Principles and Applications of Gene Technology

Lessons

Senior 6 · Biology

Principles and Applications of Gene Technology

5 lessons

Lesson 4 of 5

Gel electrophoresis and microarrays

30 min

Seeing DNA

DNA molecules are far too small to see, and a tube of DNA fragments looks exactly like a tube of water. Yet a laboratory can tell you within a day whether a PCR worked, whether a plasmid holds the right gene, who the father of a child is, or which of 20 000 genes are switched on in a tumour. Two techniques make this possible: gel electrophoresis, which sorts DNA fragments by length, and microarrays, which test a sample against thousands of known sequences at once.

Definition

Gel electrophoresis: a method that separates charged molecules, such as DNA fragments, by size, by making them move through a gel in an electric field.

DNA ladder (size marker): a mixture of DNA fragments of known lengths, run beside the samples so that their sizes can be estimated.

Short tandem repeat (STR): a short base sequence repeated many times in a row at one place in non-coding DNA; the number of repeats varies between people.

DNA profile: the pattern of fragment sizes at several STR loci, which is almost unique to each person (identical twins share one).

Probe: a short, single-stranded piece of DNA with a known sequence, used to find a complementary sequence by base pairing (hybridisation).

Microarray (DNA chip): a small glass or plastic slide carrying thousands of tiny spots, each holding many copies of one probe.

How gel electrophoresis works

A slab of agarose gel, a polysaccharide from seaweed, is cast with a row of small wells at one end and covered with a buffer solution that conducts electricity.
The DNA samples, cut with restriction enzymes or copied by PCR, are mixed with a dense, coloured loading dye and pipetted into the wells. A DNA ladder goes in one well.
A direct-current voltage is applied, with the wells at the negative electrode (cathode). The phosphate groups in DNA's backbone carry negative charges, so every fragment moves towards the positive electrode (anode).
The gel is a mesh of fibres. Small fragments slip through the pores easily and travel further; long fragments are held back. Because every nucleotide carries the same charge, charge per unit length is the same for all fragments, so size alone decides the speed.
The current is switched off before the dye reaches the end. The DNA is stained with a dye that fluoresces when bound to DNA, and the gel is viewed under ultraviolet or blue light. Each band is millions of fragments of the same length.

Distance travelled is not proportional to size. Over a useful range it is roughly proportional to the logarithm of the size, so sizes are found by plotting the ladder's distances against $\log_{10}$ of their sizes and reading the unknown from the straight line. Proteins can be separated in the same way, in a polyacrylamide gel, after treating them with a detergent that gives them a uniform negative charge.

Worked example 1: sizing a fragment

In Figure 1 the ladder's 10 000 bp band has moved 10.0 mm and its 1 000 bp band 37.0 mm. Sample A has a single band at 24.0 mm. Assuming distance is a straight-line function of $\log_{10}(\text{size})$ , estimate the size of sample A's fragment.

$\log_{10} 10\,000 = 4$ and $\log_{10} 1\,000 = 3$ . The distance rises by $37.0 - 10.0 = 27.0$ mm for each fall of 1 in $\log_{10}(\text{size})$ .
Sample A is $24.0 - 10.0 = 14.0$ mm beyond the 10 000 bp band, so $\log_{10}(\text{size}) = 4 - \frac{14.0}{27.0} = 3.48$ .
Size $= 10^{3.48} \approx 3\,000$ bp (2 s.f.).

Check. The band lies between the 5 000 bp and 2 000 bp ladder bands, as it should. Reading straight across between neighbouring bands, as if the scale were linear, would give a noticeably different answer: the log scale matters.

Using electrophoresis: DNA profiles and genetic tests

DNA profiling. Our genomes contain many STR loci, where a short sequence such as GATA is repeated, say, 8 times on one chromosome and 13 times on the homologous one. Each person inherits one allele of each STR from each parent. In profiling, about 20 STR loci are copied by PCR using fluorescently labelled primers and the products are separated by size, today usually in a thin capillary tube filled with gel. The chance that two unrelated people share all the sizes is extremely small. Profiles are used to identify victims, to link or exclude suspects, and to test relationships: every band in a child's profile must come from the mother or from the father.

Testing for an allele. A mutation can create or destroy a restriction site, changing the sizes of the fragments. The sickle-cell mutation in the β-globin gene changes one base and destroys a site for the restriction enzyme MstII. After cutting with MstII and detecting the β-globin fragments, the normal allele gives a 1.15 kilobase (kb) fragment and the sickle-cell allele a longer 1.35 kb fragment, because the missing site joins two pieces.

Worked example 2: reading a genetic test

DNA from three people, P, Q and R, is cut with MstII and the β-globin fragments are detected. P shows a band at 1.15 kb only; Q shows bands at 1.15 kb and 1.35 kb; R shows a band at 1.35 kb only. Give each person's genotype, using Hb^A for the normal allele and Hb^S for the sickle-cell allele, and say which is likely to have sickle-cell disease.

P has only the fragment from the intact site: Hb^AHb^A.
Q has one of each: Hb^AHb^S, a carrier with sickle-cell trait.
R has only the longer fragment: Hb^SHb^S, who is likely to have sickle-cell disease.

Why it works. Each person has two copies of the gene. A band appears for each allele that is present, so a heterozygote shows both, and the test can identify carriers, which a blood test of the phenotype alone cannot always do.

