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Introduction to Molecular Pathology

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  • ONE STEP: RT + DNA polymerase OR rTth (Thermusthemophilus) that works as an RT and DNA polymerase
  • Hybridization probe: Two separate, single-labeled probes anneal to target bringing donor and reporter into proximityDual-labeled hydrolysis probe: When the reporter and quencher fluorophores are in proximity on the probe – there is no signal. Once the 5’nuclease activity of the DNA polymerase hydrolyses the end nucleotides from the probe where the reporter is. The reporter emits a signal.Minor groove binding probes (MGB): like hydrolysis probes, technique to stabilize shorter probes.Molecular beacon probes: Annealing to the target after denaturation allows the reporter fluorophore to escape the quenching effect, therefore giving a signal
  • The QuantiGenePlex 2.0 System measures mRNA levels directly from a number of sources including, cultured cell lysates, tissue homogenates, dried blood spots, formalin fixed paraffin embedded (FFPE) sections or purified RNA.  It utilizes branched DNA (bDNA) technology, which relies on cooperative hybridization between the target mRNA and a specific probe set.  The probe sets consist of three types of oligonucleotides, Capture Extenders (CEs), Label Extenders (LEs), and Blocking Probes (BLs), whose sequences are selected based on the sequence of the target mRNA.  The Capture Extenders (CEs) are oligonucleotides with roughly half of the sequence being complimentary to sections of the target mRNA and the other half complimentary to the Capture probes (CP) immobilized onto the Capture beads.  The Label Extenders (LEs) are also oligonucleotides with half of the sequence being complimentary to the target mRNA and the other half complimentary to a portion of the Pre-amplifier.  The Blocking Probes (BLs) are complimentary to regions of the target mRNA not recognized by either the CE or the LE oligonucleotides and serve to reduce non-specific hybridization. The CEs provide the assay specificity by binding the target mRNA to the capture bead. Signal amplification is the result of LEs hybridizing with the target mRNA and the Pre-Amplifier sequences. The Pre-Amplifier trunk is then hybridized with multiple Amplifiers that form the branches of bDNA.   The “branches” of each amplifier have multiple biotin moieties, which will in turn bind Streptavidin-conjugated R-phycoerythrin (SAPE), the measureable signal for detection.  Using this technology, the amplification occurs with the signal rather than the target.  The signal is proportional to the target mRNA and can be achieved without purification or amplification (Figure 2).  The assay is designed to be compatible in batch or completely automated modes using BioTek’s ELx405 Magnetic Bead Washer.

Transcript

  • 1. Introduction to Molecular Pathology
    Luis E. Ferrer Torres, MD FCAP
  • 2. Molecular Pathology
    Testing of nucleic acids within a clinical context
    Helpful
    Hereditary disorders
    Oncology
    Infectious diseases
  • 3. Molecular Pathology
    Specific purposes
    Diagnosis
    Prognosis
    Prenatal testing
    Pharmacotherapy
    Pharmacogenetics
    Pharmacogenomics
  • 4.
  • 5. Watson and Crick
    The structure of DNAwas described by British Scientists Watson andCrickas 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 DNAdouble helix is stabilized by hydrogen bonds between the bases
    Doctortvrao’s ‘e’ learning series
  • 6. Watson and Crick discovers DNA / Feb 28th 1953
  • 7. Watson and Crick Builds a Model DNA 7th March 1953
  • 8. First Document on DNA published in Nature 25th April 1953
  • 9. Watson and Crick - Awarded Nobel Prize in 1962
  • 10. DNA
    A molecule contains two polynucleotide strands that form an an antiparallel double helix.
    Nucleotides:
    Nitrogenous base (AT GC,U)
    Deoxyribose
    Phosphate
  • 11.
  • 12. DNA - Structure
    The nucleotide, however, remains as the fundamental unit (monomer) of the nucleic acid polymer. There are four nucleotides: those with cytosine (C), those with guanine (G), those with adenine (A), and those with thymine (T).
  • 13. DNA
    A purine always links with a pyrimidine base to maintain the structure of DNA.
