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This lesson covers two fundamental techniques in molecular biology — the polymerase chain reaction (PCR) and gel electrophoresis — as required by the Edexcel A-Level Biology specification (9BI0, Topic 7).
PCR is a technique used to amplify (make millions of copies of) a specific segment of DNA in vitro (outside a living organism). It was developed by Kary Mullis in 1983 and has revolutionised molecular biology, forensics and medicine.
Many applications require large quantities of a specific DNA fragment, but the starting sample may contain only tiny amounts of DNA (e.g. a single hair follicle at a crime scene, ancient DNA from fossils, or a small tissue biopsy). PCR can amplify a target sequence from as little as a single molecule of DNA.
| Component | Function |
|---|---|
| Target DNA | The DNA sample containing the sequence to be amplified |
| Two primers | Short, single-stranded DNA sequences (typically 18–25 nucleotides) that are complementary to the flanking regions of the target sequence |
| Taq DNA polymerase | A heat-stable DNA polymerase enzyme (from Thermus aquaticus, a thermophilic bacterium) |
| Free DNA nucleotides (dNTPs) | Deoxyribonucleotide triphosphates (dATP, dTTP, dGTP, dCTP) — the building blocks for new DNA |
| Buffer solution | Provides optimal pH and Mg²⁺ ions for enzyme activity |
| Thermal cycler | A programmable machine that rapidly changes temperature |
Each PCR cycle consists of three temperature-controlled steps:
| Step | Temperature | Duration | What happens |
|---|---|---|---|
| 1. Denaturation | 94–98°C | 20–30 seconds | Hydrogen bonds between DNA strands break; double-stranded DNA separates into two single strands |
| 2. Annealing | 50–65°C | 20–30 seconds | Primers bind (anneal) to complementary sequences on the single-stranded DNA by hydrogen bonding |
| 3. Extension | 72°C | Depends on fragment length | Taq polymerase extends the primers by adding complementary nucleotides in the 5' → 3' direction |
Exam Tip: The annealing temperature must be specific to the primers — too high and they won't bind; too low and they bind non-specifically to the wrong sequences. In exams, you are expected to know the three steps and their approximate temperatures.
The following diagram illustrates the cyclical nature of PCR:
graph TD
A["Double-Stranded DNA"] -->|"95°C Denaturation<br/>Strands separate"| B["Single Strands"]
B -->|"55°C Annealing<br/>Primers bind"| C["Primers Attached"]
C -->|"72°C Extension<br/>Taq polymerase"| D["New DNA Strands"]
D -->|"Cycle repeats<br/>(25-30 times)"| A
After each cycle, the number of copies of the target DNA doubles:
| Cycle number | Copies of target |
|---|---|
| 0 | 1 |
| 1 | 2 |
| 2 | 4 |
| 3 | 8 |
| 10 | 1,024 |
| 20 | ~1 million |
| 30 | ~1 billion |
After n cycles, the number of copies = 2ⁿ (assuming 100% efficiency).
A typical PCR run involves 25–35 cycles and takes 2–3 hours.
Taq polymerase is used because it is thermostable — it is not denatured at the high temperatures used in the denaturation step (94–98°C). Normal DNA polymerase from E. coli would be destroyed at these temperatures.
| Application | Details |
|---|---|
| Forensic science | Amplifying DNA from crime scene samples for genetic fingerprinting |
| Medical diagnosis | Detecting pathogens (e.g. SARS-CoV-2 testing), genetic diseases |
| Paternity testing | Comparing DNA profiles |
| Archaeology | Amplifying ancient DNA from fossils or preserved specimens |
| Genetic research | Cloning genes, sequencing, mutagenesis studies |
Gel electrophoresis is a technique used to separate DNA (or RNA or protein) fragments according to their size. It is often used after PCR or after treatment with restriction enzymes.
DNA fragments carry a negative charge (due to the phosphate groups in the sugar-phosphate backbone). When placed in an electric field, they migrate towards the positive electrode (anode). Smaller fragments move through the gel matrix more easily and therefore travel further than larger fragments.
| Band position | Fragment size |
|---|---|
| Close to the well (top) | Large fragments |
| Far from the well (bottom) | Small fragments |
The DNA ladder allows you to estimate the size (in base pairs, bp) of unknown fragments by comparing their position to the known markers.
Exam Tip: When describing gel electrophoresis, always state that DNA is negatively charged, moves towards the positive electrode, and smaller fragments move further. These are the three key points that examiners look for.
Restriction endonucleases (restriction enzymes) are enzymes that cut DNA at specific recognition sequences. They are produced naturally by bacteria as a defence against bacteriophages.
| Feature | Detail |
|---|---|
| Recognition sequence | A specific short palindromic DNA sequence (typically 4–8 bp) |
| Palindromic | The sequence reads the same on both strands in the 5' → 3' direction |
| Sticky ends | Some restriction enzymes cut the two strands at different positions, creating short single-stranded overhangs |
| Blunt ends | Other restriction enzymes cut both strands at the same position, creating flush ends |
Example (EcoRI):
| Strand | Sequence |
|---|---|
| 5' → 3' | G ↓ A A T T C |
| 3' → 5' | C T T A A ↑ G |
EcoRI cuts between G and A on both strands, producing sticky ends with the overhang 5'-AATT-3'.
