Molecular

Pathology
Dr. Fahd Al-Mulla
15-11-2006
Molecular Basis of Diseases I
Fundamentals and Techniques
 Learning
Objectives
• Definitions of: Molecular Biology and Pathology
• Basics of Molecular Biology; DNA, RNA, Protein structures,
transcription and translation and genetic control.
• Basic techniques applied to DNA; PCR, Southern blotting, and
Sequencing
• Basic techniques applied to RNA; northern blots, RT-PCR
• Basic techniques applied to proteins; western blotting,
Immunohistochemistry
• In situ hybridisation (Cytogenetics), FISH
• Basic principles of Microarray
 What is Molecular Pathology?

• Pathology: is the study of diseases.

• Molecular biology: the study of molecules in biological
systems that are responsible for normal biological traits or
behaviors i.e.: DNA replication, transcription and translation in
normal cells.
• Molecular pathology: an evolving field that examines and
identifies the molecules involved in specific diseases.
• Integrates knowledge and techniques applied in molecular
biology to pathology.
 Molecular Pathology:
Rationale

• Classical pathologists examine tissue sections stained with

Haematoxilin and Eosin (H&E) and other stains, and is able to
know the issues origin, organization and what disease it
represents. However, this is an art involving human skill, not
science.
• A pathologist is unable to define the molecules and how they
interact to produce the disease represented by what is
observed microscopically. This is the job of a molecular
pathologist.
• The molecular pathologist utilizes techniques from molecular
biology to study differences between normal and diseased
tissue at the molecular level, so that the specific molecules
associated with the disease maybe identified.
Relevance of Molecular Pathology
• Provides a more comprehensive understanding of a
disease, it’s natural history, and progression.
• Elucidates the causes of the disease (viruses, hereditary,
disruptions of the normal control processes, such as the
cell-cycle, apoptosis etc…)
• Associates specific molecules or a set of molecules with
the prognosis of a disease.
• Provides an understanding of the overall complexity of
the disease.
• Enables new treatment modalities for specific diseases.
 Nucleic Acids

•Nucleic Acids: Are macromolecular structures which store and

express all the information necessary for building and
maintaining life.

•Two Types:
-DNA (DeoxyriboNucleic Acid): is the repository
of the genetic code and information.
-RNA’s (RiboNucleic Acids): are regarded as vectors
and translators of the information contained in the DNA.

•From a chemical standpoint, Nucleic Acids are giant linear

condensation polymers of Nucleotide sub-units.
 A Nucleotide consists of three basic units:
1) A nitrogenous base: Either a purine (Adenine (A) or Guanine (G)), or a
pyrimidine: (Cytosine (C) or Thymine (T) (or Uracil (U) in the case of RNA).
2) A sugar : Deoxyribose (DNA) or Ribose (RNA).
3) A phosphate group.

 A sugar and a base form a Nucleoside, and a Nucleotide is a phosphorylated

nucleoside.
 Inter-nucleotide linkages are formed by a phosphodiester bond between a
5'-phosphate group and the 3'-hydroxyl group of the next nucleotide sugar.

 The nucleotide sequence encodes the blueprint necessary to construct proteins.

 DNA: Structure
• DNA consists of two unbranched polynucleotide chains (strands) held
together in an antiparallel manner by hydrogen bonds formed between
specific pairs of bases [Adenine-Thymine] [Guanine-Cytosine].
• The sequence (code) of bases on one strand determines the sequence
of the other strand. This is also known as complementarity.
• The joined anti-parallel strands are twisted about each other in the shape
of a right-handed double helix.
• The DNA double helix can be visualized as a twisted ladder in which
rungs are bases pairing and sides are deoxyribose-phosphate chains.
 DNA: Structure
 In addition to the stabilizing effect of the hydrogen bonds on the double
helix, Van der Waals forces and hydrophobic interactions are also
involved in its stabilization. The hydrophobic bases stacked on the
inside and the hydrophilic ribose-phosphate chains are on the outside
also confer this effect.

