 Searches GenBank
 Searches can be made of nucleic acid and amino
acid sequences
 Limits and parameters can be added (type of
organisms)
THE HUMAN GENOME PROJECT (HGP)  1 single-base difference between 2 random

 Primary mission: to decipher the sequence of the individuals fopund approxiamaetly every 1,000
complete human genetic material (entire genome), bases along human DNA sequence.
identify all genes contained within the genome, and
provide research tools to analyze all this genetic
information.
 Established by National Institute of Health (NIH)
by James Watson.
 Epstein-Barr virus – 1st complete genome
sequence (1984)
 Craig Venter and colleague (Institute Genomic
Research) completed the:
o 1st sequence of a free-living organism
(Haemophilus influenzae)
o Sequence of the smallest free-living organism
(Mycoplasma genitalium)
 Sequence approach of the 2 projects:

1. NIH method: hierarchical shotgun approach
– to sequence from known regions.
2. Celera (established by Venter): whole-
genome shotgun sequencing – to sequence
random fragments.
OTHER GENOME ORJECTS
 Chromosome 21: 1st chromosome to be 1. Human Haplotype Mapping (HapMap) Project
sequenced completely.  Goal: to find blocks of sequences that are
 Size of the entire genome: 2.91 Gbp (2.91 billion inherited together.
bp)  Revealed >1,000 disease-associated regions
 AT = 54%; GC = 38%; bases still to be determined of the genome (coronary artery disease and
= 8% diabetes).
 Chromosome 2: most GC-rich (66%) 2. 1000 Genome Project
 Chromosome X: fewest GC bp (25%)  Provides a resource of structural variants in
 Number of genes: estimated between 20,000- different populations.
25,000  Over 88 million variants were verified: 84.7
 Chromosome 19: most gene-rich per unit length million SNPs, 3.6 million short insertions/
(23 genes/ Mbp) deletions, and 60,000 structural variants.
TOPIC 1: DNA POLYMORPHISMS AND
HUMAN IDENTIFICATION
1. POLYMORPHISMS
- Variations of DNA sequences (ranging
from a single base pair to thousands of
base pairs) that are shared by 1%-2%
or more of a given population
TYPES STRUCTURE DETECTION
METHODS
Single Single-nucleotide Sequencing,
nucleotide differences (1- other
polymorphism bp), may occur in
(SNPs) gene-coding
regions or in
intergenic
sequences
Long Highly repeated Sequencing
interspersed sequences (6-
nucleotide 8kbp in length),
elements containing RNA
(LINES) polymerase
promoters &
open reading
frames
Short Highly repeated Sequencing
interspersed sequences
nucleotide approximately 0.3
elements kbp in length,
(SINES) including Alu
elements
Short tandem Head-to-tail PCR
repeats (STRS) repeats of DNA
sequences with
<10-bp repeat
units
Variable-number Head-to-tail Southern Blot,
tandem repeats repeats in DNA PCR
(VNTRS) with 10-50-bp
repeat units
Restriction A sequence Southern Blot
fragment length variation that
polymorphisms results in
RFLPS) creating,
destroying, or
moving a
restriction site
(FINAL: LAB 1) :(:

2. RESTRICTION FRAGMENT LENGTH
POLYMORPHISMS (RFLPs)
- Differences in sizes & number of
fragments generated by restriction
enzyme digestion of DNA
 Nucleotide changes may also destroy,

change, or create restriction enzymes
sites, altering the number of fragments.
STEPS IN USING RFLPs:

I. Construct a restriction enzyme map of
the DNA region under investigation.
II. The number & sizes of the restriction

fragments of a test DNA region cut with
restriction enzymes are compared with
the number & sizes of fragments
expected based on the restriction map
The uniqueness of the collection of
polymorphisms in each individual is the
III. Detection of polymorphism: observe
basis for human ID at the DNA level.
fragment numbers & sizes different from
those expected from the reference RFLP protocols for human ID:
restriction map.
 North American labs use Haelll
restriction enzyme
 RFLP typing in humans required the use  European labs use Hinfl restriction
of Southern blot technique enzyme
Regulation of results from independent labs:
 Standard Reference Material (SRM)

DNA Profiling Standard for RFLP
analysis (1992)
 Supplies necessary materials for
final analysis (currently provided by
>2,000 RFLP loci have been described In National Institute of Standards and
human DNA Technology)
A. GENETIC MAPPING WITH RFLPs

- Polymorphisms can be used as
landmarks/markers in the genome to
determine the location of other genes
- More frequently a particular
polymorphism is present in persons
with a disease phenotype = more likely
(FINAL: LAB 1) :(:

an affected gene is located close to the blot multiple-locus probe (MLP)-RFLP
polymorphism system
- Single-locus probe (SLP) (1990) in
❖ Inherited breast cancer: Europe & N. America
- RFLP location: 17q21 - RFLP Southern blot technique: 100 ng
- BRCA1 gene: mapped to this position to 1 ug of DNA (1-20 kbp)
- Requires 0.7% gels
B. RFLP & PARENTAGE TESTING - Results: 5-7 days
- Fragment sizes of an individual are a
combination of those from each parent 3. SHORT TANDEM REPEATS (STRS)
- Paternity test: alleles/fragment sizes of - Head-to-tail repeats of DNA sequences
the child & the mother are analyzed with <10-bp repeat units
- Alleged fathers (AFs): provide the - Detection method: PCR
remaining alleles/fragments (inclusion) - Allelic ladders: used to determine the
no. of repeats in the locus by the size
of the amplicons
- Specimen required: 10 ng (key factor
for forensic analysis)
- Analysis: Fluorescent detection
systems (capillary electrophoresis)
- Analysis time: 24-48 hours
STR NOMENCLATURE
 International Society for Forensic