    Adenine ( A ) binds to Thymine ( T ), with two hydrogen bonds between them.
    Guanine ( G ) binds to Cytosine ( C ), with three hydrogen bonds between them.
  • 14. Chemical structure of DNA
  • 15. DNA is Endless structure
    The rungs of the ladder can occur in any order (as long as the base-pair rule is followed)
    Those 4 bases have endless combinations just like the letters of the alphabet can combine to make different words.
  • 16. DNA
    Example
    First strand GGGTTTAAACCC
    Second strand CCCAAATTTGGG
  • 17. Central Dogma of Molecular Biology
  • 18.
  • 19.
  • 20. 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.
  • 21. DNA Replication
    DNA replication is semi-conservative. That means that when it makes a copy, one half of the old strand is always kept in the new strand. This helps reduce the number of copy errors.
    So we remained what we were ?
  • 22. Transcription
    RNA polymerase II mediates transcription and generates a precursor ss-mRNA identical to the sense (coding) stand except for U for T.
    Precursor ss-mRNA is processed in nucleus by spliceosomes that catalyze intron removal and exon ligation with the regulation by exonic and intronic enhancers and silencers with production of different pre m-RNA that go across nucleus
  • 23. DNA to RNAcreates functional translations
    DNA remains in the nucleus, but in order for it to get its instructions translated into proteins, it must send its message to the ribosome's, where proteins are made. The chemical used to carry this message is Messenger RNA
    Doctortvrao’s ‘e’ learning series
  • 24. DNA – RNA – DNAa 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
    Doctortvrao’s ‘e’ learning series
  • 25. Translation
    m-RNA directs protein synthesis.
    Occurs in ribosomes (rRNA+proteins)
    Codons (three bases) are read by transfer (tRNA)
    There are 64 possible codons, therefore most of the 21 aminoacids are specified by more than 1 codon
  • 26. RNA = Ribonucleic acid.
    RNA is similar to DNA except:
    It has one strand instead of two strands. Has uracil instead of thymine3.Has Ribose instead of Deoxyribose
     
  • 27.
  • 28. Gene Expression
    DNA level expression control
    Transcriptional
    Post-Transcriptional
    Epigenetics
    DNA methylation
    Histone modification
  • 29. 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
  • 30. Gene Expression
    Post transcriptional
    Export of mRNA out of nucleus
    Alternative splicing
    mRNA stabilization
    mRNA degradation
    RNA interference or silencing
    miRNA and siRNA
  • 31. Gene Expression
    DNA level expression control
    Transcriptional
    Post-Transcriptional
    Epigenetics
    DNA methylation
    Histone modification
  • 32. 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.
  • 33. Modes of inheritance
    Mutations of single genes
    Patterns: autosomal dominant, autosomal recessive, X-linked, mitochondrial
    Anticipation
    Increased severity of a certain diseases in successive familiar generations associated with triple repeats
    Mosaicism
    At least two cell lines derived from a single zygote
  • 34. Modes of inheritance
    Genomic imprinting
    Different expression of alleles depending on parent origin
    UniparentalDisomy
    Both copies inherited from one parent
    Environmental influence
    Chronic diseases
  • 35. 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.
  • 36. 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
  • 37. 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
  • 38. 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…
  • 39. 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
  • 40. 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)
  • 41. Applications of Direct DNA sequences
  • 42.
  • 43. 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]
  • 44.
  • 45. 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
  • 46. 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
  • 47. Southern Blotworking protocol
  • 48. 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 
  • 49. 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
  • 50. 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 ~95°C
    Annealing ~45-60°C
    Extension ~72°C
  • 51. PCR
    Denaturation
    Breaks the hydrogen bonds between the ds-DNA
    Anealing
    Binding to oligonucleotide sequence (probe)
    Extension
    DNA polymerase (heat stable, Taq [Thermophilusaquaticus]) replicates the selected DNA sequence
    Xn = X0 × (1 + E)n E= 0 - 1
  • 52. RT-PCR
    To detect or quantify RNA transcripts or viral RNA
    RNA is converted to DNA
    Reverse transcriptase (Avian Myeloblastosis Virus and MoloneyMurine Leukemia virus)
    Isothermal reaction with primers: oligodT, random hexamer primers, or target specific primers
    One step vs. two steps
  • 53.