 The N-glycosic bonds (sugar-base) are not directly opposite one

another on the strands. Therefore two grooves of different width appear
between ribose-phosphate chains on the surface of the molecule.

 These are called major or minor grooves, and they allow bases to be
exposed to solvents and to other molecules, thus enabling chemical and
biochemical substances to interact with specific bases without disrupting
the double helix structure.
<>
Ha, S. C., Lowenhaupt,
K., Rich, A., Kim, Y. G. &
Kim, K. K. Nature 437,
1183-1186 (2005).
 DNA :
Packaging
• In Prokaryotic cells the DNA molecule is in the form of a circle which is
coiled into a super helix and often organized into a compact structure
containing various proteins and RNAs called Nucleoid.

• In Eukariotic cells, the DNA is packaged in Chromatin within the

nucleus.

• Nucleosomes are the fundamental structural packing units of

chromatin.

• A nucleosome is a complex of DNA tightly wrapped around basic

proteins called Histones. These help to stabilize and determine
chromatin structure. The nucleosome core consist of two tetrameric
molecules, each having four histone-subunits (H2A, H2B, H3 and H4).
 DNA:
Chromosomes
• A cell divides by means of a process called Mitosis.

• During Interphase or the “rest” phase, chromatin exist as a tangle of

fibers of 10-30 nm diameter and 0.25-2 mm length.

• The unfolded (beads-on-a-string) regions are referred to as

euchromatin and the more condensed ones as heterochromatin.

• Immediately prior to a cell division, the chromatin condenses into

metaphase chromosomes, and the DNA packaging factor increase
dramatically from 50 to about 7000.
 DNA:
Chromosomes
• A chromosome is made of two identical, symmetrical DNA molecules
called chromatids, joined by a centromere which attach them to the
mitotic spindle.

• Each chromosome contains 2 chromatids, 1 centromere, 4 telomeres

(ends of DNA molecules)

• Human cells contain 46 chromosomes (23 pairs), one set inherited

from each parent.

• There are 24 different chromosomes: 22 autosomes and two sex

chromosomes which define the gender: X for female (XX), Y for male
(XY).
 DNA: Genes
• Genes are specific sequences of nucleotides, encoding information
needed for constructing mRNA then proteins. They are the fundamental
physical units of heredity.
• A Genome is the complete set of genetic information for an organism
encoded by the DNA (or RNA in some viruses). It is made of three
different types of DNA :
1) Single Copy DNA consist of genes, found in only one or few places
in the genome. It also includes multiple copy genes such as those
coding for rRNA or Histones which exist as large clusters of multiple
copies (50-10000 copies).
2) Repetitive dispersed DNA fractions are short sequences repeated
100000 - 1,000,000 times in different places throughout the genome.
3) Satellite DNA is made of highly repetitive sequences, found in the
chromosomes centromere and telomeres.
 DNA:
Genes
• The human genome is estimated to comprise about 3 billions
nucleotide pairs and at least 30,000 genes. However it seems that
there is no correlation between complexity of an organism and the
number of nucleotide pairs (np) per haploid genome.

• Some plants and amphibian organisms have total DNA amount of

about 100 billions np (30 times more than human), even the genome
of higher eukaryotes seems to contain a large excess of DNA.

• In mammals, only about 10% of the genome is known to be

expressed in protein encoding or in regulation processing.

