Please enable JavaScript.

Coggle requires JavaScript to display documents.

HMM202, Week 10 – Probes and Hybridisation, Week 8 – Pharmacogenetics &…

- - - - Separates DNA by size with electric field
      - Smaller fragments migrate faster
    - - Cut DNA at palondromic specific sites
      - Blunt/Sticky ends
    - - Amplifies target DNA region
      - Steps: 95 Denaturation → 55 Annealing → 72 Extension
    - - Real-time quantification with fluorescent probes
    - - Determines nucleotide order
    - - Detects specific DNA sequences using probe
      - Requires electrophoresis + transfer to membrane
    - - Hybridisation to probes on chip
      - Detects many sequences simultaneously
    - - Measures DNA concentration via absorbance (260 nm)
      - Quick, minimal sample, less accurate with contaminants
      - DNA purity: 260/280 ratio (~1.8 = pure, <1.8 = protein contamination)
        260/230 ratio (~2.0 = low organic/salt contamination)
    - - Uses fluorescent dye binding DNA
      - Highly specific, more accurate at low concentrations
      - Sensitive detection of nucleic acids using dyes
    - - Zone (CZE)
        
        charge : mass
        
        Pro: high res
        Con: complex moleules harder
      - Gel (CGE)
        
        size/shape
  - - - Somatic
        
        not inherited, e.g. cancer
        
        Mutation Detection
        
        Chemical Cleavage of Mismatch (CCM)
        
        potassium permangenate (T)
        Hydroxylamin (C)
        
        Piperidine cleavage
        
        Enzymatic Cleavage of Mismatch (ECM)
        
        Exonuclease
        
        detects unknown mutations
        
        Single-Strand Conformation Polymorphism (SSCP)
        
        detects DNA conformation differences
        
        Proteomics (MS, MudPIT, DIGE)
        LCM
        H&E, IHC
        CE
        Western blot/ protein gels
        HPLC
        ED
        Biochemical assays
        CRP test
      - Germline
        
        inherited, e.g. CF
        
        PCR/ qPCR
        Sanger
        NGS(WES, WGS)
        SNP
        RE digestion + Gel electrophoresis
        Southern blot
        IN-SITU Hybridisation
        CRISPR
      - Mutation= DNA change
        Variant= alternate version
        
        Variation
    - - nonfunctional
        evolutionary insight
- - - - Bacterial Infection
    - - PCR
    - - Throat swabs
    - - Antibiotics + Vaccination
  - - - Gene=CFTR, Allele=δF508
    - - Newborn Heel prick (IRT lvls)
        [Sweat Chloride]
    - - 1/25 Aussies carriers CFTR
    - - TTT
  - - - Systemic Inflammation
    - - Long Term Infection
    - - Liver
- - - - High sensitivity
      - Detected by autoradiography (black band on X-ray film)
    - - Tag added during synthesis
      - Excited by light → emits fluorescence
  - - - Biotin linked to probe; detected using avidin/streptavidin + HRP
    - - Non-radioactive steroid label
      - Detected by chromogenic or chemiluminescent substrate
- - - - TD50
        
        toxicity in 50% of pop.
      - ED50
        
        effective in 50% of pop.
- - - - CYP2D6, CYP2C19, CYP3A4
- - - - Tm 58-65°C, 18-30 nucleotides long
- - - - extracted by restriction endonucleases
- - - - Genomics (DNA sequencing, SNP analysis)
      - Transcriptomics (RNA-seq, microarrays)
      - Proteomics (mass spectrometry, protein arrays)
      - Metabolomics (NMR, LC-MS)
    - - Pattern recognition using large datasets
      - Machine learning for predictive modelling
    - - TCGA, GEO databases for disease-related markers
    - - CRISPR/Cas9 or RNAi screens to identify disease-linked proteins
- - - - Construct phage library (diverse antibody fragments)
      - Incubate with antigen → binders retained
      - Wash to remove non-specific binders
      - Elute bound phages → amplify in E. coli
      - Repeat (biopanning) to enrich for high-affinity binders
  - - - Identify VH/VL sequences
      - Clone into plasmid vector
      - Transform/transfect host cells (E. coli, CHO, HEK293)
      - Express, purify, and test antibody
- - - - Bacteria (E. coli): fast, cheap, simple proteins
      - Yeast: allows post-translational modifications
      - Mammalian cells (CHO, HEK293): correct folding, glycosylation
      - Insect cells: high yield
- - - - Dark = AT-rich, gene-poor
        
        heterochromatic = tightly packed
        transcriptioannly inactive
      - Light = GC-rich, gene-rich
        
        euchromatic = loosely packed
        transcriptioannly active
    - - Metacentric, submetacentric, acrocentric, telocentric
- - - - Down Syndrome (21), Edwards (18), Patau (13), Klinefelter (XXY)
    - - Turner’s Syndrome (XO)
  - - - Reciprocal – exchange between non-homologous chromosomes
      - Robertsonian – fusion of acrocentric chromosomes
- - - - normal dna rep
    - - tRNA
    - - Early development
- - - - tumour supressor genes
- - - - Lentivirus
      - Adeno-associated Virus
    - - LNPs
      - Liposomes
- - - - Histopathology
        
