Biotechnology Applications
Explore the key molecular biology techniques used in modern biotechnology: PCR, gel electrophoresis, DNA sequencing, and an introduction to CRISPR gene editing.
Polymerase Chain Reaction (PCR)
PCR is a technique that amplifies (copies) a specific segment of DNA millions of times from a tiny initial sample. It is fundamental to forensics, medical diagnostics, and genetic research. PCR uses a heat-stable enzyme called Taq polymerase (from the thermophilic bacterium Thermus aquaticus).
The Three Steps of Each PCR Cycle
1. Denaturation (~95 °C)
Heat separates the double-stranded DNA into two single strands
2. Annealing (~55-65 °C)
Short DNA primers bind to complementary sequences flanking the target region
3. Extension (~72 °C)
Taq polymerase synthesises new DNA strands from the primers, doubling the target DNA
After n cycles, the number of copies = 2n. After 30 cycles: ~1 billion copies!
Gel Electrophoresis
Gel electrophoresis separates DNA fragments by size. DNA is negatively charged (due to phosphate groups), so it migrates toward the positive electrode when placed in an electric field. Smaller fragments move faster through the gel matrix.
How Gel Electrophoresis Works
Setup
- 1. DNA samples loaded into wells in agarose gel
- 2. Electric current applied (negative to positive)
- 3. DNA migrates through the gel pores
- 4. Gel stained to visualise DNA bands
Interpreting Results
A DNA ladder (known sizes) is run alongside for comparison.
DNA Sequencing and CRISPR
DNA sequencing determines the exact order of nucleotides in a DNA molecule. Modern next-generation sequencing (NGS) can read entire genomes rapidly and affordably. CRISPR-Cas9 is a revolutionary gene-editing tool that can precisely cut and modify DNA at specific locations.
DNA Sequencing
Sanger sequencing uses chain-terminating dideoxynucleotides labelled with fluorescent dyes to determine the sequence one base at a time.
Next-gen sequencing reads millions of fragments simultaneously, enabling whole genome sequencing in hours rather than years.
CRISPR-Cas9
A guide RNA directs the Cas9 protein to a specific DNA sequence. Cas9 cuts both strands at this location.
The cell's repair mechanisms can then knock out the gene (via NHEJ) or insert a new sequence (via HDR with a template).
Applications and Ethics
These technologies enable: forensic DNA profiling, prenatal genetic testing, gene therapy for genetic diseases, creation of genetically modified organisms, and personalised medicine.
Ethical considerations include germline editing (changes passed to future generations), equitable access to gene therapies, environmental impacts of GMOs, and genetic privacy concerns.
Key Vocabulary
PCR (Polymerase Chain Reaction)
A technique to amplify a specific DNA segment through repeated cycles of denaturation, annealing, and extension using Taq polymerase.
Gel Electrophoresis
A method for separating DNA fragments by size using an electric field and a gel matrix. Smaller fragments travel farther.
CRISPR-Cas9
A gene-editing system using a guide RNA to direct the Cas9 nuclease to a target DNA sequence for precise cutting and modification.
DNA Primer
A short single-stranded DNA sequence (~20 bases) that binds to the target region and provides a starting point for DNA polymerase during PCR.
Worked Examples
How many copies of a target DNA sequence will be produced after 25 cycles of PCR, starting with a single copy?
Step 1: Each cycle doubles the number of copies: 2n
Step 2: 225 = 33,554,432
Answer: Approximately 33.6 million copies after 25 cycles.
In gel electrophoresis, sample A shows bands at 500 bp, 300 bp, and 200 bp, while sample B shows a single band at 1000 bp. What can you conclude?
Step 1: Sample A has been cut into three fragments by restriction enzymes (500+300+200 = 1000 bp total).
Step 2: Sample B remains uncut -- it is a single fragment of 1000 bp.
Answer: Both samples likely contain the same 1000 bp DNA, but sample A was treated with restriction enzymes that cut at two recognition sites, while sample B was not digested.
Explain how CRISPR-Cas9 could be used to treat sickle cell disease.
Step 1: Sickle cell disease is caused by a point mutation (A→T) in the beta-globin gene, producing abnormal haemoglobin.
Step 2: A guide RNA is designed to target the mutated sequence in haematopoietic stem cells extracted from the patient.
Step 3: Cas9 cuts the DNA at the mutation site. A repair template with the correct sequence is provided for homology-directed repair.
Answer: The corrected stem cells are returned to the patient, where they produce normal red blood cells with functional haemoglobin. This is a form of somatic gene therapy.
Knowledge Check
Select the correct answer for each question. Click "Check Answer" to see if you are right.
Question 1
In PCR, the purpose of the denaturation step is to:
Question 2
In gel electrophoresis, DNA migrates toward the positive electrode because:
Question 3
The role of the guide RNA in CRISPR-Cas9 is to:
Question 4
Taq polymerase is used in PCR instead of regular DNA polymerase because it:
Question 5
Starting with one double-stranded DNA molecule, how many copies are present after 3 complete PCR cycles?
Key Concepts Summary
- ●PCR amplifies DNA through cycles of denaturation (95 °C), annealing (55-65 °C), and extension (72 °C). Each cycle doubles the DNA.
- ●Gel electrophoresis separates DNA fragments by size. Smaller fragments migrate faster. Used for DNA profiling and analysis.
- ●DNA sequencing (Sanger or next-gen) determines the exact nucleotide order, enabling whole genome analysis.
- ●CRISPR-Cas9 uses a guide RNA to direct precise DNA cutting by Cas9, enabling gene knockout or insertion.
- ●These technologies raise important ethical questions about germline editing, genetic privacy, and equitable access.