Transcript of basic concept of molecular pathology
- 1. AKASH MALI ,India Forensic medicine Vytautas Magnus
University,Lithuania.
- 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. 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. DNA A molecule contains two polynucleotide strands that form
an an antiparallel double helix. Nucleotides: Nitrogenous base (AT
GC,U) Deoxyribose Phosphate
- 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. 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
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- 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. Gene Expression DNA level expression control Transcriptional
Post-Transcriptional Epigenetics DNA methylation Histone
modification
- 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. Gene Expression Post transcriptional Export of mRNA out of
nucleus Alternative splicing mRNA stabilization mRNA degradation
RNA interference or silencing miRNA and siRNA
- 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Southern Blot working protocol
- 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Branched DNA applications Detection HIV, HBV, and HCV
Measures viral loads Less sensitive than PCR Doctortvraos e
learning series
- 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. 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. 1. P.T. Cagle, T.C. Alan (Eds.), Basic Concepts of
Molecular Pathology. Molecular Pathology Library, vol. 2, Springer
Science and Business Media, 2009.