        Role
        
        Presence/Type of disease
        
        Stage, severity, spread of disease
        
        Cellular abnormalities
        
        Tissue processing
        [FDCESSHI]
        
        Fixation
        
        Dehydration
        
        Clearing
        
        Embedding
        
        1 more item...
        
        Features of Disease
        
        Inflammation
        
        Acute
        
        neutrophils
        
        Chronic
        
        lymphocytes
        
        Neoplasia
        
        Benign
        
        non-invasive, slow growing
        
        Malignant
        
        invasive, metastasise
        
        Degeneration
        
        Necrosis
        
        swelling, membrane rupture, inflammation
        
        Apoptosis
        
        shrinkage, apoptotic bodies
        
        Infectious disease patterns
        
        Bacterial
        
        neutrophils
        
        Viral
        
        lymphocytes
        
        Fungi
        
        distinctive cell wall morph.
        
        Protein Analysis
        
        Tissue Homogenisation
        
        Detergents: SDS, CHAPS
        
        Reducing agents: DTT
        
        Denaturants: urea
        
        Laser Microdissection
        
        Isolation of specific cells from heterogeneous tissue
        
        Proteomics applications
        
        Determine proteins produced by genome
        
        Map protein localisation in tissue
        
        Identify abnormal protein distribution in disease
        
        Protein characterisation methods
        
        Gel electrophoresis: 1D, 2D, DIGE – size & charge separation
        
        HPLC – separation by polarity or binding affinity
        
        Capillary electrophoresis CE(zone/gel)– size-to-charge separation
        
        MudPIT – multidimensional separation before MS
        
        Mass spectrometry: MALDI-TOF, SELDI-TOF – mass-to-charge detection
        
        Edman degradation – sequential N-terminal sequencing
        
        Protein identification workflow
        
        Digestion → ionisation → m/z detection → sequence
        
        Proteome analysis
        
        Post-translational modifications
        
        Acetylation
        
        Phosphorylation
        
        Ubiquitination
        
        Glycosylation
        
        Methylation
        
        Gel-based separation
        
        Non-gel-based separation
        
        MS-based identification
        
        Sampling tissue
        
        Homogeneous: direct analysis
        
        Heterogeneous: laser microdissection
        
        Visualising abnormal tissue
        
        Histology (H&E)
        
        IHC
        
        Spatial proteomics
        
        Bioinformatics
        
        Gene Sequencing
        
        Determination of nucleotide order in DNA
        
        Computational analysis to assemble and annotate sequences
        
        Homology Searches
        
        Identify sequence similarity between genes/proteins
        
        BLAST – Basic Local Alignment Search Tool
        
        Compares query sequence to database
        
        High similarity suggests evolutionary/functional relationship
        
        Phylogenetics
        
        Study of evolutionary relationships between organisms/genes
        
        Sequence alignment → distance matrix → tree construction
        
        Phylogenetic trees
        
        Rooted – shows common ancestor
        
        Unrooted – shows relationships without ancestor direction
        
        Methods
        
        Neighbour-joining – distance-based
        
        Maximum parsimony – minimal evolutionary changes
        
        Maximum likelihood – statistical probability of tree
        
        MC1R Gene
        
        Encodes melanocortin-1 receptor
        
        Involved in pigmentation regulation
        
        Variants linked to red hair, fair skin, UV sensitivity
        
        Example: Arg151Cys mutation – common in Irish population, alters receptor function
        
        PCR Primer Design
        
        Short DNA sequences to initiate amplification
        
        Design considerations
        
        Target specificity
        
        GC content (~40–60%)
        
        Tm= 52 - 58 celcius
        
        Avoid secondary structures (hairpins/dimers)
        
        Adding Restriction Enzyme (RE) Sites
        
        Include specific RE site at 5′ end for cloning
        
        Add extra bases upstream for efficient cutting
        
        Tags
        
        His-tag for purification
        
        FLAG-tag for detection
        
        Bioinformatics in Diagnostics
        
        Identify disease-related mutations
        
        Predict protein function from gene sequence
        
        Compare patient data to reference genomes
- - - - 1 Fixation
        
        2 Embedding
        
        3 Sectioning
        
        4 Antigen
        Retrival (mod epitope)
        
        5 Permeabilisation
        
        6 Blocking
        
        1 more item...