Sticky ends are crucial for genetic engineering because they allow DNA fragments from different sources to be joined together. If two DNA fragments have been cut with the same restriction enzyme, they will have complementary sticky ends that can base-pair with each other. The fragments can then be permanently joined by DNA ligase.
Genetic fingerprinting uses PCR and gel electrophoresis to produce a pattern of DNA bands that is unique to an individual (except for identical twins).
| Application | How it is used |
|---|---|
| Forensics | Matching suspect DNA to crime scene samples |
| Paternity/maternity testing | A child inherits half their STR alleles from each parent |
| Immigration cases | Verifying family relationships |
| Identifying disaster victims | Matching DNA to relatives |
| Conservation | Studying genetic diversity in endangered populations |
Exam Tip: In genetic fingerprinting, it is the number of repeats at each STR locus that varies between individuals, not the sequence of the repeat itself. Each person has two alleles at each STR locus (one from each parent), producing one or two bands per locus.
Southern blotting is a technique that combines gel electrophoresis with hybridisation to detect specific DNA sequences:
| Technique | Purpose | Key principle |
|---|---|---|
| PCR | Amplify specific DNA sequences | Thermal cycling: denature, anneal, extend |
| Gel electrophoresis | Separate DNA by size | DNA is negatively charged; smaller fragments travel further |
| Restriction enzymes | Cut DNA at specific sequences | Produce sticky or blunt ends |
| Genetic fingerprinting | Identify individuals by DNA | STR variation produces unique band patterns |
Exam Tip: PCR and gel electrophoresis questions are extremely common. Be able to describe each step of PCR clearly, explain the role of each component, and describe how gel electrophoresis separates fragments by size.
This material sits in Edexcel 9BI0 Topic 8 (Grey Matter — Coordination, Response and Gene Technology), which expects candidates to describe PCR as a cyclic, in-vitro amplification of a defined DNA target using thermostable DNA polymerase (Taq), flanking primers, dNTPs and a programmed denature-anneal-extend cycle, and to describe gel electrophoresis as size-based separation of negatively charged DNA fragments through an agarose matrix toward the positive electrode. Synoptic links run backwards to lesson 1 on gene structure (the primers anneal to specific flanking sequences, so the gene structure dictates what can be amplified) and lesson 2 on transcription (PCR mimics DNA replication, not transcription — a frequent misconception); laterally to Topic 4 (biodiversity and natural resources) for DNA barcoding of species using PCR-amplified COI, Topic 6 (immunity, infection and forensics) for SARS-CoV-2 RT-qPCR diagnostics and forensic profiling, and Topic 1 (DNA structure and replication) for the antiparallel template logic that makes primer extension directional; and forwards to lesson 7 on genetic engineering (PCR is the standard way to obtain a clean, abundant copy of a gene before ligation into a vector), lesson 8 on gene therapy and screening (PCR underlies prenatal screening and CRISPR validation), and lesson 9 on genomics and bioinformatics (PCR products are the starting material for short-read sequencing libraries). Refer to the official Pearson Edexcel 9BI0 specification document for exact wording.
Question (8 marks):
(a) Describe what happens during one PCR cycle, giving the temperature, approximate duration and molecular event for each step. (4)
(b) A forensic scientist starts with one intact target DNA molecule and runs 30 PCR cycles at perfect efficiency. Calculate the theoretical number of copies of the target region produced and explain why the actual yield is lower. (4)
Solution with mark scheme:
(a) M1 (AO1) — denaturation. 95 °C for ~30 s. Hydrogen bonds between complementary base pairs break and the double-stranded DNA template separates into two single strands. The covalent phosphodiester backbone is unaffected.
A1 (AO1) — annealing. 50–65 °C for ~30 s (temperature set roughly 5 °C below the primer melting temperature, T_m). Two short single-stranded primers (~20 nucleotides) bind by complementary base-pairing to the flanking regions of the target on each separated strand, defining the boundaries of the amplicon.
A1 (AO1) — extension. 72 °C for ~30 s. Taq DNA polymerase (from Thermus aquaticus) adds free dNTPs by complementary base pairing to the 3' end of each primer, synthesising the new strand in the 5' → 3' direction along each template.
A1 (AO2) — outcome of one cycle. Each starting double-stranded template has yielded two double-stranded copies of the region between the primers — the target has doubled. Repeating denature → anneal → extend re-uses the new strands as templates for the next round.
(b) M1 (AO2) — exponential amplification. Each cycle nominally doubles the target, so after n cycles the copy number is 2ⁿ. Starting with one template, 30 cycles gives 2³⁰ ≈ 1.07 × 10⁹ copies — roughly a billion-fold amplification.
A1 (AO2) — short-product takeover. From cycle 3 onward, the dominant product is the defined-length amplicon flanked on both sides by primers; long, ragged off-target strands are diluted out exponentially.