Microarrays: thousands of tests at once

A microarray is a chip about the size of a microscope slide carrying tens of thousands of spots, each with a different probe attached. A sample is labelled with a fluorescent dye and washed over the chip. Wherever the sample contains a sequence complementary to a probe, it hybridises (base-pairs) to that spot, and the spot glows when scanned with a laser. A computer records the brightness of every spot.

Analysing genomes. Fragments of a person's or an organism's DNA are labelled and applied. The pattern of glowing spots shows which sequences or alleles are present. Microarrays are used to compare the genomes of related species or strains of a pathogen, and to detect hundreds of thousands of single-base differences (SNPs) at once, for example in searching for alleles linked to a disease.

Analysing mRNA (gene expression). The mRNA in a cell shows which genes are switched on. To compare two kinds of cell, such as normal cells and cancer cells from the same tissue:

mRNA is extracted from each and turned into cDNA with reverse transcriptase.
The cDNA from normal cells is labelled with a green dye and the cDNA from cancer cells with a red dye.
The two are mixed in equal amounts and hybridised to the same chip, where each spot carries a probe for one gene.
Each spot's colour shows the relative activity of that gene in the two samples.

Comparing gene expression in this way has shown which genes are overactive in particular cancers, and it can help doctors to choose a treatment. The spot intensities are usually compared as a ratio of red to green brightness.

Warning

Common mistakes

"DNA moves towards the negative electrode." DNA is negatively charged, so it moves towards the positive electrode (anode). Load the wells at the negative end.
"Larger fragments move further because they carry more charge." Charge per unit length is the same for all fragments; the gel holds the larger ones back, so they move less far.
"A yellow spot means the gene is not expressed." Yellow means equal expression in both samples; a dark spot means no expression.
Safety. Some DNA stains are mutagens, and ultraviolet light damages eyes and skin: wear gloves and a UV-blocking face shield, and use the safer stains and blue-light viewers chosen for schools where possible. The tank runs on a high voltage: switch off before opening the lid.

Amoeba Sisters: "Gel Electrophoresis". Watch how DNA is loaded, which way it moves, and how a ladder is used to estimate fragment sizes.

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Practice: electrophoresis and microarrays

Explain why DNA fragments move through an agarose gel towards the anode.
Explain why fragments separate according to size and not according to charge.
State the purpose of (a) the loading dye, (b) the DNA ladder.
Using the ladder in Worked example 1, estimate the size of a fragment that has travelled 40.0 mm.
A child's DNA profile has bands at 6 STR positions. Explain how this profile could be used to test whether a man is the child's father.
Using Worked example 2, predict the bands shown by the child of parents P and R, and give the child's genotype.
Describe how a microarray could compare the expression of genes in normal cells and in cancer cells.
In a two-colour microarray, a gene's spot gives a red intensity of 1 200 and a green intensity of 300. Calculate the red-to-green ratio and interpret it, given red = cancer cells and green = normal cells.

Show answersHide answers

The phosphate groups of the DNA backbone are negatively charged, so the fragments are attracted to the positive electrode.
Every nucleotide carries the same charge, so all fragments have the same charge per unit length; the gel's mesh slows longer fragments more, so smaller ones travel further.
(a) Makes the sample dense so it sinks into the well, and shows how far the run has gone. (b) Fragments of known sizes, to estimate the sizes of the sample fragments.
$\log_{10}(\text{size}) = 4 - \frac{40.0 - 10.0}{27.0} = 2.89$ ; size $= 10^{2.89} \approx 770$ bp.
Every band in the child's profile must come from the mother or the father. Bands not matched by the mother must all be present in the man's profile; if any are not, he is excluded as the father. If all match, he is very probably the father.
P gives Hb^A and R gives Hb^S, so the child is Hb^AHb^S and shows bands at both 1.15 kb and 1.35 kb.
Extract mRNA from both; make cDNA with reverse transcriptase; label normal cDNA green and cancer cDNA red; mix and hybridise to a chip with probes for many genes; scan: red spots show genes more active in cancer cells, green more active in normal cells, yellow equal.
$\frac{1\,200}{300} = 4$ . The gene is about four times as active in the cancer cells as in the normal cells.

Key points

Electrophoresis: DNA is negatively charged and moves through agarose towards the anode; smaller fragments travel further; sizes are read against a ladder on a log scale.
DNA profiling compares the lengths of STR alleles copied by PCR; every band in a child comes from one of the parents.
A mutation that removes a restriction site changes fragment sizes, so electrophoresis can detect alleles such as Hb^S.
Microarrays carry thousands of probes; labelled DNA or cDNA hybridises to matching spots.
Two-colour arrays compare gene expression: red, green, yellow (equal) or dark (neither).

Sources and credits

17.1 Biotechnology and 17.4 Applying Genomics, from Biology 2e by Mary Ann Clark, Matthew Douglas and Jung Choi, OpenStax (Rice University), licensed under CC BY 4.0. Used for gel electrophoresis and microarrays. Adapted by Ganzaa.
Photo: Gel electrophoresis 2, by Mnolf, via Wikimedia Commons, licensed under CC BY-SA 3.0.
Video: "Gel Electrophoresis", Amoeba Sisters, embedded from YouTube.

Figures 1 and 2, the worked examples and the practice questions were written by Ganzaa.

This lesson adapts CC BY-SA material and is shared under CC BY-SA 4.0.

1Seeing DNA

2How gel electrophoresis works

3Using electrophoresis: DNA profiles and genetic tests

4Microarrays: thousands of tests at once

Seeing DNA

How gel electrophoresis works

Using electrophoresis: DNA profiles and genetic tests

Microarrays: thousands of tests at once