1. 1. AKASH MALI ,India Forensic medicine Vytautas Magnus University,Lithuania.
2. 2. Molecular Pathology Testing of nucleic acids within a clinical context Helpful Hereditary disorders Oncology Infectious diseases Specific purposes Diagnosis Prognosis Prenatal testing Pharmacotherapy Pharmacogenetics Pharmacogenomics Molecular pathology employs an ever-expanding array of special techniques to study nucleic acids, genes, gene products, receptors, signaling pathways, the cell cycle, and mutations.
3. 3. Watson and Crick The structure of DNA was described by British Scientists Watson and Crick as long double helix shaped with its sugar phosphate backbone on the outside and its bases on inside; the two strand of helix run in opposite direction and are anti- parallel to each other. The DNA double helix is stabilized by hydrogen bonds between the bases Doctortvraos e learning series
4. 4. DNA A molecule contains two polynucleotide strands that form an an antiparallel double helix. Nucleotides: Nitrogenous base (AT GC,U) Deoxyribose Phosphate
5. 5. DNA makes a Copy of Self Replication is the process where DNA makes a copy of itself. Why does DNA need to copy? Simple: Cells divide for an organism to grow or reproduce, every new cell needs a copy of the DNA or instructions to know how to be a cell. DNA replicates right before a cell divides.
6. 6. DNA RNA DNA a never ending cycle RNA has the job of taking the message from the DNA to the nucleus to the ribosome's. Transcription - RNA is made from DNA Translation - Proteins are made from the message on the RNA Doctortvraos e learning series
7. 7. RNA = Ribonucleic acid. RNA is similar to DNA except: It has one strand instead of two strands. Has uracil instead of thymine 3.Has Ribose instead of Deoxyribose
8. 8. Gene Expression DNA level expression control Transcriptional Post-Transcriptional Epigenetics DNA methylation Histone modification
9. 9. Gene Expression DNA level expression control Transcriptional House keeping genes Always on Transcription factors Usually lie upstream in the promoter region Enhancer and silencer elements
10. 10. Gene Expression Post transcriptional Export of mRNA out of nucleus Alternative splicing mRNA stabilization mRNA degradation RNA interference or silencing miRNA and siRNA
11. 11. What is Gene The gene, the basic units of inheritance; it is a segment within a very long strand of DNA with specific instruction for the production of one specific protein. Genes located on chromosome on it's place or locus.
12. 12. Mutations and Polymorphisms Mutation: change in DNA sequence Polymorphism: non disease causing change in DNA or a change found at a frequency of 1% in population When evaluating changes in DNA sequence use neutral terms: sequence variant, sequence alteration or allelic variant. There may be: Missense, nonsense, deletions, insertions, frame shifts, duplications, amplifications, trinucleatide repeats.
13. 13. Single Nucleotide Polymorhisms and Haplotypes SNPs are single base differences in the DNA of individuals There are ~10 million SNPs in the human genome IMPORTANCE: Pharmacogenetics Ex. CYP (cP450) Alleles of SNPs that are close together tend to be inherited together. Haplotype: a set of associated SNPs alleles in a region of a chromosome
14. 14. Overview of Molecular Techniques and Instrumentation Standard or usual specimen flow Specimen collection (blood, tissue) Nucelic acid isolation (DNA or RNA) Nucleic acid quantification (optional) Nucleic acid storage Nucleic acid amplification (or other) Test interpretation Quality control
15. 15. Nucleic acid isolation (DNA or RNA) Manual vs. automated Cell lysis Dependent of specimen type, nucleic acid being isolated for, desired purity and application to be used in FFPE yields ~200 pairs Purification Organic: phenol-chloroform Non organic: silica, anion exchange chromatography and magnetic particles DNA or RNA Isolation RNA rapidly degrades
16. 16. Methods DNA sequencing Southern Blot PCR RT-PCR Real Time PCR Methylation-Specific PCR In-situ PCR Protein Truncation Test Transcription-Mediated Amplification Strand Displacement Amplification Nucleic Acid Sequence- Based Amplification Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
17. 17. Gene sequencing Determining the exact sequence of the four bases in a given DNA template Two methods Maxam-Gilbert Chemical degradation Sanger Chain termination Radiolabeled, Dye-prime or Dye-terminator (cycle sequencing) Pyrosequencing Sequnces a short length of DNA (~30-60 bases)
18. 18. Applications of Direct DNA sequences Clinical condition Gene HIV drug resistance HIV-protease, RT Cystic fibrosis CFTR gene Beta thalassemia Beta globin Cancer predisposition breast BRCA1 Hereditary non polyposis colon cancer TP53 MEN PTEN Ret proto-oncogene Congenital hearing loss Connexin 26 HCV genotyping 5UTR