  • 54.
  • 55. 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
  • 56.
  • 57. 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
    FlexmiRmicroRNA 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
  • 58.
  • 59.
  • 60.
  • 61. Luminex
  • 62. Real Time - PCR
    Amplifies and detects PCR product fluorescently in each well of PCR plate
    Don’t 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
  • 63.
  • 64. Real Time - PCR
    The crossing threshold or cycle threshold (Ct) is the amplification cycle number at which fluorescence is obtained
    Ct is proportional to the amount of staring template (interrogated sequence) in the sample
    Excellent for Q-PCR
  • 65. PCR
    Advantages
    Sensitivity
    Specificity
    Speed
    Versatility
    Automated
    No need for intact DNA/RNA
    Disadvantages
    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+)
  • 66. 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
  • 67. Branched DNAmakes the complicated matters simple
    The technology uses variety of branched DNA ( bDNA ) probes and signal amplification reporter molecules
    And generate Chemiluminescent signal.
    The signal correlates with target nucleic acid
    Doctortvrao’s ‘e’ learning series
  • 68. Capture Extenders (CEs), Label Extenders (LEs), and Blocking Probes (BLs)
  • 69. Branched DNA applications
    Detection HIV, HBV, and HCV
    Measures viral loads
    Less sensitive than PCR
    Doctortvrao’s ‘e’ learning series
  • 70. 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
  • 71.
  • 72.
  • 73. InvaderFRET / Cleavase
    Signal amplification
  • 74.
  • 75. Product Overview – Update
    CervistaTM HPV HR is an FDA approved test that screens for the presence of 14 high-risk HPV types
    100% detection of CIN3+ and 99.1% NPV for CIN2+
    Only FDA approved HPV screening test with an internal control
    Reduces patient call backs
    Limits QNS (only 2 ml sample volume required)
    <1% indeterminate rate
    No equivocal zone for interpretation
    75
    © 2009 Hologic, Inc. All right reserved.
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  • 76. HPV HR Indications
    Indications for use:
    • To screen patients with atypical squamous cells of undetermined significance (ASC-US) cervical cytology results to determine the need for referral to colposcopy
    • 77. In women 30 years and older the CervistaTM HPV HR test can be used with cervical cytology to adjunctively screen to assess the presence or absence of high-risk HPV types. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management
    76
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  • 78. Product Design & Chemistry
  • 79. Invader® Chemistry
    Repeating ProcessAmplifies Signal
    Cleavase® Enzyme
    Probe
    A technology protected by 128 issued U.S. patents
    Structure-specific recognition and cleavage with Cleavase® enzyme
    Signal amplification
    Isothermal reactions: no thermal cycling needed
    Fluorescence detection
    78
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  • 80. A7
    A5
    A6
    A9
    51, 56, 66
    A5/A6
    A7
    18, 36, 45, 59, 68
    A9
    16, 31, 33, 35, 52, 58
    CervistaTMHPV HR Test Design
    Developed from phylogenetictree of A-superfamily HPV strains, based on L1 region
    Cervista HPV is specific for high-risk types selected from the A5/A6, A7 and A9 virus groups:
    79
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  • 81. Primary reaction
    Secondary reaction (Simultaneous)
    Signal amplification is typically ~107 per molecule of target sequence.
