A microarray tests thousands of genes at once: which ones a genome contains, and which ones a cell is switching on. The flood of sequence data that results is stored in shared online databases that any scientist can search.
A microarray is a small slide carrying thousands of spots, each holding many copies of a single-stranded DNA probe for one gene or sequence. To study gene expression, mRNA is extracted from two samples, converted to cDNA by reverse transcriptase, and labelled with different fluorescent dyes. The cDNA hybridises to complementary probes, unbound DNA is washed off, and a scanner records the colour and brightness of every spot: which genes are expressed in each sample, and how strongly. To analyse genomes, labelled fragments of genomic DNA are used instead, showing which genes or alleles two genomes share. Bioinformatics databases store nucleotide sequences of genes and genomes, amino acid sequences of proteins and protein structures, so scientists anywhere can search, compare and use them.
Fig. 1: part of a microarray after hybridisation with cDNA from two samples; the colour of each spot shows in which sample its gene was being expressed
Section 01
Reading a microarray.
The slide holds 64 spots in an 8 by 8 grid, all dark to start. Press Add labelled cDNA and wash and watch each spot light up green, red or yellow, or stay dark.
The probes: each spot contains many identical single-stranded DNA probes, short sequences complementary to part of one particular gene. The position of every probe on the slide is known, so a glowing spot identifies a gene.
Gene expression (detecting mRNA): mRNA is extracted from two kinds of cell, for example healthy cells and cancer cells. Reverse transcriptase makes cDNA from each, and the cDNA from one sample is labelled with a green fluorescent dye and the other with red.
Hybridisation: the labelled cDNA samples are mixed and applied to the slide. Each cDNA molecule binds by complementary base pairing to the probe for its own gene. Unbound DNA is washed off, so only hybridised DNA fluoresces.
Reading the result: a laser scanner measures the fluorescence of each spot. Green means the gene was being transcribed only in sample 1, red only in sample 2, yellow in both, dark in neither. The brightness shows how much mRNA was present, so expression levels can be compared.
Analysing genomes: genomic DNA (not mRNA) is cut into fragments, denatured to single strands and labelled, then hybridised in the same way. This shows which genes are present in a genome, compares the genomes of two species or strains, and detects particular alleles or mutations, for example in genetic testing.
Why it matters: thousands of genes are tested in one experiment, so patterns such as the set of genes switched on in a tumour can be found, helping diagnosis and the choice of treatment.
Examiner trap
A dark spot in an expression study does not mean the gene is missing from the cells. It means the gene was not being transcribed, so there was no mRNA, and therefore no cDNA, to bind.
Stage 1 · Learn
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According to the label in the simulator, what does each spot on the microarray hold?
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After you press Add labelled cDNA and wash, a spot turns yellow. What does this show?
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Some spots in the simulation stay dark. What is the best interpretation in a study of gene expression?
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Why does the simulator wash the slide after adding the labelled cDNA?
Section 02
Shared databases: bioinformatics.
Sequencing and microarrays produce huge amounts of data. Bioinformatics uses computers to store, search and analyse it, through large public databases.
Database holds
Example of use
nucleotide sequences of genes and whole genomes
search a newly sequenced gene against all known sequences to identify it and predict its function; design primers for PCR and probes for microarrays
amino acid sequences of proteins
compare the same protein in different species to work out evolutionary relationships; spot changes caused by mutations
protein structures (3D shapes)
see the shape of an active site or receptor and design drugs that fit it
Benefits: data are freely available to scientists worldwide, so work is not repeated; new sequences can be compared quickly with millions of known ones; similar sequences in different organisms suggest similar functions and show how closely related species are; disease-causing alleles can be identified and genetic tests designed; pathogens can be identified from their sequences and the spread of new strains tracked.
Examples: GenBank (nucleotide sequences), UniProt (protein sequences) and the Protein Data Bank (protein structures). You do not need to learn these names, only what such databases provide.
Stage 2 · Exam
Exam-style questions
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In a gene expression study, why is mRNA converted to labelled cDNA before it is added to a microarray?
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Genomic DNA from bacterial strain P is labelled green and from strain Q red, and both are hybridised to a microarray of probes for strain P genes. A spot is green only. What does this suggest?
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Which is a benefit of databases of nucleotide sequences?
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How can a database of protein structures help in developing a new drug?
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