19. 19. Array-based Comparative Genomic Hybridization Comparative Genomic Hybridization is done in metaphases in classical cytogenetics (M-CGH) Resolution 5 Mb Bacterial Artificial Chromosome (BAC) maps the human genome therefore an Array based-CGH can be created (A-CGH). Different resolutions up to 32,000 (45 kb) cDNA-CGH Oligonucleotide-CGH Can detect Single Nucleotide Pleomorphisms (SNPs) [Gene Chip]
20. 20. Methods DNA sequencing Southern Blot PCR RT-PCR Real Time PCR Methylation-Specific PCR In-situ PCR Protein Truncation Test Transcription-Mediated Amplification Strand Displacement Amplification Nucleic Acid Sequence- Based Amplification Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
21. 21. Southern Blot Edwin M Southern, 1974 DNA extracted DNA cut into pieces (Restriction Endonucleases) Electrophoresis and size separated Blot (transferred) to a membrane Anealed with labeled (radioactive, fluorescence, chemiluminescent) probe
22. 22. Southern Blot working protocol
23. 23. Uses of Southern Blotting Southern blots are used in gene discovery and mapping, evolution and development studies, diagnostics and forensics. In regards to genetically modified organisms, Southern blotting is used as a definitive test to ensure that a particular section of DNA of known genetic sequence has been successfully incorporated into the genome of the host organism. Used in prognosis of cancer and in prenatal diagnosis of genetic diseases
24. 24. Methods DNA sequencing Southern Blot PCR RT-PCR Real Time PCR Methylation-Specific PCR In-situ PCR Protein Truncation Test Transcription-Mediated Amplification Strand Displacement Amplification Nucleic Acid Sequence- Based Amplification Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
25. 25. PCR Kary B. Mullis 1983 Target amplification Single oligonucletide Multiplexed Mimics the natural process of DNA replication, therefore, requires: DNA template, DNA polymerase, dNTPs, buffer, Mg++, two primers to flag the target sequence Thermal cycler Denaturation ~95C Annealing ~45-60C Extension ~72C
26. 26. PCR Denaturation Breaks the hydrogen bonds between the ds-DNA Anealing Binding to oligonucleotide sequence (probe) Extension DNA polymerase (heat stable, Taq [Thermophilus aquaticus]) replicates the selected DNA sequence Xn = X0 (1 + E)n E= 0 - 1
27. 27. RT-PCR To detect or quantify RNA transcripts or viral RNA RNA is converted to DNA Reverse transcriptase (Avian Myeloblastosis Virus and Moloney Murine Leukemia virus) Isothermal reaction with primers: oligo dT, random hexamer primers, or target specific primers One step vs. two steps
28. 28. PCR or RT-PCR Product analysis / detection Real Time Hybridization Membrane bound Reverse line blots Liquid Bead Array with Flow Cytometry Electrophoresis Agarose Capillary Cycle sequencer
29. 29. Multiplexed PCR and ELISA Protein Expression Profiling Cancer Markers Cardiac Markers Cellular Signaling Cytokines, Chemokines, and Growth Factors Endocrine Isotyping Matrix Metalloproteinases Metabolic Markers Neurobiology Transcription Factors/Nuclear Receptors Genomic Research FlexmiR v2 Custom microRNA Assay FlexmiR microRNA Panels Gene Expression Profiling Genotyping Genetic Disease Cystic Fibrosis Cytochrome p450 Immunodiagnostics Allergy Testing Autoimmune Disease HLA Testing Infectious Disease Vaccine Testing Newborn Screening Biodefense/Environmental
30. 30. Real Time - PCR Amplifies and detects PCR product fluorescently in each well of PCR plate Dont have to run gel afterwards Use for endpoint detection Examples Fast PCR screening without gels Locate clone or mutant of interest Genotyping SNPs Genotype individuals using allele specific primers
31. 31. PCR Advantages Disadvantages Sensitivity Specificity Speed Versatility Automated No need for intact DNA/RNA Target sequence needs to be known Target needs to be conserved among individuals (polymorphisms) Oligonucleotide length Can fail in the detection of chromosomal abnormalities like translocations, inversions, large addition or deletions Contamination (F+)
32. 32. Methods DNA sequencing Southern Blot PCR RT-PCR Real Time PCR Methylation-Specific PCR In-situ PCR Protein Truncation Test Transcription-Mediated Amplification Strand Displacement Amplification Nucleic Acid Sequence- Based Amplification Signal amplification Branching DNA Hybrid Capture Invader FISH DNA arrays and chips
33. 33. Branched DNA applications Detection HIV, HBV, and HCV Measures viral loads Less sensitive than PCR Doctortvraos e learning series
34. 34. Hybrid Capture Qiagen Signal amplification technique Denaturated DNA gets hybridized to complimentary unlabeled RNA sequences (if DNA sequence is present) Antibody bound to the well is attracted to RNA:DNA hybrids A second conjugated anti RNA:DNA hybrid antibody is added Chemiluminescent signal is generated in proportion of target DNA present
35. 35. Applications in Anatomic Pathology 1. Anatomic Pathology Testing to Detect or Characterize Neoplasia. 2. Molecular Anatomic Testing for Targeted Therapies. 3. Anatomic Pathology Testing for Infectious Agents.
36. 36. 1. P.T. Cagle, T.C. Alan (Eds.), Basic Concepts of Molecular Pathology. Molecular Pathology Library, vol. 2, Springer Science and Business Media, 2009.