• This excess of DNA is supposed to serve as a hiding package or

decoy, preventing genes from accidental mutations.
 DNA: Genes

•Genes sizes vary widely in length, from hundreds

to several thousand nucleotide pairs.
•Even in longer genes only a small part of the
sequence is used to encode information.
•The coding regions are named exons and the
non-coding interrupting sequences are called
introns.
•Generally the more complex and recently evolved
the organism, the more numerous and larger the
introns.
•Some specific DNA regions are dedicated to the
control of genes expression. These regulatory
sequences are often located at the beginning of the
gene (5‘ side), at its end (3‘ side) but also
sometimes in introns or in exons.
 RNA :
Structure
 RNA is the messenger in the cell. It carries information from
the
chromosomes to the ribosomes, the protein manufacturing
centers of the cell.
 Physically, RNA consists of a single long strand composed of
nucleotides or bases, and uses the same nucleotides as DNA
except thymine is replaced by a nucleotide called uracil (U).
 There are three classes of RNA:
1) messenger RNA (mRNA)
2) transfer RNA (tRNA)
3) ribosomal RNA (rRNA)
RNA: mRNA

mRNA is transcribed from

DNA, carrying information for
protein synthesis. Three
consecutive nucleotides in
mRNA encode an amino acid
or a stop signal for protein
synthesis. The trinucleotide
is know as a codon.
RNA: tRNA

The major role of tRNA is

to translate an mRNA
sequence into an amino
acid sequence. A tRNA
molecule consists of 70-80
nucleotides.
RNA:
rRNA
After rRNA molecules are produced
in the nucleus, they are transported to
the cytoplasm, where they combine
with tens of specific proteins to form a
ribosome. The ribosome moves one
codon down the mRNA chain in
protein synthesis (translation). This
is repeated in the next cycles of
elongation. Protein synthesis will
terminate when the ribosome arrives
at one of three stop codons.
 Techniques: PCR

• PCR was first conceived in 1983 by Kary Mullis, a molecular

biologist who received a Nobel Prize for the discovery 10 years
later
• A PCR (Polymerase Chain Reaction) is performed in order to
make a large number of copies of a gene. Otherwise, the
quantity of DNA is insufficient and cannot be used for other
methods such as sequencing.

• A PCR is performed on an automated cycler, which heats and

cools the tubes with the reaction mixture in a very short time.

• Performed for 30-40 cycles, in three major steps:

1)denaturation, 2)annealing, and 3)extension.
 Techniques: PCR

• 1) Denaturation at 94°C :
During the denaturation, the double strand melts open to single
stranded DNA, all enzymatic reactions halt.

• 2) Annealing at 54°C :
The primers are freely moving due to Brownian motion. Ionic bonds
are constantly formed and broken between the single stranded primer
and the single stranded template.

• Primers that fit exactly will have stable bonds that last longer. The
polymerase attaches onto a piece of double stranded DNA (which is
template and primer), and starts copying the template. Once there are
a few bases built in, the ionic bond is so strong between the template
and the primer, that it does not break anymore.
 Techniques: PCR

• 3) Extension at 72°C :
• This temperature is ideal for the polymerase. The primers,
which have a few bases built in, already have a stronger ionic
attraction to the template than the forces breaking these
attractions.
• Primers that are on positions with no exact match, loosen their
bonds again (because of the higher temperature) and do not
extend the fragment.
The bases (complementary to the template) are coupled to the
primer on the 3' side (the polymerase adds dNTP's from 5' to
3', reading the template from 3' to 5' side, bases are added
complementary to the template)
 Techniques: PCR