Genetics (1997)
A simplified RFLP paternity test is
shown, who do you think the father of A. STR within genes: according to gene
the child is? name
Ex:
a. THO1 = intron 1 of human tyrosine
hydroxylase gene on chromosome 11
C. HUMAN ID USING RFLPS b. TPOX = intron 10 of human thyroid

- 1st genetic tool for human ID: ABO peroxidase gene on chromosome 2
blood group antigens
- 1st human DNA profiling/fingerprinting
B. Non-gene associated STRS: D#S#
system: UK Forensic Science Service
a. D = DNA, # = chromosome location
(1985) using Sir Alec Jeffrey's Southern
of STR
(FINAL: LAB 1) :(:

b. S= unique segment, # = no. - Avuncular testing: 2 alleged relatives
registered in the International are related as either an aunt/uncle of a
Genome Database (GDB) niece/nephew
- Probability of relatedness: based on
the no. of shared alleles between the
STR & SEX ID
tested individuals
Amelogenin locus:
Amelogenin gene: located on the X & Y Y-STR
chromosomes
- STR located on the Y chromosome,
- Function of its encoded protein: paternally related men share all Y loci
required for embryonic development & - Represented only once per genome &
tooth maturation only in males
- Polymorphism in the 2nd intron (Y - Applications: forensic, lineage,
allele is larger than in X allele) population studies, kinship testing
- PCR and electrophoresis: 2 - Y-STR/paternal lineage test: 2 or more
bands/peaks for males (XY) & 1 males have a common paternal
band/peak for females (XX) ancestors
- Surname test: group of males having
the same surnames is expected to be
STR & PATTERNITY TESTING related to a common male ancestor,
Paternity test: test subject is the father/not sharing the same Y-chromosome
alleles
- Observation of shared alleles between
the alleged father & the child
- Paternity index = likelihood of 4. SINGLE-NUCLEOTIDE
paternity POLYMORPHISMS (SNPs)
- Combined paternity index (CPI) = - Single-nucleotide differences (1-bp)
summarizes & evaluates the genotype - HGP: human nucleotide sequence
information differs every 1,000-1,500 bases from 1
- CPI = 5.719 x 8.932 x 15.41 x 10.22 = individual to another
8,044.931 - International SNP Map Working Group:
- Indicate that the child is 8,045x more 2 haploid genomes differ at 1
likely to have inherited the 4 observed nucleotide per 1,331 bp
alleles from the AF than from another o 11 million sites in genome of 3
man billion bp that vary in at least 1% of
the world's population = 11 million
STR & SIBLING TESTS SNPs in each individuals
- Full-sibling test: likelihood that 2 SNPs

people tested share a common mother - Genetic mapping, disease production,
& father and human ID
- Half-sibling test: likelihood that 2 - Detection: direct sequencing
people tested share 1 common parent - Approx. 10 millions SNPs have been
- Kinship index/sibling identified (99% have no biological
index/combined sibling index: effects; over 60,000 within genes;
likelihood ratio some are associated with disease)
(FINAL: LAB 1) :(:

- Classification: according to location, - Result of environmental events: Profile
relation to coding sequences, & is unique = no 2 individuals will have
whether they cause a the same environmental exposures
conservative/nonconservative
Sequence alteration BY PAIR ACTIVITY:
SNP DATABASES
- Collections of DNA sequence variants
used as a reference for screening
genomic sequencing data
- Detection of variant: sequencing, may
already be described or associated with
a disease phenotype as noted in the
databases -
- Include short deletions, insertions, &
duplications involving more than 1
nucleotides
- dbSNP, dbVar, ClinVar, & others
OTHER ID METHODS
A. MITOCHONDRIAL DNA
POLYMORPHISMS
- Contribute to individual differences in
function & susceptibility to various
diseases such as Parkinson disease,
Alzheimer disease, bipolar disorder, &
cancer
B. PROTEIN BASED ID
- Amino acid variations
C. EPIGENETIC PROFILES
- Epigenetic alterations (DNA
methylation)
(FINAL: LAB 1) :(:

TOPIC 2: MOLECULAR DETECTION OF  Translocations, inversions, deletions,
INHERITED DISEASES duplications, marker chromosomes,
derivative chromosomes
01. Inherited Diseases
 Chromosome breakage - caused by
- Caused by mutations (changes) in
chemicals, radiation, chromosome
germ cells that are passed down
breakage syndromes (Fanconi
from parent to child
anemia, Bloom syndrome, ataxia
telangiectasia)
 Detection: karyotyping, FISH,
Mutations in Somatic Cell:
microarray technology (CGH)
1. Cancer
EXAMPLES OF DISEASE ARISING
2. Congenital malformations FROM INHERITED CHROMOSOME
(present at birth) MUTATION
- due to factors upsetting the
developmental process
CHROMOSOMAL ABNORMALITIES
 Genome mutations – abnormal chromosome #

