Biochemistry chapters
Biotechnology and human disease
The laboratory tools of molecular biology and how they diagnose disease: restriction enzymes, gel electrophoresis, Southern, Northern and Western blots, PCR and real-time PCR, DNA sequencing, microarrays, ELISA, recombinant DNA products, gene therapy and CRISPR, with worked PCR copy numbers.
- Restriction endonucleases cut DNA at specific palindromic sequences, leaving sticky or blunt ends; DNA fragments are joined into vectors (plasmids) with ligase to make recombinant DNA (human insulin, growth hormone, hepatitis B vaccine, clotting factors).
- Blots: Southern = DNA, Northern = RNA, Western = protein ("SNoW DRoP"); ELISA detects antigens or antibodies with enzyme-linked antibodies (HIV screening, hepatitis serology); Western blot was the classic confirmatory HIV test.
- PCR amplifies a DNA segment by cycles of denaturation (about 94–95 °C), primer annealing (about 50–65 °C) and extension (72 °C) by heat-stable Taq polymerase, doubling the target each cycle (2ⁿ copies after n cycles); RT-PCR starts from RNA (viral loads, COVID-19), real-time PCR quantifies as it runs (GeneXpert for TB and rifampicin resistance).
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Recombinant DNA technology
Restriction endonucleases
- Bacterial enzymes ("molecular scissors") that cut double-stranded DNA at specific recognition sites, usually palindromes (read the same 5′→3′ on both strands, e.g. GAATTC for EcoRI).
- Leave sticky (staggered) or blunt ends.
- Bacteria use them to destroy invading viral DNA; their own DNA is protected by methylation.
Making recombinant DNA
- Cut the human gene and a vector (plasmid, bacteriophage, cosmid, artificial chromosome) with the same restriction enzyme.
- Join them with DNA ligase.
- Put the vector into a host (bacteria, yeast, mammalian cells) and select the cells that took it up (antibiotic resistance markers).
- The host makes the human protein.
For proteins, genes are copied as cDNA (made from mRNA by reverse transcriptase), because bacteria cannot remove introns.
Products: human insulin, growth hormone, erythropoietin, hepatitis B vaccine (HBsAg made in yeast), HPV vaccine, clotting factors VIII and IX, interferons, tissue plasminogen activator, monoclonal antibodies.
Hybridisation and blotting
A probe is a labelled single-stranded piece of DNA or RNA that binds (hybridises) to a complementary sequence.
Gel electrophoresis separates DNA, RNA or protein by size (smaller pieces move further towards the positive electrode, as DNA is negatively charged).
| Blot | Detects | Probe | Uses |
|---|---|---|---|
| Southern | DNA | DNA probe | Gene deletions, RFLP, sickle cell (historically), fingerprinting |
| Northern | RNA | DNA or RNA probe | Gene expression (mRNA levels) |
| Western | Protein | Antibody | Confirming HIV antibodies (classic), Lyme disease, prion detection |
| Southwestern | DNA-binding proteins | DNA probe | Transcription factors |
"SNoW DRoP": Southern = DNA, Northern = RNA, Western = Protein.
ELISA (enzyme-linked immunosorbent assay)
- Detects antigen or antibody using an antibody linked to an enzyme that makes a colour change.
- Very sensitive; used for screening: HIV, hepatitis B (HBsAg) and C, pregnancy tests, hormone levels.
Polymerase chain reaction (PCR)
Amplifies a specific DNA sequence millions of times from a tiny sample.
Ingredients: template DNA, two primers (short DNA pieces flanking the target), dNTPs, a heat-stable DNA polymerase (Taq polymerase from Thermus aquaticus), buffer with magnesium.
Each cycle:
- Denaturation at about 94–95 °C: strands separate.
- Annealing at about 50–65 °C: primers bind.
- Extension at 72 °C: Taq polymerase builds new strands.
Each cycle doubles the target; 25–35 cycles are usual.
Copies after n cycles = starting copies × 2ⁿ
1 copy, 10 cycles: 2¹⁰ = 1024 copies
1 copy, 20 cycles: 2²⁰ = 1,048,576 (about 1 million)
1 copy, 30 cycles: 2³⁰ = 1,073,741,824 (about 1 billion)
Start with 100 copies, 20 cycles:
100 × 2²⁰ ≈ 100 × 1,048,576 ≈ 105 million copies
(Ideal doubling; real efficiency is a little lower.)
