Principles and Applications of Gene Technology

Lessons

Senior 6 · Biology

Principles and Applications of Gene Technology

5 lessons

Lesson 2 of 5

Artificial DNA synthesis and the polymerase chain reaction

30 min

Finding one gene among twenty thousand

The human genome holds about 3 billion base pairs and roughly 20 000 protein-coding genes. A gene you want to move into a bacterium is a tiny fraction of that, and in human cells it is broken up by introns that a bacterium cannot remove. Gene technologists therefore rarely cut a gene straight out of a chromosome. Instead they make the gene, either by copying it from the cell's mRNA or by building it chemically from its known sequence, and then they copy it millions of times with the polymerase chain reaction. This lesson explains both.

Definition

Reverse transcriptase: an enzyme, first found in retroviruses such as HIV, that makes DNA using an RNA template.

Complementary DNA (cDNA): DNA made from an mRNA template by reverse transcriptase; it has no introns.

Oligonucleotide: a short, single-stranded chain of nucleotides, usually made chemically.

Polymerase chain reaction (PCR): a method that copies a chosen section of DNA many millions of times by repeated cycles of heating and cooling.

Primer: a short single-stranded DNA sequence that pairs with one end of the target sequence and gives DNA polymerase a starting point.

Taq polymerase: a heat-stable DNA polymerase from Thermus aquaticus, a bacterium of hot springs; it is not denatured at 95 °C and works best at about 72 °C.

Making a gene from mRNA

A cell that makes a lot of one protein contains many copies of its mRNA. The β cells of the pancreas, for example, are full of insulin mRNA. That mRNA has already had its introns removed, so it is the ideal template:

The double-stranded cDNA is the coding sequence of the gene with no introns, so a bacterium can transcribe and translate it correctly. Short linkers or primers carrying restriction sites are added to its ends so that it can be cut with the same enzyme as the plasmid.

Making a gene chemically

If the base sequence of a gene is known, from a genome database or worked out from the protein's amino acid sequence, the gene can be built from scratch. A gene-synthesis machine makes short oligonucleotides, adding one nucleotide at a time in a programmed order. Overlapping oligonucleotides are then annealed and joined, and the full gene is copied by PCR. Building a gene this way lets scientists:

change codons to those the host cell uses most, so the protein is made efficiently;
add restriction sites and a promoter at the ends;
make genes that do not exist in nature, or that come from an organism nobody has a sample of.

The first human insulin made in bacteria, at the end of the 1970s, used two chemically synthesised genes, one for each of the insulin chains.

Worked example 1: working back from a protein

Part of a protein has the amino acid sequence Met–Trp–His–Lys. Met and Trp each have one codon; His has two (CAU, CAC) and Lys has two (AAA, AAG). How many different mRNA sequences could code for this part of the protein, and why does this matter when designing a gene?

Multiply the number of codons for each amino acid: $1 \times 1 \times 2 \times 2 = 4$ possible sequences.
For a protein of 50 amino acids with an average of 3 codons each, the number would be about $3^{50} \approx 7 \times 10^{23}$ .

Why it matters. Because the genetic code is degenerate, a protein sequence does not tell you the gene's exact base sequence. When a gene is synthesised for a bacterium, the designer simply chooses, for each amino acid, the codon the bacterium uses most.

The polymerase chain reaction

Kary Mullis devised PCR in 1983. It copies a chosen section of DNA in a test tube, without any living cells. The tube contains:

the template DNA containing the target sequence, even a trace from a drop of blood or a single hair root;
two different primers, each about 20 nucleotides long, complementary to the 3′ ends of the two strands of the target;
free nucleotides carrying the four bases A, T, C and G;
Taq polymerase and a buffer containing magnesium ions, which the enzyme needs.

A machine called a thermal cycler heats and cools the tube through the same three steps again and again:

Denaturation (about 95 °C): the hydrogen bonds between the two strands break and the strands separate.
Annealing (about 55 to 65 °C): the primers bind by base pairing to their complementary sequences at the 3′ end of each target strand. Two different primers are needed because the strands are antiparallel.
Extension (about 72 °C): Taq polymerase adds nucleotides to the 3′ end of each primer, building a new complementary strand.

Each cycle doubles the number of copies of the target. Starting from one molecule, the number after $n$ cycles is $2^{n}$ , so 30 cycles give $2^{30} \approx 1.1 \times 10^{9}$ copies in an hour or two. The primers fix the ends of the product: from the third cycle onwards, most new strands run exactly from one primer to the other, so the copies are all the same length. Eventually the reaction slows as primers and nucleotides run out.

PCR is used to copy genes for cloning, to produce DNA profiles from tiny forensic samples, and to diagnose infections. In RT-PCR, reverse transcriptase first turns a virus's RNA into DNA so that it can be amplified; this is how HIV viral load and SARS-CoV-2 infection are measured.