1. Polyploidy = more than 2
2. Aneuploidy = gain (trisomy) or loss
(monosomy)
❖ Detection: karyotyping, ploidy
analysis, flow cytometry, & FISH
 Mosaicism
- 2 or more genetically distinct 02. Patterns of Inheritance in Single-
populations of cells from 1 zygote in Gene Diseases
- Single-gene diseases affect structural
an individual
proteins, cell surface receptor proteins,
- Results from mutation events growth regulators, and enzymes
affecting somatic/germ cells
DOMINANCE RELATIONSHIPS
EXAMPLES OF GENOME DISORDERS
1. Complete dominance
- Heterozygous phenotype (child = Tt)
- Homozygous phenotype (1 parent =
TT) Example: height
2. Partial/incomplete dominance
- Offspring phenotype is variably
intermediate (combine) between the
homozygous & heterozygous
parentals
- Example: gene affecting hair texture
3. Codominance
- Simultaneously demonstrate the
CHROMOSOMAL ABNORMALITIES phenotype of both parents
 Chromosomal mutations - Example: ABO blood group
- abnormalities in structure
(FINAL:LEC 2) :):
PATTERNS OF INHERITANCE IN 3. Sex-Linked Transmission
SINGLE GENE DISEASES - Mostly X-linked
A. X-linked recessive transmission:
- A.K.A. transmission patterns/mode of
- Common
inheritance
- Always expressed in males
- The manner in which a genetic trait,
o inherit the trait from
disorder, or risk of disorder is passed
heterozygote/homozygote mother
from one generation to the next
- Females are carriers and can only be
- Determined by examination of family
expressed if the causative allele is
histories
present in 2 copies
- Pedigree: diagram of family
o inherit the trait from affected father
phenotype/genotype
and affected heterozygote mother
- 3 main patterns: autosomal dominant,
- Ichthyosis, colorblindness, hemophilia
autosomal recessive, sex-linked (X-
linked)
1. Autosomal Dominant Transmission

- Criteria:
a. Males and females can be
affected. Male-to-male
transmission may occur.
b. Males and females transmit the
trait with equal frequency.
c. Successive generations are B. X-linked dominant transmission:
affected. - Rare
d. Transmission stops after a - Always expressed in females
generation in which no one inherits o Passed from male to all daughters
the mutation. but to no sons
- Affected individual = has dominant - Expressed also in males, with more
allele severe effects
- Parent 1 (affected) x Parent 2 - Rickets, Rett syndrome, incontinentia
(unaffected) = 50%-100% pigmenti, congenital hypertrichosis
risk/likelihood of expressing the
disease phenotype on the child
PENETRANCE VS. VARIABLE
2. Autosomal Recessive Transmission EXPRESSIVITY
- Criteria:
a. Males and females can be 1. Penetrance
affected. - Freq. of expression of disease
b. Affected males and females can phenotype in individuals with a gene
transmit the gene, unless it lesion
causes death before reproductive a. Complete penetrance -
age homozygous recessive
c. The trait can skip generations. b. Incomplete penetrance
d. Parents of an affected individual
are heterozygous or have the 2. Variable Expressivity
traits - Range of phenotypes in individuals
- Affected individuals =homozygous with the same gene lesion
recessive genotypes
- Carriers heterozygotes/asymptomatic
(FINAL:LEC 2) :):
03. Molecular Basis of Single-Gene FACTOR V LEIDEN
Diseases
- Cause: Single point mutation in the
Detection: Molecular methods, coagulation factor V gene F5 (1q23) at
morphological studies, clinical chemistry exon 10 (GA at nucleotide 1691,
R506Q)
Final Diagnosis: physiological,
- Genotype: heterozygous form (4%-
morphological and laboratory results
8% of the general population) &
SINGLE-GENE DISORDERS homozygous (0.06%-0.25%)
- Thrombophilia: inherited blood
clotting disorder
- Treatment for blood clot/deep venous
thrombosis: anticoagulants
- Molecular methods: PCR-RFLP,
SSP-PCR
- Other methods: Invader technology,
clot- based methods, family history
PROTHROMBIN
- Precursor to thrombin in the
coagulation cascade
- Autosomal-dominant increased risk
of thrombosis: mutation in the 3'
untranslated region of the gene that
codes for prothrombin or coagulation
factor II, F2 (11p11-q12)x
- Laboratory tests: F2 & F5 mutations
- Molecular methods: multiplex PCR-
RFLP
- Phenotypic methods: thrombin time,
prothrombin time, platelet count, CBC
LYSOSOMAL STORAGE DISEASE
- Automated systems: measure
- Cause: incompletely digested changes in light transmittance during
macromolecules due to loss of clot formation generating a curve
enzymatic degradation (acid - Other: sequencing of factors XI & XIII
hydrolases)
- Defects in proteins required for normal
lysosomal function METHYLENETETRAHYDROFOLATE
→ physical abnormalities REDUCTASE
- Screening: gene product testing
- Molecular testing: genes that code - Hyperhomocysteinemia: autosomal
for the enzymes & their subunits recessive disorder caused by
- Detection of mutation: direct deficiency of the 5,10-
sequencing methylenetetrahydrofolate reductase
(MTHFR) gene product
o Increased homocysteine levels →
predisposition to venous & arterial
thrombosis
(FINAL:LEC 2) :):
- Genetic polymorphisms: 677C>T metabolize & detoxify compounds
(p.A222V) & 1298A>C (p.E429A) → (drugs)
deficiencies in folate metabolism - Polymorphisms affect the metabolism
- Detection: standard/multiplex PCR of hormones, caffeine,
with RFLP (HinfI & MboII) or chemotherapeutic drugs,
sequencing, multiplex qPCR, HR-MCA antidepressants, & oral contraceptives:
o Tests are used to predict the
response to drugs
HEMOCHROMATOSIS
- Detection of polymorphisms: allele-
- Autosomal recessive condition, specific PCR
overabsorption of iron from food → - Screening tests: microarray, bead
pancreas, liver, & skin damage; heart array, sequencing
disease; diabetes
- Diagnosis: measurement of blood iron 04. Single-Gene Disorders with Non-
levels, transferrin saturation, liver Classical Patterns Of Inheritance
biopsy
CONDITIONS THAT DO NOT FOLLOW
- Molecular cause: dysfunction of the
MENDELIAN RULES OF INHERITANCE:
hemochromatosis type I HFE or HLA-
H gene product (C282Y, H63D, S65C) a. Mitochondrial gene mutations
- Indications for mutation testing: b. Genomic imprinting
clinical symptoms & increased serum c. Gonadal mosaicism
ferritin & transferrin-iron saturation d. Nucleotide-repeat expansion
- C282Y mutation detection: PCR- disorders
RFLP e. Multifactorial inheritance
CYSTIC FIBROSIS
A. MITOCHONDRIAL (mt) GENE
- Life-threatening autosomal recessive MUTATIONS
disorder that causes severe lung - Maternally inherited
damage & nutritional deficiencies - mtDNA
- Affects cells that produces mucus, o Circular, 16,569 bp, with 37 genes,
sweat, saliva, & digestive juices → 1000-nt control region
secretions become thick & sticky
- Cause: loss of function of the CFTR
gene (3-bp deletion F508del & 1,900
other mutations such as G542X,
- Mutations affect energy production →
G551D, N1301K, R117H, W1282X,
muscles & nervous system
1717- 1G>A)
- Heteroplasmy: mutated mt & normal
- Molecular tests for mutation
mt in the same cell
detection: RFLP, PCR-RFLP, HA,
- Molecular methods:
temporal-gradient gel electrophoresis,
o Large deletions: Southern blot
SSCP, SSP-PCR, cleavase, bead
o Point mutations: PCR-RFLP
array technology, & direct sequencing
Disease Resulting from mutations in

