The shape of the lesson
By the end of the lesson, learners can
- Reading a microarray: The slide holds 64 spots in an 8 by 8 grid, all dark to start.
- Shared databases: bioinformatics: Sequencing and microarrays produce huge amounts of data.
- mRNA → cDNA on the array = which genes are being expressed.
- Genomic DNA on the array = which genes are present.
- Green / red = one sample only; yellow = both; dark = neither.
- Databases: nucleotide sequences, amino acid sequences, protein structures.
One picture every idea returns to
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.
Forty-five minutes, phase by phase
| Time | Phase | What happens in the room | Grouping |
|---|---|---|---|
| 0 to 5 min | Hook: predict | Open with the question in the title, Thousands of genes on one slide.. A microarray tests thousands of genes at once: which ones a genome contains, and which ones a cell is switching on. Put the figure on the board: it is the one picture every idea in the lesson returns to. | Whole class, then pairs |
| 5 to 17 min | Teach and check: Reading a microarray | The slide holds 64 spots in an 8 by 8 grid, all dark to start. Project tab 5 of the lab (Genetic technology lab, microarrays and databases) and let a student drive it while the class predicts. | Whole class, then pairs |
| 17 to 30 min | Teach and check: Shared databases: bioinformatics | Sequencing and microarrays produce huge amounts of data. | Mix-Pair-Share |
| 30 to 36 min | Check | The four Stage 1 questions from the student lesson, answered on mini whiteboards or by show of fingers. Re-teach the one most get wrong. | Individual, then whole class |
| 36 to 43 min | Plenary | The exam-style questions from Stage 2, one at a time: thirty seconds alone, then a random call. The feedback lines in the key give the examiner's wording. | Individual, random call |
| 43 to 45 min | Exit and homework | Set the worksheet below (6 questions, 18 marks). Learners self-assess against the objectives before they leave. | Individual |
Protect the parts that carry the learning
The sequence carries about fifty minutes of material, so the lab is driven by one student while the class predicts rather than explored freely. Times are a guide.
Protected: the check at 30 minutes. It is the only individual read on whether the key idea landed, and it is not cut.
Cut first if the build overruns: the last check in Shared databases: bioinformatics, never the check or the plenary.
In a 60 minute block: run worksheet questions 1 to 3 in class as a Rally Coach set and model both the hardest worked example and its check live.
Mix-Pair-Share
Students mix around the room until you call pair; they pair with the nearest classmate, discuss the prompt, then mix again. Three prompts of rising difficulty, three different partners.
The full structure, with its variations and the other twelve, is on the cooperative-learning page.
What to head off, and how
| Trap learners fall into | Teaching move that pre-empts it |
|---|---|
| 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. |
Microarrays and bioinformatics: worksheet
6 questions, 18 marks. Written in the style of the papers; no past paper question is reproduced.
Describe the structure of a microarray.
Outline how a microarray can be used to compare the genes expressed in cancer cells with those expressed in healthy cells of the same tissue.
cDNA from healthy cells was labelled green and from tumour cells red. Four spots gave these results: gene W green, gene X red, gene Y yellow, gene Z dark. Explain what the results show about genes W, X and Z, and suggest which gene might be a target for a new cancer drug.
Explain how a microarray can be used to compare the genomes of two closely related species, rather than their gene expression.
Outline three benefits of databases that provide information about the nucleotide sequences of genes and genomes, and the amino acid sequences and structures of proteins.
A researcher sequences an unknown gene from a soil bacterium. A database search shows its amino acid sequence is 85 % identical to an enzyme from another bacterium, and its predicted structure has a very similar active site. What can the researcher conclude, and what must still be done?
Answer key and marking notes
- A small glass slide (chip) with thousands of spots in a grid, at known positions.
- Each spot holds many copies of a single-stranded DNA probe, complementary to part of one particular gene or sequence.
- Extract mRNA from the cancer cells and from the healthy cells.
- Use reverse transcriptase to make cDNA from each mRNA sample.
- Label the two cDNA samples with different fluorescent dyes (for example red and green) and mix them.
- Apply to the microarray: cDNA hybridises by complementary base pairing to probes for the same gene; wash off unbound cDNA.
- Scan the slide: the colour of each spot shows whether the gene is expressed in one sample, both or neither, and the brightness shows how much mRNA was present.
- W is expressed in healthy cells only (switched off in the tumour); X is expressed in tumour cells only.
- Z is not expressed in either type of cell (no mRNA, though the gene is still present).
- Gene X is a possible target, because its product is made only by tumour cells, so a drug against it may affect tumour cells but not healthy cells.
- Genomic DNA (not mRNA) is extracted from each species, cut into fragments and made single-stranded.
- Each species' DNA is labelled with a different fluorescent dye and hybridised to probes for known genes.
- Spots that fluoresce with both labels show genes present in both genomes; spots with only one label show genes present in only one species (or very different sequences).
Any three of: data are shared freely worldwide so research is not repeated; a new sequence can be searched against known sequences to identify the gene or predict its function; sequences from different species can be compared to find evolutionary relationships; protein structures help in designing drugs that fit active sites or receptors; disease-causing alleles can be identified and genetic tests or probes and primers designed; pathogens can be identified and new strains tracked.
- The gene probably codes for a similar enzyme with a similar function (the two bacteria may share a common ancestor).
- The prediction must be tested by experiment, for example by expressing the gene and testing the activity of the protein.
8 multiple-choice questions on this lesson
From the topic question bank. Assign them in the app for a marked score in your gradebook, or run the topic as a class game.
