Genetic Technology: Question 8
Syllabus 19.1
A DNA microarray (gene chip) is a slide onto which thousands of microscopic spots have been fixed, each spot containing many copies of a single-stranded DNA probe of known, different base sequence, at a known position.
(a) A hospital laboratory uses a microarray to test a patient's DNA sample for the presence of several different disease-associated alleles at the same time. Describe how the patient's DNA sample is prepared and applied to the microarray, and explain how the results reveal whether any of the disease-associated alleles are present. [4]
(b) In a separate investigation, mRNA extracted from a tumour sample is converted to cDNA and labelled with a red fluorescent marker, and mRNA from a healthy tissue sample is converted to cDNA and labelled with a green fluorescent marker. Equal amounts of both labelled samples are applied together to the same microarray. Explain how the colour produced at a particular spot would show that the corresponding gene is expressed more highly in the tumour sample than in the healthy sample. [3]
Show worked solution Hide worked solution
Worked solution
Part (a): Screening a patient’s DNA for disease-associated alleles
A microarray works by exploiting complementary base pairing between a known probe and an unknown sample.
- Sample preparation. A sample of the patient’s DNA is extracted, cut into fragments (for example using restriction enzymes or by PCR amplification of specific regions), and its strands separated so the DNA is single-stranded. Each fragment is labelled with a fluorescent marker.
- Application to the array. The labelled sample is washed over the surface of the microarray, where it comes into contact with every probe spot. Wherever a fragment in the sample has a base sequence complementary to the probe fixed at a particular spot, the fragment hybridises (base-pairs) with that probe and becomes bound there. Fragments with no matching probe do not bind and are washed away in a rinsing step.
- Detection. The slide is scanned, typically with a laser, to detect which spots fluoresce and how brightly.
Because the base sequence, and therefore the disease association, of the probe fixed at every position on the array is known in advance, a spot that fluoresces identifies exactly which disease-associated allele (or alleles) the patient’s DNA contains, allowing several different alleles to be screened for simultaneously in a single test.
Part (b): Comparing gene expression with a two-colour microarray
Here, the microarray is used differently: instead of testing for the presence of an allele, it compares how much a gene is being transcribed in two different samples.
- mRNA is a direct product of transcription, so the amount of mRNA (and therefore of the cDNA made from it) for a particular gene reflects how highly that gene is being expressed in the tissue it came from.
- If a gene is transcribed more in the tumour sample than in the healthy sample, more red-labelled tumour cDNA than green-labelled healthy cDNA will be available to hybridise with the probe for that gene.
- At that probe’s spot, more red label than green label ends up bound, so the spot fluoresces predominantly red (rather than green, or an even/yellow mix of both colours, which would indicate similar expression in both samples).
By scanning the whole array and comparing the balance of red and green fluorescence at every spot, many genes can be compared for their relative level of expression between the tumour and healthy tissue in a single experiment.
Final answers
- (a) The patient’s labelled, single-stranded DNA sample hybridises only with complementary probes at known positions; a fluorescent spot after washing identifies that the corresponding disease-associated allele is present in the patient’s DNA.
- (b) A spot fluorescing predominantly red shows that more red-labelled tumour cDNA than green-labelled healthy cDNA hybridised there, meaning more mRNA for that gene was present in (and so it is expressed more highly in) the tumour sample.