CYTOCHROME P-450
Mitochondrial Genes
- Group of mono-oxygenase enzymes
localized to the ER
- Present in high concentrations in the
liver & small intestine → enzymes
(FINAL:LEC 2) :):
C. GONADAL MOSAICISM
- Generation of new mutations in
germline cells → giving rise to
eggs/sperm carrying the mutation
which then becomes a heritable
phenotype
- Expected when phenotypically normal
Genes that control mitochondrial
parents have more than 1 affected
functions are found in nuclear genome
child
- Example: osteogenesis imperfecta
D. NUCLEOTIDE-REPEAT EXPANSION
DISORDER
- Nucleotide repeats, such as STRS
(1-10 bp repeating units) can expand
in length during DNA replication &
meiosis
- Triplet-repeat mutations: expansions
of STR w/3-bp repeating units in the
gene sequence
o Fragile X syndrome
B. GENOMIC IMPRINTING o Huntington disease
- Only 1 copy of a gene in an individual o Idiopathic congenital central
(either from mother or father) is hypoventilation syndrome
expressed, while the other copy is (CCHS)
suppressed
o Example: mules (male donkey x a. Fragile X Syndrome
female horse) & hinnies (male - CGG expansion (up to >2,000
horse x female donkey) repeats) in the noncoding region 5' to
- Cause: transcriptionally silencing the FMR-1 gene
through histone/DNA modification - Symptoms (increase in severity
- Genetic disorders: 1 or other allele of with each generation): learning
a gene is lost (uniparental disomy) disorders & mental retardation
- Examples (IQ~20), long face, large ears,
o Prader-Willi syndrome = paternal macroorchidism
del(15)(q11q13) - Detection:
o Angelman syndrome = same o Karyotyping
region, maternal o PCR
- Cytogenetic methods: o Southern blot
o Translocations & some deletions: o Capillary electrophoresis
standard karyotyping
o Microdeletions: HR-karyotyping b. Huntington Disease
o FISH with labeled probes - CAG expansion (9-37 repeats to 38-
- Molecular methods: 86 repeats) in the huntingtin structural
o PCR-RFLP/STR analysis gene (4p16.3)
o Methylation-specific PCR - Symptoms: impaired judgment,
o Southern blot using methylation- slurred speech, difficulty in swallowing,
specific restriction enzymes chorea, personality changes,
o Assays developed for CNV depression, mood swings, unsteady
detection: FISH, array-based CGH, gait, intoxicated appearance
NGS
(FINAL:LEC 2) :):
- Detection: - Prognostic & diagnostic value of
o Standard PCR methods gene mutation analysis:
o Capillary electrophoresis o Annotation of demographics
(ethnicity/gender, lifestyles)
c. Idiophatic Congenital Central
Hypoventilation Syndrome (CCHS)
- Gene mutations in PHOX2b gene in
chromosome 4: insertion of multiple
alanine residues
- Inadequate breathing while asleep,
hypoventilation while awake
- Occurs in association with an intestinal
disorder (Hirschsprung disease) &
symptoms of ANS
dysregulation/dysfunction
- Detection:
❖PCR w/32P-labeled primer &
polyacrylamide gel electrophoresis
❖Standard PCR & agarose gel
electrophoresis
OTHER EXAMPLES OF REPEAT
EXPANSION DISEASE Limitations of Molecular Testing
- Phenotypic methods: treatment is
directed to the phenotype
- Genes with variable expressivity
o Gene mutation may not predict the
severity of the phenotype
- Clotting time & transferrin saturation
o Better guides for anticoagulant
treatment
- Molecular testing
o May discover genetic lesions in the
absence of symptoms
o Offer only a diagnosis, not a cure
E. MULTIFACTORIAL INHERITANCE
- Disorders (& normal conditions)
controlled by multiple genetic &
environmental factors
(nutritional/chemical exposures)
- Phenotypes: conditioned by the no. of
controlling genes inherited
- Detection:
o HR-array methods
o NGS
- Interpretation:
o Databases (ClinVar & dbSNP)
(FINAL:LEC 2) :):
MOLECULAR ONCOLOGY
OSICO-2E
ONCOLOGY
Study of tumors/neoplasm
Growth of tissue that exceeds and is not
coordinated with normal tissue
• Benign — not recurrent; suffix —

