IMAT Biology · Microorganisms and Biotechnology

PCR, Gel Electrophoresis, Probes and Sequencing

PCR copies a stretch of DNA by doubling it each cycle, gel electrophoresis sorts the pieces by size, probes find a sequence by base pairing, and sequencing reads the bases in order.

All Microorganisms and Biotechnology notes

Why this matters

Only one past question sits here: a 2015 item on which probes of a DNA microarray bind a sample, which is base pairing. PCR, gels and sequencing have not been asked in this chapter yet, but they are standard syllabus and easy marks.

Concept 1 of 4: The polymerase chain reaction (PCR)

PCR is DNA replication in a test tube, repeated over and over. Heat replaces the helicase that would unzip DNA in a cell, and a heat-proof polymerase builds the new strands. Each cycle copies every strand present, so the number of copies doubles each time: a single molecule becomes about a billion after 30 cycles.

Definition

A PCR tube contains the template DNA, two short single-stranded DNA primers (one for each end of the target), free nucleotides (dNTPs), a heat-stable Taq polymerase (from Thermus aquaticus, a hot-spring bacterium) and a buffer with magnesium ions. Each cycle has three steps:

Denaturation, about 95 °C: hydrogen bonds break and the two strands separate.
Annealing, about 50 to 65 °C: the primers bind (by base pairing) to their complementary sequences. This is the coolest step.
Extension, about 72 °C: Taq polymerase adds nucleotides to the 3' end of each primer, copying the template.

Uses: forensic samples, diagnosing infections, cloning a gene. RT-PCR first copies RNA into DNA with reverse transcriptase (used to detect RNA viruses such as SARS-CoV-2). PCR was invented by Kary Mullis (1983).

Copies after n cycles (ideal)

N = N_0 \times 2^{n}

$N_0$ number of double-stranded copies at the start
$n$ number of cycles
$N$ number of copies after n cycles

Worked example

A forensic sample contains a single copy of a target DNA sequence. About how many copies are there after 25 cycles of PCR, assuming perfect doubling?

Practice this conceptself-check · 4 quick reps

PCR needs no helicase, and its primers are DNA

In PCR, heat separates the strands, so no helicase is added. The primers are short pieces of DNA made in the laboratory, whereas in a living cell the primers for replication are RNA made by primase. And annealing, not extension, is the lowest temperature in the cycle.

Concept 2 of 4: Gel electrophoresis and DNA profiling

Every nucleotide carries one negative phosphate group, so every DNA molecule has the same charge per unit length. In an electric field all fragments are pulled towards the positive electrode equally hard. The gel is a tangle of fibres that holds back long pieces more than short ones, so the fragments end up sorted by size alone.

Definition

DNA is loaded into wells at the end of an agarose gel next to the negative electrode (cathode).
DNA is negatively charged (phosphate groups), so it moves towards the positive electrode (anode).
Smaller fragments move further; large ones stay near the wells.
Sizes are read by comparing the bands with a DNA ladder of known sizes run in the next lane.
DNA is invisible, so the bands are shown with a stain under UV light, or with a labelled probe.
DNA profiling compares regions that vary greatly between people, such as short tandem repeats (STRs). In a paternity test, every band of the child must match a band of the mother or of the father.
Proteins can be separated the same way, in polyacrylamide gels.

Feature	What happens	Why
Direction of movement	Towards the positive electrode	Phosphate groups make DNA negative
Distance travelled	Short fragments travel furthest	The gel slows long molecules more
Fragment nearest the wells	The largest one	It is held back most by the gel
Size of an unknown band	Read against the DNA ladder	Same gel, same conditions
Child in a paternity test	Each band matches the mother or the father	Half of each person's DNA comes from each parent

Practice this conceptself-check · 4 quick reps

DNA runs to the positive electrode, smallest first

DNA is negative, so it moves to the anode (positive), away from the wells at the cathode. The shortest fragments go furthest. Options with DNA moving to the negative electrode, or large fragments moving fastest, are wrong.

Concept 3 of 4: Complementary base pairing, probes and microarrays

A probe is a short single strand of DNA with a label on it. It sticks only where the bases on the other strand pair with its own, A with T and G with C, all along its length. So a probe works as a search tool: if it lights up, the matching sequence is there.