Worked example 2: how many copies?

A forensic sample contains 50 copies of a target sequence. (a) How many copies would there be after 25 cycles of PCR if every cycle doubled the DNA? (b) Repeat the calculation for a more realistic efficiency in which each cycle multiplies the DNA by 1.9. (c) If each cycle takes 2.5 minutes, how long does the run take?

(a) $50 \times 2^{25} = 50 \times 33\,554\,432 = 1.7 \times 10^{9}$ copies (2 s.f.).
(b) $50 \times 1.9^{25} = 4.7 \times 10^{8}$ copies (2 s.f.), less than a third of the ideal.
(c) $25 \times 2.5 = 62.5$ minutes, about one hour.

Interpretation. Even with less than perfect efficiency, a trace of DNA becomes hundreds of millions of copies in about an hour, which is enough to analyse on a gel (lesson 4). The same power is a weakness: one stray molecule of contaminating DNA is amplified too, so laboratories use separate rooms, gloves and negative controls.

Warning

Common mistakes

"Primers bind at 95 °C." At 95 °C nothing can base-pair; primers bind when the mixture is cooled to the annealing temperature.
"Any DNA polymerase will do." A human or E. coli polymerase would be denatured in the first 95 °C step. Taq polymerase survives it.
"cDNA is the same as the gene in the chromosome." cDNA has no introns, and no promoter or other regulatory sequences.
"PCR copies the whole genome." It copies only the region between the two primers.

Amoeba Sisters: "PCR (Polymerase Chain Reaction)". Watch for what happens at each of the three temperatures, and why the number of copies doubles every cycle.

✦

Practice: making and copying genes

Name the enzyme that makes DNA from an RNA template, and the group of viruses it was first found in.
Explain why cDNA made from mRNA, rather than DNA cut from a chromosome, is used to make a human protein in bacteria.
Give two advantages of making a gene chemically.
Describe what happens in each of the three steps of a PCR cycle, giving the temperature.
Explain why Taq polymerase is used in PCR.
Calculate the number of copies of a target sequence produced from 1 000 starting molecules after 20 perfect cycles. Give your answer in standard form.
A peptide has the sequence Met–Phe–Cys–Tyr. Phe, Cys and Tyr each have two codons. How many mRNA sequences could code for it?
A PCR run gives no product. Suggest three possible reasons.

Show answersHide answers

Reverse transcriptase; retroviruses (such as HIV).
Human genes contain introns, which bacteria cannot remove. mRNA has already been spliced, so cDNA has only the coding sequence; and cells making the protein contain many copies of its mRNA, so it is easy to obtain.
Any two: codons can be chosen to suit the host; restriction sites and a promoter can be built in; no tissue sample is needed; the sequence can be changed or designed.
Denaturation, about 95 °C: strands separate. Annealing, about 55 to 65 °C: primers bind to the 3′ ends of the target strands. Extension, about 72 °C: Taq polymerase adds nucleotides to the primers, making new strands.
It comes from a hot-spring bacterium, so it is not denatured at 95 °C and works best at 72 °C; it does not have to be replaced each cycle.
$1\,000 \times 2^{20} = 1\,048\,576\,000 \approx 1.0 \times 10^{9}$ .
$1 \times 2 \times 2 \times 2 = 8$ .
Any three: primers do not match the template; annealing temperature too high; Taq polymerase or nucleotides missing or damaged; no Mg²⁺; no template DNA, or the target sequence absent from it; thermal cycler fault.

Key points

Reverse transcriptase makes cDNA from mRNA; DNA polymerase makes the second strand. cDNA lacks introns, so bacteria can express it.
Genes can be synthesised from oligonucleotides when their sequence is known, with codons chosen for the host.
PCR: denature at 95 °C, anneal primers at 55 to 65 °C, extend with Taq polymerase at 72 °C; repeat 25 to 35 times.
Copies after $n$ cycles $=$ starting copies $\times 2^{n}$ (in theory).
Uses: cloning, DNA profiling, diagnosis (RT-PCR for RNA viruses).

Sources and credits

17.1 Biotechnology, from Biology 2e by Mary Ann Clark, Matthew Douglas and Jung Choi, OpenStax (Rice University), licensed under CC BY 4.0. Used for PCR, reverse transcriptase and cDNA. Adapted by Ganzaa.
Photo: CSIRO ScienceImage 610 PCR Machine in Genetics Laboratory, by K. Aken, CSIRO, via Wikimedia Commons, licensed under CC BY 3.0.
Video: "PCR (Polymerase Chain Reaction)", Amoeba Sisters, embedded from YouTube.

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

1Finding one gene among twenty thousand

2Making a gene from mRNA

3Making a gene chemically

4The polymerase chain reaction

Finding one gene among twenty thousand

Making a gene from mRNA

Making a gene chemically

The polymerase chain reaction