What is a probe on a DNA microarray?
- Aa short single-stranded DNA sequence complementary to part of one target gene
- Ba fluorescent dye that binds to any double-stranded DNA
- Can enzyme that copies mRNA into complementary DNA
- Da protein that recognises and cuts a specific base sequence
When labelled cDNA hybridises to a probe on a microarray, what holds the two together?
- Ahydrogen bonds between complementary bases
- Bphosphodiester bonds formed by DNA ligase
- Cpeptide bonds between the dye and the probe
- Dionic attraction between their phosphate groups
The table shows microarray results for four genes. cDNA from healthy cells was labelled green and cDNA from tumour cells red. Which gene shows the greatest increase in expression in the tumour cells?
- Agene B
- Bgene A
- Cgene C
- Dgene D
Before designing PCR primers to amplify a gene from a newly discovered species of fish, a scientist searches a nucleotide sequence database. What is the most useful thing the search can provide?
- Athe sequence of the same gene in related species, so primers can match regions that are similar in all of them
- Bthe name of the restriction endonuclease that was used to sequence the gene
- Cthe temperature at which Taq polymerase is denatured in fish cells
- Dthe 3D structure of the protein, which decides the primer sequences
Why does measuring mRNA show which genes are being expressed in a cell?
- AmRNA is made only when a gene is transcribed, so its amount reflects how active the gene is
- BmRNA contains a copy of every gene in the genome, whether or not it is used
- Cevery gene is transcribed into the same amount of mRNA in all cells
- DmRNA is identical in every cell of a multicellular organism
In a two-colour gene expression experiment, sample 1 is labelled green and sample 2 red. One spot glows orange: clearly more red than green, but with some green. What does this show about the gene?
- Ait is expressed in both samples, but more strongly in sample 2
- Bit is expressed only in sample 2
- Cit is present in the genome of sample 2 only
- Dit is expressed equally in the two samples
Labelled genomic DNA from a chimpanzee and from a mouse was hybridised to two microarrays carrying probes for 1000 human genes. Chimpanzee DNA bound strongly to 980 probes; mouse DNA bound strongly to 850. Which conclusion is best supported?
- Amore chimpanzee sequences are similar enough to human genes to hybridise, suggesting a closer relationship
- Bmice have 150 fewer genes than humans
- Cchimpanzees express 98 % of human genes in all their cells
- Dmice evolved from chimpanzees and lost some genes
A mutation changes one amino acid in an enzyme. How could a database of protein structures help to predict the effect of the mutation?
- Ait shows whether the changed amino acid lies in or near the active site, where it could alter the shape or binding
- Bit gives the mRNA sequence, so the mutation can be reversed by editing the RNA
- Cit shows which tissues of the body express the enzyme
- Dit lists the restriction sites in the gene, which shows whether the mutation is harmful
Quiz key
Each spot holds many copies of a single-stranded DNA probe whose base sequence is complementary to part of one gene, so only that gene's labelled DNA binds there. The fluorescent label is attached to the sample cDNA, not the probe. Copying mRNA into cDNA is the job of reverse transcriptase. A protein that cuts a specific sequence is a restriction endonuclease.
Hybridisation is complementary base pairing: A with T and C with G, held by hydrogen bonds, which is why a wash removes poorly matched DNA. Phosphodiester bonds join nucleotides within a strand; no ligase is used. Peptide bonds join amino acids. Phosphate groups are all negatively charged, so they repel rather than attract.
Gene B has a very low signal in healthy cells (40) and a very high one in tumour cells (950), so it is switched on in the tumour. Gene A has the largest total signal but similar values in both samples, so its expression hardly changes. Gene C shows the opposite pattern: it is switched off in the tumour. Gene D is barely expressed in either.
Closely related species usually have similar sequences for the same gene, so primers designed from conserved regions in database sequences are likely to bind the new species' DNA. Restriction enzymes are not used to sequence DNA. Taq polymerase is a bacterial enzyme used in the tube, unrelated to fish cells. Primer design depends on the DNA base sequence, not on the shape of the protein.
A gene is expressed when it is transcribed, so the mRNA population shows which genes are active and roughly how strongly. Genomic DNA, not mRNA, contains every gene. Transcription rates differ greatly between genes and between cells. Different cell types transcribe different sets of genes, so their mRNA differs.
Both labelled cDNAs hybridised, so the gene is transcribed in both samples; the stronger red signal means more mRNA in sample 2. Expression in sample 2 only would give a pure red spot. An expression study uses cDNA from mRNA, so it shows transcription, not whether the gene is present in the genome. Equal expression gives yellow.
Strong hybridisation needs closely matching sequences, so more matches suggest more similar genomes and a more recent common ancestor with humans. Weak binding could mean a gene is missing or simply that its sequence has diverged, so it cannot be concluded that mice lack 150 genes. Genomic DNA shows sequence similarity, not expression. Mice and chimpanzees share a common ancestor; one did not evolve from the other.
A 3D structure shows where each amino acid sits; a change in or near the active site, or one that disrupts folding, is likely to affect activity, whereas a change on the surface far from it may have little effect. Protein structure databases hold shapes, not mRNA sequences, and do not reverse mutations. Tissue expression comes from studies of mRNA such as microarrays. Restriction sites say nothing about protein function.
What to project
The student lesson embeds these tabs. Open them full screen on the projector; every slider has a drag handle on the stage.
Slides for this lesson
A projectable deck for every pack is on the way. Until then, present the student lesson full screen: its sections, figure and lab tabs follow the same order as the plan above.