Recent Advances in oral pathology

RECENT ADVANCES IN ORAL PATHOLOGY: 

RECENT ADVANCES IN ORAL PATHOLOGY DATE OF PRESENTATION: PRESENTED BY: DR. SOURAB KUMAR.

Molecular Techniques: 

Molecular Techniques Flow Cytometry Elutriation Magnetic Bead Separation Enzymatic Separation Laser Capture Microscopy Electrophoresis(Gel, Free Zone Capillary) Chromosome Banding Techniques(Solid Staining,G Banding, Q banding,C banding,R banding,NOR ) Fluorescent in situ hybridization(FISH): Fiber FISH. Twenty-Four Color Painting. Color Banding Comparative Genomic Hybridization. DNA Microarrays (RNA Arrays) Southern Blot Analysis

PowerPoint Presentation: 

Restriction Fragment (Variable number of tandem repeats – VNTR, Micro-Satellites : Small tandem repeats, SNIPs) Polymerase Chain Reaction Northern Blot Analysis RNA Mapping Ribonuclease Protection Assay(RPA) S1 nuclease mapping In Situ Hybridization Immunohistochemistry Enzyme-linked immunoassay Protein Electrophoresis(Two-dimensional, Western blot) Mass Spectroscopy Chromatography(Ion-exchange, Size exclusion, Hydrophobic interaction, Affinity, Fas , Reverse phase high pressure liquid) Nuclear Magnetic Resonance(NMR) 1D, 2D.

Flow Cytometry: 

Flow Cytometry Principle/ Mechanism Sort cells based on their surface markers Target Cells Probe Fluorescent Labels, Coulter Machine(Flow Sorter) Visualization Separated viable or nonviable cells, digital output.

PowerPoint Presentation: 

Laser optics Laser Beam Flow chamber Sheath Sample Y X Z Y Z X

PowerPoint Presentation: 

Each cell generates a quanta of fluorescence PE FL FITC FL 488nm Sct Confocal Lens Dichroic Lenses Photomultiplier Tubes (PMT’s) Discriminating Filters Forward Light Scattering Detector

PowerPoint Presentation: 

http://www.bdbiosciences.com

The electronics system takes the data from collection to computer: 

The electronics system takes the data from collection to computer http://www.bdbiosciences.com

Elutriation: 

Elutriation Principle/ Mechanism Sort cells or subcellular organelles based on their size and density. Target Cells Probe Centrifuge Visualization Fractionated layers/ phases analyzed further.

Magnetic Bead Separation: 

Magnetic Bead Separation Principle/ Mechanism Uses magnetic beads tagged to cell specific antibody that is then isolated by a strong magnet. Target Cells Probe Magnetic beads tagged to antibodies. Visualization Isolated cells are analyzed/cultured as desired.

Labeling Of Cells With Beads: 

Labeling Of Cells With Beads Bead Monoclonal Ab

Magnetic Separation: 

High Gradient Magnetic Field Trapped Labeled Cells Magnetic Separation

Magnetic Separation: 

Magnetic Separation Cells Of Interest

Indirect Labeling: 

Indirect Labeling

Depletion Strategy: 

Depletion Strategy

Depletion Strategy: 

Depletion Strategy

Depletion Strategy: 

Depletion Strategy

Enzymatic Separation: 

Enzymatic Separation Principle/ Mechanism Differential Susceptibility of tissue components to enzymatic digestions. Target Tissues. Probe Proteolytic digestion like trypsin , collagenase . Visualization Separated components are analyzed further.

Laser Capture Microscopy: 

Laser Capture Microscopy Principle/ Mechanism Uses laser beams as ‘optical tweezers’ to isolate cells/matrix components. Target Tissues. Probe Laser Beams. Visualization Microscopy.

Electrophoresis: 

Electrophoresis Principle/ Mechanism GEL Free Zone Capillary Separation of molecules based on their mobility(mass and charge) in an electrical field. Agarose (DNA,RNA) and Acrylamide (Proteins) Performed in Fused-Silica capillaries. Target DNA, RNA, Proteins or their complexes. Probe Electrical Field and Matrix gels or capillaries. Visualization Direct staining, radioactive labels, Chemiluminescence .

