Molecular Diagnostics

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Molecular Diagnostics

• Modern medicine and agriculture depend on
  accurate and rapid detection of organisms,
  proteins, small molecules or specific genetic
  alleles
• Optimal detection strategy
• Specific
• Sensitive
• Quick
• Simple
• Low cost

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Comparison of Methods Used to Diagnose Parasite
Infections
3
ELISA

• Enzyme-linked immunosorbent assay
• Protocol
• Bind sample to support
• Add primary antibody
• Add secondary antibody-enzyme conjugate
• Add substrate (color reaction)

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Antigens and Antibodies

• Most antigens have many antigenic determinants or
  epitopes
• Injecting antigens into mammal produces serum of
  polyclonal antibodies
• Bind to many different epitopes and with
  different affinities
• A uniform, high affinity, highly specific
  antibody preparation would be preferable
• Monoclonal antibodies

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Monoclonal Antibodies

• Each B cell produces only one antibody (all of
  the molecules produced are identical)
• A clone of B cells therefore all produce
  identical antibodies
• Identify the proper clone and the culture of
  these cells produces a monoclonal antibody
  against the desired epitope

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HAT Selection

• Cells obtain purines and pyrimidines by either of
  two ways
• De novo biosynthesis requiring dihydrofolate
  reductase (DHFR) activity
• Salvage requiring HGPRTase (hypoxanthine guanine
  phosphoribosyl transferase) for purines
• Drug aminopterin inhibits DHFR forcing cells to
  utilize salvage pathways
• HGPRTase (-) cells cannot salvage purines
  (hypoxanthine or guanine)

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HAT Selection Procedure

• HAT
• Aminopterin
• Hypoxanthine
• Thymidine
• Protocol
• Immunize mouse with Ag
• Isolate spleen (B cells)
• Fuse with HGPRT- myeloma tumor cells
• Select for actively dividing HGPRT cells on HAT
  medium

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Screening for Monoclonal Antibody Production

• HGPRT clones screened by immunoassay (use in
  ELISA protocol)
• Clones that produce antibody binding to target
  antigen cultured and further characterized

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Targets for Monoclonal Antibody-based Diagnostic
Tests
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DNA Diagnostic Systems

• Presence of an organisms genetic material is a
  strong indicator of the presence of the organism
  or infectious agent
• General nucleic acid hybridization scheme
• Bind single-stranded DNA (denatured) to membrane
• Add single-stranded probe
• Wash away unbound probe
• Detect hybridized probe
• Radioactivity
• Nonradioactive detection method

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DNA Diagnostic Probes

• Protocol at left
• Useful for detection of parasites
• Distinguish readily between subtypes
• Distinguish readily between present and past
  infections (not necessarily easily done by some
  ELISA protocols)
• PCR makes highly sensitive

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Nonradioactive Detection Procedures

• Chemiluminescent detection of bound DNA probe
• Biotin-labeled nucleotides
• Streptavidin (SA) binds biotin
• Alkaline phosphatase conjugated to biotin also
  binds SA
• AP cleaves small molecule releasing light

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Detection by Fluorescent Dyes

• Fluorescent dye attached to PCR primer(s)
• PCR product now fluorescent-labeled
• Detect following laser activation

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Molecular Beacons

• Probe
• Palindromic region
• Fluorophore
• Quencher
• Binding of probe to target sequence separates
  quencher from fluorophore
• Laser activation for detection

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Molecular Beacons

• Molecular beacons with different fluorophores
  allow for simultaneous testing for multiple
  sequences

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Detection of Heterozygotes

• Use of multiple fluorophores and lasers

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DNA Profiling

• Restriction Fragment Length Polymorphism (RFLP)
• Shown to left uses a multilocus probe
  (barcoding)
• Presently virtually all identity profiling is
  done using PCR and STR loci

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RFLP and VNTR Loci

• Variable Number Tandem Repeat (VNTR) loci were
  the first loci widely used for human identity and
  parentage testing
• Repeat units generally 15-30 bp in length and the
  number of different alleles per locus can exceed
  100
• Most testing today uses PCR and STR (short tandem
  repeat) loci

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RAPD Analyses

• Random amplified polymorphic DNA (RAPD)
• Use a short (6-10 nucleotides) primer which binds
  randomly throughout genome
• Only locations with two primer binding sites
  facing each other at a distance of a few thousand
  bp or less yield amplified products
• Sequence variations between individuals result in
  unique patterns of products upon gel
  electrophoresis

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RAPD Analyses

• Used primarily for analyses of uncharacterized
  species
• Still has applications in population studies and
  forensic analyses

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Bacterial Biosensors

• Insert lux genes (for bioluminescence) into
  constitutive (but nonessential) gene in test
  strain
• Pollutants which inhibit strains metabolism
  reduce florescence
• Chemical analyses then identify toxic compound

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Genetic Screening

• PCR amplify region to be screened
• Sickle cell mutation eliminates a CvnI site
• Cut with CvnI
• Electrophoretic size analysis

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PCR/OLA Analysis

• PCR amplify target region
• Add two oligonucleotides which bind to test
  region (one with biotin, other with digoxigenin)
• Add DNA ligase, ligation only if perfect match
• ELISA type assay follows

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Padlock Probes

• Probe complementary to target only at ends
• Bind probe
• Ligate (if perfect match)
• Ligated form remains attached to target which is
  attached to matrix (96-well plate)

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PCR Using Florescence-labeled Primers

• Primer ends at SNP locus to be analyzed
• Mismatch gives no PCR product
• Perfect match gives florescent product