oma to the tissue of origin
• Malignant — invasive and tending
to recur at multiple sites (cancer)
Metastasis — movement of tumor cells
from the original (primary) site of the tumor
to other locations
Molecular oncology — study of cancer at
the molecular level
CANCER
1. Solid tumors — an abnormal mass
of tissue that usually does not What causes cancer?
contain cysts or liquid areas
Cancer is caused by nonlethal mutations in
• Carcinoma - tumor of epithelial
DNA affecting 2 types of genes that control
tissue origin
the cell division cycle and cell survival:
• Sarcoma - tumor of bone, cartilage,
muscle, blood vessels, or fat ONCOGENES
• Teratocarcinoma — multiple cell
• Promote cell division
types
• Include cell membrane receptors,
2. Hematological malignancies — • that are bound by growth factors.
abnormal cells in the blood grow out • hormones. and other extracellular
of control • signals
• Leukemia — large nos. of WBCs • Support cell survival by inhibiting
populate the bone marrow & • apoptosis
peripheral blood • >100 in the human genome
• Lymphoma — neoplasm of TUMOR-SUPPRESSOR GENES
lymphocytes that forms discrete
tissue masses • Factors that control transcription-
• Plasma cell neoplasms — abnormal translation of genes required for cell
plasma cells or cells form tumors in /division
the bones/soft tissues of the body
MOLECULAR ONCOLOGY
OSICO-2E
• Participate in repairing DNA damage • DNA, RNA, proteins or other

& in promoting apoptosis molecules. from these targets
• Slow down/stop cell division by
Examples: DNA/RNA from
counteracting the movement of the
cell from Gl-to S (Gl checkpoint) or 1. Cytokeratin genes in gastric cancer
02 to M phase (G2 checkpoint) > 30
2. Carcinoembryonic antigen in breast
in the human genome
cancer
In cancer cells:
TUMOR-SPECIFIC TARGETS
ONCOGENES
Genetic structures resulting from mutations
Gain-of-function mutations resulting in oncogenes & tumor-suppressor genes that
are associated with tumor development
from:
Solid tumors, Leukemias, lymphomas
• Amplification/translocation of DNA
regions containing the genes Mutated cell free nucleic acid/circulating
• Activating mutations that cause tumor cells can be detected in blood & other
aberrant activity of the proteins body fluids
TUMOR-SUPPRESSOR CENES Examples: surface receptors. mutated

proteins, tumor-associated antigen,
Loss-of-function mutations metabolic markers.
• Inactivation of the gene products
(deletion, translocation, mutation of GENE & CHROMOSOMAL
the genes) MUTATIONS IN SOLID TUMOR
Molecular diagnosis and treatment: 1. Human epidermal growth factor

