DNA chips

DNA chips

1. Intro

   • use to study mutations or gene expression (included in Genomics - study of genes)
     • Single Nucleotide Polymorphisms (SNP)= mutations affecting one nucleotide in a sequence - useful biomarkers of disease
   • other concep> electropheresis - access to size but not able to differentiate mutated strands (which have the same number of base pairs).
   • . They consist of thousands of probes—each representing a different gene—immobilized on “chips” or slides, and rely on complementary hybridization to evaluate gene expression in different biological conditions.
   • microarray biochip (similar to Northern blotting)
     • How do it work?
       • are arrays of DNA spots, containing single stranded DNA that can bind (hybridise) with its complementary strand. If the complementary strand is present in the solution that is put onto the array, the single strand becomes a double strand, by capturing its complementary partner. The partner is labeled with a dye, making the spot visible.

2. Process

   • explained using an example: Gene expression
     • understand which genes are expressed more than others in a particular condition >> mRNA of a cell type compared with that of a control
   • 1.Printing the DNA array (probes) either printed (similar to ink-jet printing) or synthesised.
   • 2.mRNA is extracted from the cells : cells are lysed and the RNA purified, usually using the different solubility of RNA in different solvents.
   • 3. mRNA is less stable than DNA so it is ‘reverse transcribed’ into DNA, using a reverse transcriptase
     • + dye (usually fluorescent)
       • Result = TARGET
   • 4.Hibridisation. The target solution is dispensed on the array and hybridise with the probes on the specific spots.
   • 5. . Imaging. The array is analysed by taking pictures in the different colors. Where a spot is green or red, only one of the cell type expressed the gene, whereas it is yellow when both cells expressed it.

3. Fabricating the array

   • consist on the design of the small length probes (through complex bioinformatics analysis) by selecting short strands of DNA that represent each genes specifically.
   • types
     • A. Printing
       • 1. The DNA to put on the spots is extracted and amplified using PCR (requiring gene-specific primers obtained through bioinformatics tools)
         • probe lenghts varies (usually 10s and 100s base parings)
       • 2. DNA dispensed using a robot (arrayer) onto a slide
       • 3. slide is coated with chemical anchors that retain the DNA (covanlently or using less stringent forces)
         • spot size around 100 um
     • B. In situ synthesis – using photolithography
       • DNA probes on the surface of a chip are built base by base, using chemical synthesis
       • At each step, the synthesised probe is protected with a light sensitive ‘cap’ preventing elongation.
       • next base is added by deprotecting the molecule, via exposure to light (usually UV) through a mask to select which spot will receive the next base.
       • not rely on PCR BUT reaps the benefits of the resolution of photolithography – micro-sized spots