MODERN METHODS IN MOLECULAR PATHOLOGY

Presented By: Safia Nisar

Sumaiya Gul

What makes them Important?

Increasing Knowledge of Molecular basis of the disease

Advances in technology for analyzing nucleic acids and gene products

Recent introduction of array technology help to analyze multiple genes, transcripts and Proteins

Before Moving on…

• A pathologist needs to have adequate Knowledge of

Human Genetics

Molecular Biology

Microbiology

Molecular Methods may be Used for

• Infectious Disease Testing• Molecular Oncology• Molecular Genetics• Histocompatibilty

Infectious Disease Testing

• Performed most frequently• Quantification of Infectious agents• Identification of difficult or impossible to

cultivate agents• Identification of antiviral or antibiotic

resistance genes• Identification of toxin genes

Molecular Oncology

• Advances in the Knowledge of biology of leukemias and lymphomas

• Understanding the molecular genetics of solid tumors

• Genetic alterations associated with the occurrence of neoplasia

Molecular Genetics

• Sub-discipline of Medical genetics• Involves discovery and Laboratory testing for

DNA that underlie single gene disorders• May also be used in the diagnosis of

syndromes involving epigenetic abnormalities

Single Gene Disorders

Achondroplasia Cystic FibrosisDuchene Muscular distrophy

Huntington Disease

Hereditary Breast Cancer

SYNDROMES

Angleman Syndrome

Beckwith-Wiedeman syndrome

Prader-willi Syndrome

Uniparental disomy

Histocompatibility

• Transformation from Serologic testing to DNA based identification

• More precision and accuracy• Sequence specific PCR• Sequence specific oligonucleotide

hybridization• DNA sequencing of PCR products

DNA EXTRACTION

• Routine procedure to collect DNA for molecular or Forensic analysis

• Steps Involved

Cell LysisRemoval of membrane

lipids

Removal of Proteins

Removal of RNA

Precipitation of DNA by

alcohol

RNA Extraction

• Purification of RNA• Somewhat complicated• Guanidinium thiocyanate-phenol-chloroform

extraction

The Methods

• PCR (Polymerase Chain Reaction)• LCR (Ligase Chain Reaction)• SDA (Strand Displacement Amplification)• Hybridization• Diagnostic Markers• BDA (Branched DNA Assay)• TMA (Transcription Mediated Amplification)

Polymerase Chain Reaction

• Makes large copies of a gene• Generally three step; Denaturation, Anealing

and Extension• Amount of DNA is insufficient otherwise• Diagnosis of Disease: Linkage analysis,

detection of mutant alleles, diagnosing infectious agents, epidemiological studies

Ligase Chain Reaction

• DNA Amplification Technique• Amplification of the nucleic acid used as probe• For each of the two DNA strands, two partial probes

are ligated to form the actual one; uses DNA Polymerase and DNA Ligase

• Each cycle results in the doubling of target nucleic acid molecule

• Greater Specifity• Used for Single base mutations and genetic disorders

Strand Displacement Amplification

• An isothermal nucleic acid amplification method• Primer contains a restriction site annealed to template• Amplification primers are then annealed to 5' adjacent

sequences (form a nick) and start amplification at a fixed temperature

• Newly synthesized DNA are nicked by a restriction enzyme

• Polymerase starts amplification again, displacing the newly synthesized strands

• 109 copies of DNA can be made in one reaction

Hybridization

• In situ hybridization (ISH) is a powerful technique for localizing specific nucleic acid targets within fixed tissues and cells, allowing you to obtain temporal and spatial information about gene expression and genetic loci.

How it works

• The nucleic acid probe is synthesized, labeled, purified, and annealed with the specific target

• The difference is the greater amount of information gained by visualizing the results within the tissue.

TWO TYPES

• Today there are two basic ways to visualize RNA and DNA targets in situ:

1. Fluorescence (FISH) and 2. Chromogenic (CISH) detection.

Fluorescence In Situ Hybridization (FISH)

• Multiplex fluorescence in situ hybridization (FISH) exemplifies the elegance that only fluorescence-based strategies offer: the ability to assay multiple targets simultaneously and visualize co-localization within a single specimen. Using spectrally distinct fluorophore labels for each different hybridization probe, this approach gives you the power to resolve several genetic elements or multiple gene expression patterns in a single specimen, with multicolor visual display.

Chromogenic In Situ hybridization

• A process in which a labeled complementary DNA or RNA strand is used to localize a specific DNA or RNA sequence in a tissue specimen. CISH methodology may be used to evaluate gene amplification, gene deletion, chromosome translocation, and chromosome number.

Diagnostic Markers

• Also known as a biomarker, it is a measurable characteristic that reflects the severity or presence of some disease state. More generally a biomarker is anything that can be used as an indicator of a particular disease state or some other physiological state of an organism.

• A biomarker can be a substance that is introduced into an organism as a means to examine organ function or other aspects of health. For example, rubidium chloride is used as a radioactive isotope to evaluate perfusion of heart muscle. It can also be a substance whose detection indicates a particular disease state, for example, the presence of an antibody may indicate an infection. More specifically, a biomarker indicates a change in expression or state of a protein that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment.

BDA: Branched DNA Assay

• Branched DNA assay is a signal amplification assay (as opposed to a target amplification assay) that is used to detect nucleic acid molecules.

Steps in BDA

1. From the base up, a branched DNA assay begins with a dish or some other solid support (e.g., a plastic dipstick). The dish is peppered with small, single stranded DNA molecules (or chains) that 'stick up' into the air. We'll call these "capture probe" DNA molecules.

2. An "extender" DNA molecule is added. Each "extender" has two domains, one that hybridizes to the capture DNA molecule and one that "hangs out" in the air. The purpose of the extender is two-fold. First, it creates more available surface area for target DNA molecules to bind, and second, it allows the assay to be easily adapted to detect a variety of target DNA molecules.

3. Once the capture and extender molecules are in place and they have hybridized, the sample can be added. Target molecules in the sample will bind to the extender molecule. So we have a base peppered with capture probes, which are hybridized to extender probes, which in turn are hybridized to target molecules.

4. At this point, signal amplification takes place. A "label extender" DNA molecule is added that has two domains (similar to the first extender). The label extender hybridizes to the target and to a "pre-amplifier" molecule. The preamplifier molecule has two domains. First, it binds to the label extender and second, it binds to the amplifier molecule. An example amplifier molecule is an oligonucleotide chain bound to the enzyme alkaline phosphatase.

TMA: Transcription Mediated Amplification

• Transcription-Mediated Amplification (TMA) is an RNA transcription-mediated amplification system using two enzymes to drive the reaction: RNA polymerase and reverse transcriptase.

Properties of TMA

• TMA is isothermal; the entire reaction is performed at the same temperature in a water bath or heat block. This is in contrast to other amplification reactions such as PCR that require a thermal cycler instrument to rapidly change the temperature to drive reaction.

• TMA can amplify either DNA or RNA, and produces RNA amplicon, in contrast to most other nucleic acid amplification methods that only produce DNA.

• TMA has very rapid kinetics, resulting in a billion-fold amplification with 15-60 minutes. TMA can be combined with HPA for endpoint detection or with molecular torches for real-time detection.

• There are no wash steps, and no amplicon is ever transferred out of the tube, which simplifies the procedure and reduces the potential for contamination.