    Invader® Chemistry Overview - Summary
    80
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  • 82. HPV-specific targets
    Human DNA-specific target
    Cleavage Site
    Cleavage Site
    Probe
    Probe
    C
    A
    Invader® Oligo
    Invader® Oligo
    G
    T
    Released 5´ Flap
    Released 5´ Flap
    C
    A
    Cleavage
    Site
    Cleavage
    Site
    F2
    Q
    F1
    C
    A
    FRET Cassette 2
    FRET Cassette 1
    F2
    F1
    Invader® HPV Biplex Reaction Format
    Q
    81
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  • 83. Cervista™ HPV HR Test Workflow
    Genfind Extraction
    Pellet Cells
    Read & Analyze
    Reaction Setup
    Incubation
    MAGNET
    82
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  • 84. Cervista™HPV HR Test Process
    Full 4-hour walk-away time enhances productivity.
    83
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  • 85. Interpretation of Results
    Data Analysis Software
    User-friendly
    Intuitive user interface
    Screen-by-screen walk- through of process steps
    Flexible
    Multiple reporting options
    84
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  • 86. Clinical Performance
  • 87. Cervista™HPV Clinical Trial Overview
    Screened >50,000 women; enrolled approximately 4,000
    Samples collected from 89 sites in 23 states, providing diversity
    Included both ASC-US and normal cytology arms
    ASC-US: (>1,300 women)
    Primarily intended to show that women with abnormal Pap test results and negative HPV results have < 1% probability of cervical disease (CIN2/3), or conversely >99% NPV
    Also intended to show a sensitivity ≥ 90% for detecting cervical disease
    Normal cytology (NILM) arm: (>2,000 women)
    Intended to establish that HPV-positive subjects are more likely than HPV-negative subjects to develop cervical disease (CIN2+) over a 3-year period
    All clinical objectives achieved
    86
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  • 88. Clinical Performance
    CIN3+ detection:
    Cervista™ HPV HR versus Colposcopy/Consensus Histology results (CIN3+) among women with ASC-US cytology
    100%
    detection
    ‡No CIN, CIN1 or CIN2 by Central Histology or Colposcopy without Central Histology.
    87
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  • 89. Clinical Performance
    CIN2+ detection:
    Cervista™ HPV HR versus Colposcopy/Consensus Histology results (CIN2+) among women with ASC-US cytology
    93%
    detection
    ‡No CIN or CIN1 by Central Histology or Colposcopy without Central Histology
    88
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  • 90. Clinical Performance
    Comparison of Cervista™HPV HR clinical trial and ALTS
    ASC-US/LSIL Triage Study (ALTS)
    Organized and funded by the NCI
    Included over 5,000 patients
    Benchmark ASC-US triage study
    a Cervista™ HPV HR multicenter clinical trial, 2006–2008. Clinical and analytical data on file, Hologic, Inc.
    b Immediate colposcopy arm of ALTS.
    c Number of subjects with known disease status and Cervista™ HPV HR results.
    d Referral rate for women 30 years of age and older was 43%.
    89
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  • 91. CervistaTMHPV HR Benefits
  • 92. Confidence of an Internal Control
    Test Contains an Internal Control
    CervistaTM HPV HR: The only FDA-approved HPV test with an internal control
    Example Invader Call ReporterTM Output
    Only FDA approved HPV screening test with an internal control
    Confirms the presence of adequate cellular material for testing
    Confirms that no inhibitory substances are present
    Minimizes false-negatives due to insufficient sample cellularity
    © 2009 Hologic, Inc. All right reserved.
    91
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  • 93. Minimizes False Positives
    Cross-reactivity to these common low-risk HPV types causes false-positive results, which can lead to unnecessary colposcopies
    1Hybrid Capture® 2 High-Risk HPV DNA Test® package insert #L00665, Rev. 2, 2007
    2Castle PE, Solomon D., et al. A Comparison of Two Methods to Determine the Presence of High-Risk HPV Cervical Infections. Am J Clin Pathol 2008;130:401-408.
    92
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  • 94. Substantially Reducing Patient Call Backs
    Requires only half the sample volume of other HPV tests (2 ml vs. 4 ml)
    Increases the likelihood of useful sample volume remaining for additional testing
    Providing clear results without an equivocal (gray) zone
    Reduces the indeterminate rate to <1%, compared with 4% or more for other HPV tests1
    Minimum Sample amount Required to Perform Test1
    4 ml
    2 ml
    hc2
    Indeterminate Rate
    4.7%
    <1%
    hc2
    1Solomon et al, JNCI, 2001.
