Biotechnology analysis methods

Microarrays
1. analyses change in patterns of gene expression of mRNA
2. "on/off switch" and "volume control"
3. core principle is the hybridisation between two DNA strands
  1. DNA/DNA
  2. RNA/RNA
  3. DNA/RNA
4. is a solid support where small sequences are immobilised
  1. each spot is a different gene sequence
    1. many copies of same gene sequence
    2. each spot can be a different colour representing a different status
      1. green = control DNA
      2. red = sample DNA
      3. yellow = combination of control and sample DNA
      4. black = neither control or sample DNA
5. strands are usually PCR products
6. can be DNA, cDNA or oligo
7. procedure
  1. 1. DNA chip prepared
  2. 2. fluorescently labelled hybridisation mixture prepared
  3. 3. chip and mixture incubated
  4. 4. laser scanning of fluorescent label
  5. 5. computational analysis
8. important
  1. large number of genes can be studied quickly
  2. small samples can be used
  3. Gene expression patterns in different cell types can be compared
9. three basic types that differ in their immobilised (target) DNA
  1. changes in gene expression level (transcriptomic)
    1. expression chips
    2. immobilised DNA is cDNA derived from RNA
    3. can be used to study ......
      1. diseased vs healthy gene expression
      2. development changes in gene expression
      3. changes to an altered metabolic environment
  2. genomic gains and losses (genomic)
    1. DNA repair genes
    2. comparative genomic hybridisation
    3. immobilised DNA are large pieces of genomic DNA with a known chromosomal location
    4. fluorescence reflects copy number
  3. mutations in DNA
    1. detects mutations and polymorphisms
    2. immobilised DNA is a single gene with many variants
High-throughput screening
1. method used for scientific experimentation such as drug discovery
2. allows a researcher to conduct millions of biochemical, genetic and pharmacological testing
3. can identify active compounds, antibodies or genes which modulate a particular biomolecular pathway
4. results provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology
5. target validation
  1. makes sure the role of the inhibitor in the disease is thoroughly known and the regulation of the target cell has the expected affect
    1. allows the identification of compounds whose inhibition results in cell apoptosis or the inhibition of cell proliferation
6. primary screening
  1. large scale primary screening in living cells to select the best hits among thousand of compounds
7. secondary screening
  1. panel of assays for focused secondary screening in cell-based models of human diseases or in primary cell cultures
8. lead optimisation
  1. identify compounds suitable for testing in a clinical setting
GFP-Tag
1. GFP = Green Fluorescent Protein
2. originally isolated from the jellyfish Aequorea victoria
3. 30 members of GFP-like family from Anthozoans
4. naturally occurring 27 kD protein that fluoresces green when exposed to blue light
5. useful in fluorescence microscopy techniques
Anticytokines
1. cytokines are important components of the immune system
2. imbalance of the cytokine process (production, receptor expression and dysregulation) contributes to many pathological disorders
3. there are pathways from gene expression to protein effect
  1. there are also many potential points of intervention
4. is being analysed for possible therapeutic uses
Image analysis
1. designed to provide continuous, time‐lapse images of live cells from within your own standard cell‐culture incubator
2. can produce a graph showing kinetic proliferation curves (proliferation rates against time)
FACS
1. FACS = Fluorescence Activation Cell Sorting
2. flow cytometryis a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus
  1. allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second
3. useful in finding stem cells

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Biotechnology analysis methods

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