Biotechnology analysis methods

Biotechnology analysis methods

1. Microarrays

   • analyses change in patterns of gene expression of mRNA
   • "on/off switch" and "volume control"
   • core principle is the hybridisation between two DNA strands
     • DNA/DNA
     • RNA/RNA
     • DNA/RNA
   • is a solid support where small sequences are immobilised
     • each spot is a different gene sequence
       • many copies of same gene sequence
       • each spot can be a different colour representing a different status
         • green = control DNA
         • red = sample DNA
         • yellow = combination of control and sample DNA
         • black = neither control or sample DNA
   • strands are usually PCR products
   • can be DNA, cDNA or oligo
   • procedure
     • 1. DNA chip prepared
     • 2. fluorescently labelled hybridisation mixture prepared
     • 3. chip and mixture incubated
     • 4. laser scanning of fluorescent label
     • 5. computational analysis
   • important
     • large number of genes can be studied quickly
     • small samples can be used
     • Gene expression patterns in different cell types can be compared
   • three basic types that differ in their immobilised (target) DNA
     • changes in gene expression level (transcriptomic)
       • expression chips
       • immobilised DNA is cDNA derived from RNA
       • can be used to study ......
         • diseased vs healthy gene expression
         • development changes in gene expression
         • changes to an altered metabolic environment
     • genomic gains and losses (genomic)
       • DNA repair genes
       • comparative genomic hybridisation
       • immobilised DNA are large pieces of genomic DNA with a known chromosomal location
       • fluorescence reflects copy number
     • mutations in DNA
       • detects mutations and polymorphisms
       • immobilised DNA is a single gene with many variants

2. High-throughput screening

   • method used for scientific experimentation such as drug discovery
   • allows a researcher to conduct millions of biochemical, genetic and pharmacological testing
   • can identify active compounds, antibodies or genes which modulate a particular biomolecular pathway
   • results provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology
   • target validation
     • makes sure the role of the inhibitor in the disease is thoroughly known and the regulation of the target cell has the expected affect
       • allows the identification of compounds whose inhibition results in cell apoptosis or the inhibition of cell proliferation
   • primary screening
     • large scale primary screening in living cells to select the best hits among thousand of compounds
   • secondary screening
     • panel of assays for focused secondary screening in cell-based models of human diseases or in primary cell cultures
   • lead optimisation
     • identify compounds suitable for testing in a clinical setting

3. GFP-Tag

   • GFP = Green Fluorescent Protein
   • originally isolated from the jellyfish Aequorea victoria
   • 30 members of GFP-like family from Anthozoans
   • naturally occurring 27 kD protein that fluoresces green when exposed to blue light
   • useful in fluorescence microscopy techniques

4. Anticytokines

   • cytokines are important components of the immune system
   • imbalance of the cytokine process (production, receptor expression and dysregulation) contributes to many pathological disorders
   • there are pathways from gene expression to protein effect
     • there are also many potential points of intervention
   • is being analysed for possible therapeutic uses

5. Image analysis

   • designed to provide continuous, time‐lapse images of live cells from within your own standard cell‐culture incubator
   • can produce a graph showing kinetic proliferation curves (proliferation rates against time)

6. FACS

   • FACS = Fluorescence Activation Cell Sorting
   • 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
     • allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second
   • useful in finding stem cells