A1 (AO3) — why the real yield is lower. Efficiency falls below 100% because primer-dimer and non-specific products consume reagents; dNTPs and primers become limiting at high copy number; Taq activity declines after many heating cycles; and the high product concentration favours template-template re-annealing over primer-template annealing. This produces the plateau phase of a real PCR run.
A1 (AO3) — significance. The exponential nature is the whole point — a single cell, an ancient bone fragment, a forensic trace, or a single virion in a swab becomes detectable. PCR turns "is this DNA present?" into a yes/no question with a sensitivity floor approaching one molecule.
Total: 8 marks (M2 A6).
Question (6 marks): A clinical laboratory uses RT-qPCR to detect SARS-CoV-2 in a nasopharyngeal swab. The protocol uses reverse transcriptase to convert viral RNA to cDNA, then 40 cycles of PCR with primers specific to the viral N gene and a fluorescent TaqMan probe. After the cycle the products are visualised by gel electrophoresis, showing a single band at ~120 bp in positive samples. Explain how RT-qPCR amplifies the target with sequence specificity, why a single band of defined size confirms a positive result, and why gel electrophoresis separates DNA fragments by size.
Mark scheme decomposition by AO:
| Mark | AO | Earned by |
|---|---|---|
| 1 | AO1.1 | Stating that reverse transcriptase synthesises a complementary DNA (cDNA) copy of the single-stranded viral RNA, which then serves as the PCR template |
| 2 | AO1.2 | Describing the denature-anneal-extend cycle with Taq polymerase, N-gene primers and dNTPs, doubling the amplicon each cycle |
| 3 | AO2.1 | Explaining that specificity comes from the primers — only sequences flanked by complementary primer-binding sites are amplified, so a positive band is itself diagnostic |
| 4 | AO2.7 | Explaining that gel electrophoresis separates DNA in an agarose matrix because DNA is uniformly negatively charged (phosphate backbone) and migrates toward the positive electrode, with smaller fragments threading the matrix faster and travelling further |
| 5 | AO3.1 | Interpreting a single band at the predicted size as evidence of a clean, sequence-specific product (no primer-dimer, no off-target), with size-marker comparison giving the molecular ruler |
| 6 | AO3.2 | Synoptic — connecting the diagnostic to lesson 1 (DNA structure underlies primer hybridisation), lesson 7 (PCR products feed cloning), and Topic 6 (PCR is the dominant pathogen-identification platform) |
Total: 6 marks (AO1 = 2, AO2 = 2, AO3 = 2). Edexcel reliably tests PCR through "explain why a clinical/forensic application works" prompts; candidates who treat the primers as merely "starting points" rather than as sequence-specific defining boundaries, or who say gel electrophoresis "separates by charge" rather than "by size given uniform charge density", lose AO2 marks. The mark scheme rewards candidates who explicitly link primer specificity → sequence-defined amplicon → diagnostic band of predicted size.
Lesson 1 (gene structure and the genetic code). Primers are designed against the flanking sequence of the gene of interest. Their length (~20 nt) and GC content set the T_m that determines the annealing temperature. The base-pairing chemistry that holds the double helix together (lesson 1) is the same chemistry that makes primer hybridisation specific.
Lesson 2 (transcription). PCR is often confused with transcription. PCR makes DNA from DNA using DNA polymerase and dNTPs; transcription makes RNA from DNA using RNA polymerase and NTPs. RT-PCR is the bridge — reverse transcriptase makes a DNA copy of an RNA template before PCR amplifies it.
Lesson 7 (genetic engineering and recombinant DNA). PCR is the routine way to obtain a clean, sequenceable copy of a gene before ligation into a plasmid vector. Adding restriction-site tails to the primers gives the amplicon ready-cut ends for cloning. Without PCR, classical cloning required laborious genomic library screening.
Topic 4 (biodiversity — DNA barcoding). Universal primers against conserved regions of COI (animals) or rbcL/matK (plants) PCR-amplify a short, species-diagnostic fragment from any specimen. Comparison against a reference database identifies the species — central to taxonomy, biosecurity, and conservation.
Topic 6 (immunity, infection — pathogen detection). SARS-CoV-2 RT-qPCR was the dominant COVID diagnostic. Real-time fluorescence (with TaqMan probes or SYBR green) tracks amplicon accumulation each cycle, giving a quantitative Ct value inversely related to starting viral load. Same chemistry underlies HIV, hepatitis, tuberculosis and meningococcal panels.
Topic 6 (forensics — STR profiling). Multiplex PCR amplifies short tandem repeats (STRs) at standard loci (e.g. CODIS in the US, NDNAD in the UK). Capillary gel electrophoresis then separates the products by size. Allele lengths at ~13–20 loci give a profile with a random-match probability of ~1 in 10¹⁵ — "DNA fingerprinting".
Lesson 9 (genomics and bioinformatics). PCR products are starting material for Sanger sequencing and for short-read library prep (Illumina). Whole-genome sequencing has reduced reliance on PCR for some applications, but targeted amplicon sequencing remains a workhorse for clinical panels.
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