Detection of abnormalities in specific tumor receptor 2, HER2/neu/erb-b2 1 (17q21.1)
suppressors or oncogenes
• Tyrosine-kinase activity
ANALYTICAL TARGETS OF • Normal activity: cell growth and
division
MOLECULAR TESTING
• Overexpressed in 25%-30% of
TISSUE-SPECIFIC TARGETS human breast
• cancers
Molecular characteristics of the tissue from
which a tumor arose Detection methods:
Detection and monitoring the presence of • IHC: uses antibodies (fresh/frozen
tumor: tissues)
• Southern. northern. western blots:
• abnormal amounts or locations of
gene amplification - increased RNA
& proteins
MOLECULAR ONCOLOGY
OSICO-2E
• FISH: direct detection of increased ras, N-ras (Ip13); & Harvey rat sarcoma
copy nos. of the gene in DNA viral oncogene homolog, H-ras (11p15)
• Chromogenic in situ hybridization
• Small GTP-binding proteins that
(CISH): standard bright-field
receive signals from the cell surface
microscope, genome & chromosome
proteins & activate the initial steps of
mutation detection
the mitogen-activated protein kinase
• Silver-enhanced in situ hybridization
(MAPK) pathway cascade
(SISH)
• Gain-of-function mutations in ras
2. Epidermal growth factor receptor, proto oncogenes → cancers
• Mutations in these genes are the
EFGR (7p12)
most common oncogene mutations in
• Normal activity: cell signaling human cancers (codons 12. 13. 22. &
pathways that control cell division 61 in exons 2 & 3 of the KRAS
and survival gene)
• Overexpressed in solid tumors Detection methods of mutation:
Detection methods:
• Direct sequencing,
• IHC Pyrosequencing
• qPCR: to assess gene copy no.
through increased gene expression
• SSP-PCR. SSCP. direct sequencing.
NGS: mutations
3. Kirsten rat sarcoma viral oncogene

homolog, K-ras (12p12); neuroblastoma
MOLECULAR ONCOLOGY
OSICO-2E
b) molecular methods: RT-PCR. With

amplification control (GAPDH or 18S
RNA) on tissue/liquid biopsies
5. Synovial sarcoma transloca ion,

chromosome 18 — Synovial sarcoma
breakpoint 1 & 2, SYT-SSX/, SYT-SSX2
(p11.2;q11.2)
Synovial sarcoma: rare type of cancer of
the muscle. fat, fibrous tissue, blood vessels,
or other supporting tissue of the body
Detection methods:
• FISH
• RT-PCR
• Semi-nested PCR
• Agarose gel electrophoresis &
EtBr staining
• (PCR product detection)
4. Ewing sarcoma, EWS(22q12)

Ewing sarcoma: group of tumors arising
from primitive neuroectodermal tissue
(PNET)
Diagnosis and prognosis:
a) cytogenetic methods
MOLECULAR ONCOLOGY
OSICO-2E
8. Ataxia telangiectasia mutated gene,

ATM (11q22)
ATM protein: DNA repair and/or control of
the cell cycle
Ataxia telangiectasia (AT): neurological
disorder that affects the part of the brain that
controls motor movement & speech;
predisposition to cancer
Carriers (autosomal recessive mutations) →
develops leukemia (B-cell lymphocytic
leukemia, T-cell prolymphocytic leukemia),
lymphoma (mantle cell lymphoma). or other
types of cancers
Detection methods:
6. Paired box-fokhead in • Direct DNA sequencing
Rhabdomyosarcoma, PAX3-FKHR, • SSCP
PAX7-FKHR, t(1;13), t(2;13) • Functional test for the repair of
• Rhabdomyosarcoma (RMS): most dsbreaks
common soft tissue sarcoma of • induced by irradiation
childhood (10% of all solid tumors in • Test for other family members
children) alveolar RMS. embryonal 9. Breast cancer 1 gene, BRC ( q 1 , breast
(RMS-E), & primitive (RMS-P) cancer 2 gene, BRCA2 (13q12)
Detection methods: FISH, RT-PCR, qPCR, • Women: mutation in BRCAI
RNA sequencing lifetime risk of breast/ovarian
7. Tumor protein 53, TP53(17p13) cancer)
• Men: mutation in BRCA2 (increased
• p53: gene product of TP53, DNA- risk of breast/colon/prostate cancer)
binding protein that controls • Gene products are involved in DNA
expression of other genes and dsbreak repair by homologous
participates also in the arrest of cell recombination
division in the event of DNA damage • Screening tests for mutation: SSCP,
• Mutations in this gene is found in all protein truncation testes,
types of cancers and also aids in the chromosome breakage tests, etc.
diagnosis of Li- Fraumeni syndrome • Clinical application method: direct
sequencing
MOLECULAR ONCOLOGY
OSICO-2E
• Other detection methods: SSP- • Transcription of n-myc through

PCR, allele- specific oligomer qPCR
hybridization
10. von Hippel-Lindau gene, VHL (3p26)

12. V-Ros avian UR2 sarcoma virus
• Functions as a tumor-suppressor oncogene homolog 1 (ROSO proto-
gene, promoting cell differentiation
• Von Hippel-Lindau syndrome
• genetic condition involving
abnormal growth of blood vessels in
organs (eyes, kidneys, adrenal
glands, etc.)
• predisposition for renal cell
carcinoma & other cancers
• deletions, point mutations, splice-
site mutations
• Somatic mutations: renal cell
carcinoma & tumors of the adrenal
glands
oncogene (6q22.1) & rearranged during
Detection method: direct sequencing
transfection (REI) proto-oncogene
11. V-myc myelocytomatosis viral-related (10q11)
oncogene, neuroblastoma-derived, MYCN
• ROSI oncogene: coding for a
or n-myc (2p24)
membrane receptor tyrosine kinase
• Myc family proteins: increase the • Rearranged in a variety of human
expression of several genes cancers (1%-3% lung
adenocarcinomas)
1. c-myconcogene (8q24.21): amplified in
• RET proto-oncogene: its gene
breast & ovarian cancer. lymphomas, &
product participates in sending
leukemias
signals to the nucleus
2. I-myc (1p34.2): amplified in oral cancer • Translocations resulting in gene
overexpression: thyroid papillary
3. n-myc (2p24): amplified in
carcinomas
neuroblastoma & retinoblastoma
• Point mutations activating the gene:
Detection methods: inherited multiple endocrine
neoplasia (MEN) syndromes
• FISH
• Loss of function mutations:
• Sequencing
Hirschsprung disease
• Array analysis
MOLECULAR ONCOLOGY
OSICO-2E
• Detection methods: IHC. FISH. • Inherited cancer predisposition