• At the end of a PCR, the product must be checked before it is used in

further applications. This is to confirm:
• There is a product formed: Not every PCR is successful. There is
a possibility that the quality of the DNA is poor, that one of the
primers doesn't fit, or that there is too much starting template.
• The product is of the right size: It is possible that there is a
product, for example a band of 500 bases, but the expected gene
should be 1800 bases long. In that case, one of the primers probably
fits on a part of the gene closer to the other primer. It is also possible
that both primers fit on a totally different gene.
• Only one band is formed: As in the description above, it is possible
that the primers fit on the desired locations, and also on other
locations. In that case, you can have different bands in one lane on a
gel.
The ladder is a mixture of fragments with known size to compare with
the PCR fragments. Notice that the distance between the different
fragments of the ladder is logarithmic. Lane 1 : PCR fragment is
approximately 1850 bases long. Lane 2 and 4 : the fragments are
approximately 800 bases long. Lane 3 : no product is formed, so the
PCR failed. Lane 5 : multiple bands are formed because one of the
primers fits on different places.
• Applications of PCR:
• 1) Diagnosis of Disease: Linkage analysis, detection of mutant
alleles, diagnosing infectious agents, epidemiological studies
• 2) Forensics: paternity testing, DNA typing for identification, criminal
investigations.
• 3)Recombinat DNA engineering
• 4) DNA sequence determination
• 5) new gene isolation
• 6) Anthropological studies: population genetics, migration studies.
• 7) Evolution studies
 Techniques: RT-PCR
 An RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly
sensitive technique for the detection and quantitation of mRNA (messenger
RNA).
 The technique consists of two parts:
1) The synthesis of cDNA (complementary DNA) from RNA by
reverse transcription (RT)
2) The amplification of a specific cDNA by PCR.
Compared to Northern blot analysis and RNase protection assay used to
quantify mRNA, RT-PCR can be used from much smaller samples. It is
sensitive enough to enable quantitation of RNA from a single cell.
 Real-time RT-PCR is the method of choice for quantitating changes in gene
expression. Furthermore, real-time RT-PCR is the preferred method for
validating results obtained from array analyses and other techniques that
evaluate gene expression changes.
 Techniques: Southern Blot

• Southern Blotting (named after Ed Southern, the inventor) is the

detection of specific sequences of DNA on a gel by hybridisation with a
labelled DNA probe.

• DNA is first transferred out of a gel by capilliarity (the "blot") to a thin

membrane which can be incubated with a probe and washed.

• By hybridising at different temperatures, and washing to different ionic

strengths ("stringencies") it is possible to tune the process to pick up
sequences that are either similar, or exactly identical, to the probe.
 Techniques: Southern Blot
• Applications:

• 1) To confirm the presence of a gene, often in conjunction with PCR.

• 2) To test for the presence of a specific allele of a gene (i.e. human
disease genetics).
• 3) To estimate gene complexity, before you have the gene sequence.
• 4) To detect Restriction Fragment Length Polymorphism (RFLP) and
Variable Number of Tandem Repeat Polymorphism (VNTR). The
latter is the basis of DNA fingerprinting.
 Techniques: Southern Blot
• Other uses for Southern blotting:

• It is the standard way to screen either a genomic or cDNA library

("plaque lifts"). Similarly, it can be used to identify a bacterial colony
carrying a desired plasmid / insert ("colony lifts").

• If genomic DNA is cut with several restriction enzymes, and the gel
probed for a specific gene, the number of bands in each lane gives an
indication as to whether there are single or multiple copies of the gene
in the genome.
Techniques: Southern Blot
Techniques:
Southern Blot
Technique: Southern Blot
 Techniques: Northern Blots

 Northern blots are similar to Southern, except that RNA from different
tissues is run out on a gel, and probed with a DNA or RNA probe
corresponding to a particular gene.

 Northern blotting is used for detecting and quantitation of RNA

fragments, instead of DNA fragments. The technique is exactly like
Southern Blotting. It is called "Northern" simply because it is similar
to "Southern", not because it was invented by a person named
"Northern".
 RNA samples are first separated by size via electrophoresis in an
agarose gel under denaturing conditions. The RNA is then
transferred to a membrane, crosslinked and hybridized with a labeled
probe.
 Techniques: Western Blot
• Western blot analysis can detect one protein in a mixture of any
number of proteins while giving you information about the size of the
protein.
• Allows investigators to determine with a specific primary antibody, the
relative amounts of the protein present in different samples.
 Western blots are analogous to Northern and Southern, except that
proteins are run out in an SDS polyacrylamide gel, and are detected
with specific antibodies.
 In clinical settings, Western Blotting is routinely used to confirm
serious diagnosis suggested by ELISA such as HIV seroconversion
 Nucleic Acid Hybridization