Definition

In DNA, A pairs with T (two hydrogen bonds) and G pairs with C (three hydrogen bonds). In RNA, U replaces T, so A pairs with U.
The two strands run in opposite directions (antiparallel): one 5' to 3', the other 3' to 5'.
A DNA probe is a short, labelled (fluorescent or radioactive), single-stranded DNA that hybridises (pairs) with its complementary sequence.
A DNA microarray is a chip with thousands of different probes in spots. A labelled sample is washed over it; the spots whose probes are complementary to part of the sample light up. Used to test many genes or mutations at once.
Because of pairing, double-stranded DNA has as much A as T and as much G as C (Chargaff's rule).

Base pairing

A \leftrightarrow T \quad (\text{RNA: } A \leftrightarrow U) \qquad G \leftrightarrow C

Worked example

A target strand reads 5'-ACGGTTCA-3'. Write the probe that binds to it along its whole length, first lined up under the target, then written 5' to 3'.

Practice this conceptself-check · 4 quick reps

A DNA probe or sticky end contains T, never U

Uracil is found only in RNA. When asked for the bases of a DNA sticky end or a DNA probe, any answer containing U is wrong, even if the pairing is otherwise right. Work out each base by pairing with the opposite strand: A with T, G with C.

Concept 4 of 4: DNA sequencing in outline

Sequencing reads the order of bases. The classic Sanger method makes copies of a strand that stop at random points, each ending with a labelled base. Line the copies up by length and the ending of each one, from shortest to longest, spells out the sequence.

Definition

Sanger (chain termination) sequencing (1977): DNA polymerase copies the template using normal nucleotides plus a small amount of labelled dideoxynucleotides (ddNTPs).
A ddNTP has no 3'-OH group, so once it is added no further phosphodiester bond can form and the chain stops.
This gives fragments of every length, each ending with a coloured base. They are separated by size (capillary electrophoresis), and the colours read from shortest to longest give the sequence of the new strand, which is the complement of the template.
Next-generation sequencing reads millions of fragments in parallel, so a whole genome takes days and costs little.
The Human Genome Project finished in 2003: about 3 billion base pairs and about 20,000 protein-coding genes.

Method or project	Key idea	What to remember
Sanger sequencing	Chain termination by ddNTPs	ddNTPs lack the 3'-OH; fragments sorted by size
Next-generation sequencing	Millions of fragments read at the same time	Whole genomes quickly and cheaply
Human Genome Project	First full human genome sequence, 2003	About 3 billion base pairs, about 20,000 protein-coding genes
Bioinformatics	Computers store and compare sequences	Finds genes, mutations and relationships between species

Practice this conceptself-check · 4 quick reps

Sanger sequencing reads the new strand

The labelled fragments are copies made against the template, so the colours give the complementary strand. To get the template sequence, pair each base back.

Summary — formulas & gotchas at a glance

A revision cheat-sheet for the formulas and gotchas above. Click any concept name to jump back to its full explanation.

Formulas (2)

The polymerase chain reaction (PCR)
Copies after n cycles (ideal)
$N = N_0 \times 2^{n}$
Complementary base pairing, probes and microarrays
Base pairing
$A \leftrightarrow T \quad (\text{RNA: } A \leftrightarrow U) \qquad G \leftrightarrow C$

Reference tables (2)

Gel electrophoresis and DNA profiling5 rows

Feature	What happens	Why
Direction of movement	Towards the positive electrode	Phosphate groups make DNA negative
Distance travelled	Short fragments travel furthest	The gel slows long molecules more
Fragment nearest the wells	The largest one	It is held back most by the gel
Size of an unknown band	Read against the DNA ladder	Same gel, same conditions
Child in a paternity test	Each band matches the mother or the father	Half of each person's DNA comes from each parent

DNA sequencing in outline4 rows

Method or project	Key idea	What to remember
Sanger sequencing	Chain termination by ddNTPs	ddNTPs lack the 3'-OH; fragments sorted by size
Next-generation sequencing	Millions of fragments read at the same time	Whole genomes quickly and cheaply
Human Genome Project	First full human genome sequence, 2003	About 3 billion base pairs, about 20,000 protein-coding genes
Bioinformatics	Computers store and compare sequences	Finds genes, mutations and relationships between species

Watch out for (4)

PCR needs no helicase, and its primers are DNA
→ The polymerase chain reaction (PCR)
DNA runs to the positive electrode, smallest first
→ Gel electrophoresis and DNA profiling
A DNA probe or sticky end contains T, never U
→ Complementary base pairing, probes and microarrays
Sanger sequencing reads the new strand
→ DNA sequencing in outline

Drill the past-year questions on this topic

Open the bank with this topic pre-filtered.

Open in the bank