    93
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  • 95. Cervista™ HPV vs. hc2®and RCS®
    © 2009 Hologic, Inc. All right reserved.
    94
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  • 96. Summary
    95
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  • 97. 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
  • 98. In Situ Hybridization
    Probe types:
    Centromeric or CEP (chromosome enumeration probe)
    Whole chromosome probes or paints [metaphase only]
    Locus specific probe or identifier (LSI)
    Section pretreatment.
    The labeled probe is first denatured (by heating or under alkaline conditions) into single DNA strands
    Hybridized to the target DNA (~Southern blotting) or RNA (~northern blotting) immobilized on a membrane (blotting) or in situ.
    Metaphase and Interphase cells
  • 99. ISH
    Advantages
    Interphase nuclei
    Archive material
    Can detect anomalies hidden to other methods
    Detects polysomy, losses, amplifications, translocations
    Disadvantages
    Only provides information of the specific target
    Work together with classic cytogenetics
    Minimal Residual Disease
    Do not provide allele-specific information
    Do not detect small changes
  • 100. In Situ Hybridization
    More in use Chromosomal translocations
    Useful in CMV, HSV, VZV
    Sub types of papilloma virus
    Useful in Mycobacteria, fungi and parasites
    Helicobacter pylori from gastric biopsies
    Legionella pneumophila
    Pneumocystis jiroveci
    Tests done on paraffin embedded specimen
    Need applications in Infectious diseases
    Doctortvrao’s ‘e’ learning series
  • 101.
  • 102.
  • 103. ISH - PNA probes
    Traditionally either cloned probes or synthesized oligonucleotide probes have been used for hybridization.
    Peptide nucleic acid (PNA) probe, a nucleotide analogue capable of binding to DNA/RNA in a sequence-specific manner obeying the Watson-Crick base pairing rules.
    In PNA, the sugar phosphate backbone of DNA/RNA has been replaced by a synthetic peptide backbone keeping the distances between bases exactly the same as in DNA/RNA.
    Further, the PNAs are very stable molecules. Experiments have shown virtually no degradation by DNases, RNases, proteinases or peptidases.
    The PNA probes are labeled with fluorescein and detected using a sensitive PNA ISH Detection Kit (colorimetric).
  • 104. Clinical Uses for ISH
  • 105. ISH - Examples
    Genotyping of Neoplasms
    Polysomy and other gains
    Trisomy 12 in B-CLL
    Losses
    del 1p / del 19q
    Amplification
    HER2/neu
    Translocations
    t(9,22)(q34;11) BCR/ABL in CML
  • 106. ISH - Examples
    Constitutional Molecular Genetics
    Sex chromosome enumeration
    Gender
    Polysomy and other gains
    Trisomy 21 in Down’s
    Losses
    del(22q11.2) in DiGeorge syndrome
  • 107. ISH - PNA probes
    EBER
    EBV lytic
    Kappa and Lambda
  • 108.
  • 109. ISHUrinary Cytopathology
  • 110.
  • 111. Urovision™
    >60,000 new cases of bladder cancer
    Recurrence of Urothelial carcinomas 50-80%
    Follow up cytoscopy and urine cytology (sensitivity ~48%)
    Aneuploidy 3, 7, 17 and loss of 9p21 (CDKN2A p16)
    Sensitivity 96% in HG UCs
    Interpretation: 35 abnormal cells
    ≥4 cells with aneuploidy in ≥2 chromosomes
    Loss of 9p21 in ≥12 cells
  • 112. ISHSolid Tumors
  • 113. 1p / 19q - Oligodendroglioma
  • 114. Dual Fusion Translocation Probe
    Break Apart Translocation Probe
  • 115. ISH – Solid Tumors
  • 116. ISH – Solid Tumors
  • 117. ISH - Lymphomas
  • 118. ISH - Lymphomas
  • 119. ISH - Lymphomas
  • 120. ISH - Leukemia