• The Basic Process of Binding a Single Strand of Nucleic Acid (DNA or

RNA) to Its Complementary Strand Is Called Nucleic Acid
Hybridization.
• Double-stranded DNA Can Be Denatured by Agents Such As Heat or
High PH. When Denatured, the Two Strands Separate Into Single
Strands and Diffuse Away From Each Other. If Conditions Are Then
(Slowly) Reversed (Lower the Temperature or Return the PH to
Neutrality) Then the DNA Will renature.
• If the Temperature Is Slowly Decreased, Then Each Strand of DNA
Will Find Its Corresponding Mate: the Complementary Strands of the
DNA Will Anneal and Re-form the Double Strand With Correct Watson-
crick If a Radioactively Labeled Probe Corresponding to a Part of the
Sequence of One of the Fragment Is Included in the renaturation
Mixture, It Will Participate in the renaturation, Finding and Annealing to
Its Complementary Partner
Nucleic Acid Hybridization

Probe present
No probe
A
C
C
C
T
G
C
G
 FISH
• Fluorescence In-Situ Hybridization is a method used to identify specific
parts of a chromosome. For example, if you know the sequence of a
certain gene, but you don't know on which chromosome the gene is
located, you can use FISH to identify the chromosome in question and the
exact location of the gene.
• If you suspect that there has been a translocation in a chromosome, you
can use a probe that spans the site of breakage/translocation. If there has
been no translocation at that point, you will see one signal, since the
probe hybridizes to one place on the chromosome. If, however, there has
been a translocation, you will see two signals, since the probe can
hybridize to both ends of the translocation point.
• To use FISH efficiently, you have to know what you're looking for, i.e. you
usually suspect a particular defect, based on the appearance of certain
chromosomes, etc.
 FISH

• Method:

• Make a probe complementary to the known sequence. When making

the probe, label it with a fluorescent marker, e.g. digoxigenin, by
incorporating nucleotides that have the marker attached to them.
• Put the chromosomes on a microscope slide and denature them.
• Denature the probe and add it to the microscope slide, letting the probe
hybridize to its complementary site.
• Wash off the excess probe and look at the chromosomes in a
fluorescence microscope. The probe will show as one or more
fluorescent signals in the microscope, depending on how many sites it
can hybridize to.
 FISH

• Applications

• Diagnosis in clinical and cancer cytogenetics.

• Interspecies studies of evolutionary divergence.
• Analysis of aberrations in animal models of human diseases.
• Many more applications. THINK
Interphase FISH
Metaphase FISH
FISH
 Techniques: Microarray

 DNA microarrays allow researchers to analyze the expression

of thousands of genes simultaneously.
 DNA microarrays contain thousands of individual gene
sequences in microscopic spots of ≈1-kb DNA sequences
representing thousands of genes bound to the surface of glass
microscope slides.
 Provide a means for analyzing gene expression patterns on a
genomic scale.
 Provides a medium for matching known and unknown DNA
samples based on base-pairing rules and automating the
process of identification.
Techniques: Microarray
 Techniques: Microarray
• Applications
• Gene discovery
• Disease diagnosis
• Drugs and toxicological research : The goal of pharmacogenomics is to
find correlations between therapeutic responses to drugs and the genetic
profiles of patients.
• Expression screening. The focus of most current microarray-based
studies is the monitoring of RNA expression levels which can be done by
using either cDNA clone microarrays or gene-specific oligonucleotide
microarray
• Screening of DNA variation. There is also huge potential for assaying in
drug development and patient susceptibility, as well as for mutations in
known disease genes such as cardiovascular disease and cancer as seen
in the case of the breast cancer susceptibility gene, BRCA1.
• In addition, there have been vigorous efforts to identify and catalog
• human single nucleotide polymorphism (SNP) markers.
Thank you
• Thanks to Altaf, Waleed and Sindhu
• Concentrate on the basic information
• Any questions?