Chromosome Banding Techniques: 

Chromosome Banding Techniques Principle/ Mechanism Banding due to differences in base composition, replication time, chromatin conformation and gene density Target Cell nucleus. Probe Stains like Giemsa , Leishman’s , Silver nitrate, quinacrine , diamino phenyl indole (DAPI), etc. Visualization Microscopy fluorescence microscopes, digital outputs.

Chromosome Banding Techniques: 

Chromosome Banding Techniques Solid Staining : Chromosome breakage and fragile site studies. G Banding : Highly condensed heterochromatin, AT rich most commonly used. Q Banding : Fluorescence technique using the ability of quinacrine stain to bind DNA by intercalation of external ionic banding to study polymorphic variants on acrocentric chromosomes (1,3,9,16) C Banding: Dark staining of heterochromatin located at centromeres following DNA denaturation with alkali, also used to study chromosomal polymorphism. R Banding: Reverse Pattern of G Bands, Stains the lighter G bands more darkly, GC rich, less condensed chromatin, stains telomeric regions of several chromosomes dark. Nucleolar Organizer Region(NOR) Staining : Selective staining of active transcription sites of acrocentric chromosomes(13,14,15,21 and 22) encoding the constitutive genes 18S and 28S, used to study polymorphism and identifying small bisatellited marker chromosomes, and increased transcriptional activity of tumors. DAPI/ Distamycin Staining : Uses stains Distamycin A-4 and 6-diamino-2-phenylindole to bind to AT rich regions, study small marker chromosomes derived from chromosome 15 and heterochromatin regions of other chromosomes

Fluorescent in situ Hybridization (FISH): 

Fluorescent in situ Hybridization (FISH) Principle/ Mechanism Used with interphase ( nondividing ) nucleus, gives molecular and cytogenetic information like characterizing chromosome and structural rearrangements, numerical abnormalities, microdeletions , duplications. Target Formalin Fixed or Frozen Tissues, Smears. Probe Fluorochrome coupled single stranded complementary DNA probe. Visualization Fluorescence microscopy, digital imaging.

Twenty – Four Color Painting: 

Twenty – Four Color Painting Principle/ Mechanism Visualizes all 1-22 chromosomes and the sex chromosomes X,Y on the basis of their color, used to analyze markedly abnormal(tumor) cells and their derivative and marker chromosomes, marked chromosome rearrangements. Target Fixed or frozen tissues and smears. Probe Uses five or more fluorochrome coupled single stranded complementary DNA probe. Visualization Fluorescence microscopy or inferometer (called spectral karyotyping ) with digital imaging.

Color Banding: 

Color Banding Principle/ Mechanism Complement G banding to detect rearrangements, deletions or duplications and further identify chromosome segment involved. Target Fixed or frozen tissues and smears. Probe Fluorochrome coupled to Gibbon chromosomes which bind by complementarity to human chromosomes in a unique pattern. Visualization Fluorescence microscopy with digital imaging.

Comparative genomic hybridization: 

Comparative genomic hybridization Principle/ Mechanism Changes in DNA sequence copy numbers in terms of amplifications or deletions are tested by comparative hybridization of normal genomic reference DNA with test (tumor derived/diseased) genomic DNA to normal metaphase chromosome. Target Fixed or frozen tissues, smears genomic DNA. Probe Differentially fluorochrome labeled reference and tumor. Visualization Fluorescence microscopy with digital imaging.

Array Based Comparative Genomic Hybridization: 

Array Based Comparative Genomic Hybridization Goal: to detect copy number alterations using a gene chip Ideally, the signal intensity is proportional to copy number Several genomes can be compared simultaneously Daniel Pinkel & Donna G Albertson (2005) Nature Genetics, 37:s11-17 Daniel Pinkel & Donna G Albertson (2005) Nature Genetics, 37:s11-17

DNA Microarrays: 

DNA Microarrays Principle/ Mechanism Comparative hybridization on slides or membranes labeled with complementary DNA or Oligonucleotides representing altered gene expression(DNA activity) Target mRNA from tissues to cells Probe Spotted complementary sequences binding to differentially labeled (Cy3 or Cy5) reference and analytical mRNA. Visualization Fluorescence detectors with digital imaging.

