1. Presented By: Safia Nisar Sumaiya Gul

2. 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

3. A pathologist needs to have adequate Knowledge of Human Genetics Molecular Biology Microbiology

4. Infectious Disease Testing Molecular Oncology Molecular Genetics Histocompatibilty

5. 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

6. 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

7. 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

8. AchondroplasiaCystic Fibrosis Duchene Muscular distrophy Huntington Disease Hereditary Breast Cancer

9. Angleman Syndrome Beckwith- Wiedeman syndrome Prader-willi Syndrome Uniparental disomy

10. Transformation from Serologic testing to DNA based identification More precision and accuracy Sequence specific PCR Sequence specific oligonucleotide hybridization DNA sequencing of PCR products

11. Routine procedure to collect DNA for molecular or Forensic analysis Steps Involved Cell Lysis Removal of membrane lipids Removal of Proteins Removal of RNA Precipitation of DNA by alcohol

12. Purification of RNA Somewhat complicated Guanidinium thiocyanate-phenol-chloroform extraction

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

14. 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

15. 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

17. 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 10 9 copies of DNA can be made in one reaction

18. 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.

19. 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. 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.

20. Today there are two basic ways to visualize RNA and DNA targets in situ: 1.Fluorescence (FISH) and 2.Chromogenic (CISH) detection. Today there are two basic ways to visualize RNA and DNA targets in situ: 1.Fluorescence (FISH) and 2.Chromogenic (CISH) detection.

21. 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.

22. 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.

24. 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.

25. 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.

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

27. 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.

28. 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.

29. 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.

30. 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.

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

33. 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.

34. 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. 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.