Variants
- RT-PCR: reverse transcriptase first makes cDNA from RNA; used for RNA viruses (HIV viral load, hepatitis C RNA, COVID-19) and gene expression.
- Real-time (quantitative) PCR: measures product as it forms (fluorescence); quantifies the starting amount. GeneXpert MTB/RIF detects M. tuberculosis and rifampicin resistance in about 2 hours; widely used in Pakistan's TB programme.
- Multiplex PCR: several targets at once.
- Allele-specific PCR / ARMS: detects point mutations (β-thalassaemia mutations in prenatal diagnosis).
Uses of PCR: infectious disease diagnosis (TB, HIV, hepatitis, COVID-19), prenatal diagnosis (thalassaemia from chorionic villus sample), detecting mutations, forensic analysis from tiny samples, paternity testing, monitoring leukaemia (BCR-ABL).
DNA sequencing and analysis
- Sanger (chain-termination) sequencing: uses dideoxynucleotides (no 3′-OH), which stop chain growth at specific bases.
- Next-generation sequencing: many fragments sequenced in parallel; whole-genome and exome sequencing, cancer panels.
- RFLP (restriction fragment length polymorphism): variation in fragment sizes after cutting; old method for tracking genes in families (and sickle cell, which abolishes an MstII site).
- DNA fingerprinting: compares short tandem repeats (STRs) or VNTRs, which vary greatly between people: forensics, paternity, identifying remains.
- Microarrays (DNA chips): thousands of probes test expression or copy-number changes at once.
Cytogenetic techniques
- Karyotyping: chromosome number and large changes (Down, Turner, Klinefelter).
- FISH (fluorescence in situ hybridisation): fluorescent probes find specific sequences: microdeletions (22q11 DiGeorge), HER2 amplification in breast cancer, translocations (BCR-ABL), rapid aneuploidy tests.
Gene therapy and genome editing
- Gene therapy: inserting a working gene into a patient's cells, usually with a viral vector (adenovirus, adeno-associated virus, lentivirus). Successes include some forms of SCID (ADA deficiency), spinal muscular atrophy, haemophilia B and inherited retinal disease. Risks include immune reactions and insertional cancers.
- CRISPR-Cas9: a bacterial defence system adapted to cut DNA at a chosen site guided by an RNA; used to edit genes (an approved therapy edits blood stem cells for sickle cell disease and β-thalassaemia).
- Transgenic animals and knockout mice model human disease.
- Stem cells: embryonic (pluripotent), adult (multipotent), and induced pluripotent stem cells.
- Monoclonal antibodies: made from hybridomas (B cell fused with myeloma cell); used in diagnosis and treatment.
Expect: restriction enzymes and palindromes, steps of recombinant DNA and its products (insulin, hepatitis B vaccine), cDNA, blot types (SNoW DRoP), ELISA uses, PCR steps, temperatures, Taq polymerase and 2ⁿ, RT-PCR and real-time PCR (GeneXpert), Sanger sequencing (dideoxynucleotides), DNA fingerprinting (STRs), FISH uses, and gene therapy and CRISPR.
Quick revision
- Restriction enzymes cut palindromes; ligase joins; plasmid vectors.
- cDNA from mRNA by reverse transcriptase (no introns).
- Southern DNA, Northern RNA, Western protein.
- ELISA: screening (HIV, HBsAg).
- PCR: denature (94–95 °C), anneal (50–65 °C), extend (72 °C); Taq; 2ⁿ copies.
- RT-PCR for RNA; real-time PCR quantifies; GeneXpert for TB and rifampicin resistance.
- Sanger: dideoxynucleotides. Fingerprinting: STRs. FISH: microdeletions, HER2.
Written by The House Job from standard biochemistry references (Lippincott Illustrated Reviews: Biochemistry).
FAQ
Which blotting technique detects RNA?
Northern blotting.
What are the steps in each PCR cycle?
Denaturation of the DNA at about 94–95 °C, annealing of primers at about 50–65 °C, and extension by Taq polymerase at 72 °C.
Why is Taq polymerase used in PCR?
It comes from a heat-loving bacterium, so it survives the repeated high temperatures needed to separate DNA strands.
What do restriction enzymes recognise?
Specific short DNA sequences, usually palindromes, where they cut the DNA.