Sequencing syndrome, 3%- 5%of colon cancers
& 3% endometrial cancers
13. Anaplastic lymphoma receptor
• Increased risk of developing other
tyrosine kinase (ALK) proto-oncogene,
cancers
2p23.1
• MMR mutations in MSH2 & MLHI
• ALK: receptor tyrosine kinase genes
• Chromosomal rearrangements (most
Detection methods: direct sequencing,
common). mutations, or
IHC, MSI analysis. PCR. gel/capillary
amplifications tumors (lymphomas,
gel electrophoresis (more bands/peaks in
neuroblastoma, & non- small cell
the tumor tissue than the normal)
lung cancer)
• Detection methods: FISH & • National Cancer Institute:
sequencing recommended the screening of
BAT25 & BAT26: & D5S346.
14. V-Kit Hardy-Zuckerman 4 feline
D2S123. & D17S250 for MSI
sarcoma oncogene homolog, KIT, c-K/T
determination
(4q 12)
• Kit protein: transmembrane receptor LOSS OF HETEROZYGOSITY

with tyrosine kinase activity
(LOH)
• Gene mutation: gastrointestinal
stromal tumors (GIST s). mast cell • Deletion/inactivation of a function
disease, & AML and allele, leaving a mutated allele
Detection methods: Detection methods:
• IHC: increased KIT protein • PCR & capillary electrophoresis:

• Sequencing: missense mutations amplification of heterozygous
STR/VNTR loci closely linked to the
disease gene
Formula:
MICROSATELLITE INSTABILITY
(MSI)
• Contraction & expansion of
nucleotide repeat sequences in DNA
• Cause: dysfunction of 1 or more
components of mismatch repair
(MMR) systems • Ratio <0.5 or >2 indicates LOH
Lynch syndrome or hereditary LIQUID BIOPSY
nonpolyposis colorectal cancer (HNPCC)
MOLECULAR ONCOLOGY
OSICO-2E
• A laboratory test done on a sample of immunoglobulins and T-cell

blood (plasma/serum). urine. or other receptors
body fluid to look for cells & nucleic
DETECTION OF CLONALITY
acids released by the tumors into a
person's body fluids • Gene rearrangements occur
independently in each
2 approaches:
lymphocyte
1. Cell-free or circulating free nucleic • Normal lymphocytes: polyclonal
acids (cfDNA/cfRNA), exosome (polytypic) with respect to their
rearranged lg or T-cell receptor
a) targeted: directed at specific mutations
genes
(sequencing/PCR methods)
• Lymphoma/leukemia:
b) untargeted: whole exomes or gene overrepresentation of a single
panels (sequencing & CCH) rearrangement in a specimen cell
population
Plasma DNA — most frequent source
material 1. Molecular analysis of lg heavy-chain
gene clonality
2. Circulating tumor cells (CTC)
• Protein analysis
• Mechanism of tumor metastases
• Nucleic acid analyses:
from I tissue site to another (2 to >50
cells) a) Southern blot, using BatnHI, EcoRI, &
• Isolation methods: antibody
Hind III
capture. negative depletion of
leukocytes ➢ 20-30 ug of high-quality DNA
• DNA: allele-specific PCR or other ➢ Normal: reveal the expected
sensitive methods fragments generated from the
germline DNA
GENE REARRANGEMENTS
Interpretation of results:
➢ positive = presence of extra bands
➢ negative = only germline bands are
• Series of intrachromosomal present
recombination events mediated
by recombinase enzymes that b) PCR
recognize specific sequences
• Forward primers complementary to
flanking the gene segments
the variable region & reverse primers
V(D)J recombination to the joining/constant regions of the
B-cell rearranged genes
• Normal intrachromosomal
• Normal: amplicons consisting of
breaking & joining of DNA in
fragments of different lengths =
the genes coding for
cloud/smear of bands/series of peaks
MOLECULAR ONCOLOGY
OSICO-2E
• Monoclonal: predominant/single Monoclonal population: bands

band or peak (positive result) detectable in addition to germline band
seen in normal control
c) Patient-specific primers
• PCR: targeting the TCRX gene
• Consensus primer is used to amplify
the rearranged gene from a positive Positive sample: product consistent w/ a
specimen single-gene rearrangement
• Fewer/none of the gene
Normal: polyclonal pattern
rearrangements from normal cells are
amplified • Sequencing
• Heteroduplex analysis
2. Molecular analysis of lg light-chain