PowerPoint Presentation: 

Our genome encodes an enormous amount of information about our beings our looks our size how our bodies work our health our behaviors … who we are!

RNA Arrays: 

RNA Arrays Principle/ Mechanism Direct Hybridization on slides or membranes spotted with complementary single stranded DNA or Oligonucleotides sequences. Target mRNA from tissues or cells Probe Spotted complementary sequences binding to chemically or radiolabeled mRNA Visualization Autoradiography or chemiluminescence .

Southern Blot Analysis: 

Southern Blot Analysis Principle/ Mechanism Complementarity of nucleic acid target to probe. Target Deoxyribonucleic acid (DNA) Probe Labeled Single stranded DNA Oligonucleotide (18-30 base pairs) Visualization Gel Electrophoresis Film, digital imaging.

Restriction Fragment: 

Restriction Fragment Principle/ Mechanism Variable Number of Tandem Repeats Microsatellites Single Nucleotide Polymorphism(SNIPs) DNA heterogeneity due to allelic length polymorphisms. Polymorphism, which may be inherited, makes an individual’s genomes amenable to bacterial ‘restriction nuclease’ digestions into specific patterns. Detects single/ multiallelic mutations, similar inheritance patterns or genetic finger printing(forensics). Larger repeats of 10-60 nucleotides. Small individual repeats of 1-5 nucleotides. Microsatellite instability(MSI) is used in detecting genetic analysis of malignancies. Individual Nucleotide variations in DNA sequences. Target Deoxyribonucleic acid (DNA) Probe Labeled Single stranded DNA Oligonucleotide (18-30 base pairs) Visualization Gel Electrophoresis Film, digital imaging.

Polymerase Chain Reaction.: 

Polymerase Chain Reaction. Principle/ Mechanism Uses compositional units of nucleic acids(nucleotides) with a DNA( Taq ) Enzyme to amplify and visualize them. Target DNA or RNA (Reverse transcriptase – PCR) enzyme. Probe Nucleotides, Polymerase Visualization Ethidium bromide agarose gel electrophoresis.

Northern Blot Analysis: 

Northern Blot Analysis Principle/ Mechanism Complementarity of nucleic acid target to probe Target Support-bound mRNA extracted from tissues or cells. Probe Labeled Single stranded nucleotide (18-30 base pairs) Visualization Gel Electrophoresis, Film, Digital Imaging

RNA Mapping: 

RNA Mapping Principle/ Mechanism Ribonuclease Protection Assay(RPA) S1 Nuclease Mapping Both are based on hybridization of RNA and antisense DNA protecting it from nuclease digestion RPA uses A and T1 ribonuclease digestion for quantitative mRNA analysis. S1 Mapping used S1 Ribonuclease and is used for qualitative mRNA analysis like structure, intron-exon regions,5’ or 3’ ends, mutational analysis. Target Low abundant mRNA Probe Complementary nucleic acid sequences. Visualization Nuclease protected fragments are visualized by electrophoresis or sequencing.

In Situ Hybridization: 

In Situ Hybridization Principle/ Mechanism Complementarity of nucleic acid target to probe, used for chromosome painting, analyze RNA localization, detection of viruses. Target Cell or Tissues. Probe Enzyme Labeled Antibodies Visualization Chemiluminescence .

Immunohistochemistry: 

Immunohistochemistry Principle/ Mechanism Antibodies directed against antigenic immunocytochemistry determinant of a part or whole protein. Target Support bound mRNA in tissue, cells or embryos Probe Radioactive or enzyme labeled complementary nucleic acid sequences. ( Oligos ) Visualization Autoradiography, Fluorescence, Chemiluminescence .

Enzyme-Linked Immunoassay: 

Enzyme-Linked Immunoassay Principle/ Mechanism Antibodies directed against antigenic determinant of the part or full protein. Target Protein from cell or tissue lysates , fluids like serum or plasma, secreted saliva, GCF Probe Enzyme Labeled Antibodies Visualization Chemiluminesence .

