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 Watsoncrick 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?

Sign up to vote on this title
UsefulNot useful