gene clonality
• Protein analysis
• Nucleic acid analyses:
MUTATIONS IN HEMATOLOGICAL
a) Southem blot MALIGNANCIES
b) PCR
c) RT-PCR 1. Lymphoid malignancies
• Detection of clonality at the 18 locus a) t(14;18) q(32;q21)

on chromosome 22 is useful for
• Dysregulates the BCL2 gene on
confirming/ monitoring diagnosis of
chromosome 18q21.3 (B-cell
B-cell leukemias & lymphomas
leukemia & lymphoma 2 or B-cell
• Forward primers complementary to
CLL/Iymphoma 2)
VR gene segments & reverse primers
to the JR & CA gene segments are Follicular lymphoma
frequently used
Detection methods:
• Southern blot
3. Molecular analysis of T-cell receptor • PCR or qPCR with gel
gene clonality electrophoresis or qPCR probe
• Southern blot b) t(11;14) (q13;q32)
Probes complementary to the V, J, & D CCNDI gene on chromosome 11 is attached
to the long arm of chromosome 14
Normal: absence of rearrangements
Mantle cell lymphoma, chronic lymphocytic
leukemia, B-prolymphocytic leukemia,
MOLECULAR ONCOLOGY
OSICO-2E
plasma cell leukemia, multiple myeloma.

splenic lymphoma
Detection methods:
• Southern blot
• PCR & RT-PCR with gel/capillary
electrophoresis
• FISH or flow cytometry analysis
c) FMS-related tyrosine kinase 3 (FLT3),
13q12
Mutations in FLT 3 gene aberrantly activate
the FLT 3 kinase & predict prognosis in
AML
Detection methods:
• PCR with agarose/capillary gel
electrophoresis: internal tandem
duplications (ITDs)
• PCR-RFLP using EcoRV restriction
enzyme or RT-PCR with FRET
probes: D835 (aspartic acid)
mutations
d) Janus kinase 2 (JAK2). 9p24
• Dominant mutation causing a valine-
to phenylalanine substitution of the
Jak2 protein (617th position)
• Polycythemia vera, essential
thrombocythemia, idiopathic
myelofibrosis
Detection methods:
• multiplex SSP-PCR
• Direct sequencing
• NIRCA

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RNA: Transcription, Types, Polymerases, TRANSCRIPTION

Enzymes and Regulation of

Nitrogen bases: o Catalyzed by RNA polymerase occurs

o Synthesized as single strand rather

1. Ribosomal RNA (rRNA)

o Copied, or transcribed from DNA

 Transcription Initiation - Some are responsive to protein

- Transcription catalyzed by poll: - Synthesized from highly repeated

Types of RNAs found in the cell:

1. Ribosomal RNA (rRNA)

All are synthesized from the same gene.

- Monocistronic (having only 1 protein In eukaryotes, the newly made RNA

4. Transfer RNA (tRNA)

Robert Holley & colleagues at Cornell

In 1964, solved the 1st tRNA sequence

- 1 multisubunit enzyme for all types of

- Production of RNA and protein using a

- Usually 4-20 base pairs in length

 Lac operon in the presence of lactose

Several factors affecting the stability of

 RNA sequence structure

NUCLEIC ACID EXTRACTION LATER ROUTINE LABORATORY PROCEDURES OF DNA

PURPOSE IS TO RELEASE NUCLEIC ACID FROM THE CELL

1. Detecting a specific pathogen (bacteria and

TARGET NUCLEIC ACID  Yield of DNA from Different Specimen Sources

 Free from contamination with macromolecules

OVERVIEW OF HOW THE NUCLEIC ACID IS REMOVED

FIRST EXTRACTION OF NUCLEIC ACID

Friedrich Miescher in 1869

EARLY ROUTINE LABORATORY PROCEDURES OF DNA

Developed from density-gradient centrifugation strategies

:) I BMLS 2D | MOLBIO MIDTERM 1

C. Nucleated Cells in Suspension (Blood & Bone Marrow

White blood cells (WBCs) II. DNA ISOLATION CHEMISTRIES

:) I BMLS 2D | MOLBIO MIDTERM 2

a. Buccal swabs (cotton swabs/

III. PROTEOLYTIC LYSIS OF FIXED MATERIAL

3. Genomic DNA Extraction from Urine Sample

IV. NONINVASIVE HUMAN DNA ISOLATION

1. Genomic DNA Purification from Hair

:) I BMLS 2D | MOLBIO MIDTERM 3

Before isolation of RNA

VII. ISOLATION OF MITOCHONDRIAL DNA

II. PREPARATION OF SPECIMEN MATERIAL

 Reticulocytes in blood & bone marrow samples

:) I BMLS 2D | MOLBIO MIDTERM 4

III. RNA ISOLATION CHEMISTRIES

 Cell lysis V. PROTEOLYTIC LYSIS OF FIXED MATERIAL

 Begins with similar steps as described for organic

VI. ISOLATION OF polyA (MESSENGER) RNA

 RNA often required for analysis: mRNA (2.5%-5%

 1 million eukaryotic cells or 10-50 mg of tissue

:) I BMLS 2D | MOLBIO MIDTERM 5

MEASUREMENT OF THE QUALITY & QUANTITY OF

 Separation of particles through a solution or

 Nucleic acids absorb light at 260 nm through the

 DNA and RNA can be analyzed for quality

 Beer-Lambert Law: A=𝜖bc