PowerPoint Presentation: 

The Enzyme-Lined Immunosorbent Assay (ELISA)

PowerPoint Presentation: 

bind protein to membrane or 96-well microplate neg. (and pos.) controls purity? incubate with 1 o and 2 o antibodies use soluble chromogenic substrates in 96-well plates quantify Ag or Ab Conventional ELISA

PowerPoint Presentation: 

measure absorbance with ELISA microplate reader

An example of an ELISA experiment: 

An example of an ELISA experiment Before starting the work read kit instruction carefully 1- The 96 well plate is labeled carefully and the first wells are used to draw the standard curve

An example of an ELISA experiment-Cont: 

An example of an ELISA experiment-Cont The sample is added to plate in duplicate or triplicate and then the mean result is calculated The quality control sample which is provided with the kit is treated as the test samples

Results : 

Results After reading the results the standard curve is drawn were the concentration is blotted on the X-axis and the absorbance on the Y-axis Concentration ng/ml Absorption nm

Results-cont: 

Results-cont The standards concentrations is specified on the x-axis and the reading of each standard is specified on the y-axis and the standard curve is drawn

Results-cont: 

Results-cont This standard curve is used to determine the unknown concentration of each sample by finding the opposite concentration to the absorbance Concentration ng/ml Absorption nm

Results-cont: 

Results-cont The quality control sample concentration is determined from the standard curve and if the result is in the range given by the kit manufacturer the results could be accepted

Protein Electrophoresis: 

Protein Electrophoresis Principle/ Mechanism Two Dimensional Western Blot Based on differential migration of proteins depending on their isoelectric point in an electrical field. Target Cell or tissue lysates Probe Use of Specific antibodies. Visualization Direct Staining of Gels, Chemiluminesence .

Molecular Weight Determination: 

Molecular Weight Determination Method 1: Amino Acids approx 110 daltons # residues x 110 dalton/residue = MW Method 2: Run SDS PAGE with known standards (MW markers) Graph Measure distance unknown protein travelled Compare on standard curve

Mass Spectroscopy: 

Mass Spectroscopy Principle/ Mechanism Produces gas-phase ions from sample ionization and separated on the basis of their mass-to-charge ratio. Target Proteins. Probe Ionization by chemicals, lasers, electrical fields. Visualization Digital read-outs.

How does a mass spectrometer work?: 

How does a mass spectrometer work? Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif. Create ions Separate ions Detect ions Mass spectrum Database analysis

Mass Spectrometry: 

Mass Spectrometry Analytical method to measure the molecular or atomic weight of samples

Mass Spec Principles: 

Mass Spec Principles Ionizer Sample + _ Mass Analyzer Detector

Chromatography: 

Chromatography Principle/ Mechanism (Ion-Exchange, Size exclusion, Hydrophobic interaction, affinity, Gas, Reverse Phase high Pressure liquid(RPHPLC) Broad range of physical methods used to separate and/or analyze complex mixtures, components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed. Target Lysates from cells or tissues. Probe Different Techiques Visualization Digital read-outs.

PowerPoint Presentation: 

The separation of small molecules by paper chromatography. After the sample has been applied to one end of the paper (the "origin") and dried, a solution containing a mixture of two or more solvents is allowed to flow slowly through the paper by capillary action. Different components in the sample move at different rates in the paper according to their relative solubility in the solvent that is preferentially adsorbed onto the fibers of the paper. Paper chromatography

PowerPoint Presentation: 

The separation of molecules by column chromatography. The sample is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose, immersed in solvent. Then a large amount of solvent is pumped slowly through the column and is collected in separate tubes as it emerges from the bottom. Various components of the sample travel at different rates through the column and are thereby fractionated into different tubes. Column chromatography

Nuclear Magnetic Resonance(NMR): 

Nuclear Magnetic Resonance(NMR) Principle/ Mechanism 1D 2D Measures chemical shifts of the atoms’ nuclei in the protein when it is placed in a powerful magnetic field allowing determination of its 3D structure. Target Tissues Probe Electrical Field Visualization Digital read-outs.