Molecular biology techniques

Choosing a technique

• Every lab method answers one of four questions: amplify a sequence (PCR), separate molecules (electrophoresis, chromatography), detect a specific molecule (blots, ELISA, FISH, immunohistochemistry) or read a sequence (Sanger, next-generation sequencing).
• The answer to most exam questions comes from asking which molecule is the target (DNA, RNA or protein) and what resolution is needed (whole chromosome, a few megabases, a single base).

Blotting

• Molecules are separated by gel electrophoresis, transferred (blotted) to a membrane, then detected with a labelled probe that binds only the target.

Remember Southern is DNA (named after its inventor); Northern is RNA; Western is protein.

Polymerase chain reaction

• PCR copies a chosen DNA segment exponentially: about 2ⁿ copies after n cycles.
• Each cycle has three steps: denaturation at about 94–95 °C (strands separate), annealing at about 50–65 °C (two primers bind either side of the target) and extension at about 72 °C by heat-stable Taq polymerase from *Thermus aquaticus*.
• Ingredients: template DNA, two primers, the four dNTPs, Mg²⁺ and buffer.
• RT-PCR: reverse transcriptase first copies RNA into cDNA. Used for RNA viruses (SARS-CoV-2, HIV viral load, HCV RNA) and fusion transcripts such as BCR-ABL.
• Real-time (quantitative) PCR reads fluorescence each cycle; the cycle threshold falls as starting amount rises.
• Uses: infection (TB GeneXpert, which also detects rifampicin resistance), prenatal diagnosis, forensic fingerprinting, mutation detection, minimal residual disease.
• A positive anti-HCV antibody shows exposure, not necessarily active infection; HCV RNA by PCR confirms current viraemia.
• PCR is so sensitive that contamination causes false positives.

Exam trap PCR amplifies DNA; it does not read the sequence. Sequencing reads it.

Electrophoresis and spectrophotometry

• Molecules move through a gel or on a strip in an electric field according to charge and size. DNA is negatively charged and runs to the anode; small fragments move fastest.
• Serum protein electrophoresis gives albumin, α1, α2, β and γ bands. A narrow spike in the γ region (M band) means a monoclonal gammopathy: many copies of one immunoglobulin from one plasma-cell clone (myeloma, MGUS, Waldenström). A broad polyclonal rise occurs in chronic infection, autoimmune disease and cirrhosis (β–γ bridging). A low α1 band suggests α1-antitrypsin deficiency. Immunofixation identifies the heavy and light chain.
• Haemoglobin electrophoresis or HPLC detects HbS, HbC, HbF and a raised HbA2 in β-thalassaemia trait.
• Lipoprotein electrophoresis: chylomicrons stay at the origin; LDL forms the β band, VLDL the pre-β band, HDL the α band. A broad β band marks remnant accumulation in type III dyslipidaemia.
• Spectrophotometry measures absorbance of light by a solution; it obeys the Beer–Lambert law (absorbance is proportional to concentration and path length).

ELISA

• An antibody linked to an enzyme converts a substrate to a coloured product, so colour intensity is proportional to the amount of antigen or antibody.
• Formats: direct, indirect (detects antibodies), sandwich (captures antigen between two antibodies) and competitive (small molecules such as hormones).
• Uses: HIV screening (fourth generation detects p24 antigen plus antibody), hepatitis B surface antigen, hCG, hormone assays.
• ELISA is highly sensitive, so it is used for screening; positives are confirmed by a more specific test.

Cytogenetics: karyotype, FISH and microarray

• For karyotyping, dividing cells (blood lymphocytes, amniocytes, chorionic villi) are cultured and arrested in metaphase by colchicine, then stained by G-banding (Giemsa).
• Couples with recurrent miscarriage or several stillbirths: karyotype both parents. A balanced translocation (often Robertsonian) leaves the carrier healthy but produces unbalanced gametes. A carrier of a balanced 14;21 Robertsonian translocation risks a child with translocation Down syndrome.
• Karyotype notation: total number, then sex chromosomes. Turner 45,X; Klinefelter 47,XXY (tall, small testes, gynaecomastia); Down 47,XX or XY,+21; mosaicism when two cell lines coexist, such as 45,X/46,XX (milder Turner). A virilised 46,XX infant with uterus and ovaries usually has congenital adrenal hyperplasia; a girl with absent uterus, blind vagina and normal breasts may be 46,XX Müllerian agenesis or 46,XY androgen insensitivity.

Sequencing and microarrays

• Sanger (chain-termination) sequencing uses a small proportion of dideoxynucleotides (ddNTPs), which lack a 3′-OH group. When one is added, the next phosphodiester bond cannot form, so chains stop at each base; fragment lengths reveal the order. Accurate for one gene and used to confirm variants.
• Next-generation sequencing reads millions of fragments in parallel: gene panels, whole exome or genome, tumour profiling and non-invasive prenatal testing on cell-free fetal DNA.
• DNA microarrays carry thousands of probes on a chip and detect variation in DNA sequence (SNPs, copy number) and gene expression at once; used for tumour expression profiling.

Recombinant DNA and other tools

• Restriction endonucleases cut DNA at specific palindromic sites; RFLP analysis compares the fragment patterns.
• Recombinant DNA: the human gene is inserted into a plasmid vector and expressed in bacteria (E. coli) or yeast. Human insulin (1982) was the first recombinant drug approved; others include growth hormone, erythropoietin, factor VIII and hepatitis B vaccine.
• Flow cytometry: fluorescent antibodies count cells by surface markers (CD4 counts, leukaemia and lymphoma typing, PNH by loss of CD55/CD59).
• Immunohistochemistry: antibodies on tissue (cytokeratin for carcinoma, vimentin for sarcoma, S-100 for melanoma, CD markers for lymphoma, HER2, oestrogen receptor).
• Chromatography: HPLC (HbA1c, haemoglobin variants); gas chromatography–mass spectrometry for drugs and toxins. CRISPR–Cas9 edits genes at chosen sites.

Genetic terms that go with these tests

• Variable expressivity: people with the same mutation show different severity (neurofibromatosis type 1 in one family). Incomplete penetrance: some carriers show no features at all.
• Mosaicism: two genetically different cell lines in one person, from an error after fertilisation.
• Hardy–Weinberg: if disease frequency is q², carrier frequency is about 2q. For cystic fibrosis at 1 in 2,500, q = 1/50, so carriers are about 1 in 25.

Exam trap Variable expressivity is different severity; incomplete penetrance is no disease at all despite the gene.

How FCPS asks about this

• Which blot detects RNA / protein: Northern / Western; Southern detects DNA.
• Enzyme and temperatures in PCR: Taq polymerase; denature about 95 °C, anneal 50–65 °C, extend 72 °C.
• Narrow spike in the gamma region: monoclonal gammopathy (myeloma, MGUS).
• Couple with repeated stillbirths and normal history: karyotype both parents for a balanced translocation.
• How Sanger sequencing stops chains: dideoxynucleotides lack a 3′-OH group.
• How clinical human insulin is made: recombinant DNA in microbes (bacteria or yeast).
• Same mutation, different severity in NF1 family members: variable expressivity.