Biotechnology & Molecular Techniques

Molecular biology techniques have transformed biological research and medical practice. From diagnosing infectious diseases to engineering therapeutic proteins and editing the human genome, these tools are indispensable in modern biomedicine.

Polymerase Chain Reaction (PCR)

PCR (Kary Mullis, 1983, Nobel Prize 1993) amplifies specific DNA sequences exponentially using thermocycling. A PCR reaction contains: template DNA; two oligonucleotide primers flanking the target sequence (typically 18–25 bp, Tm ~55–65°C); heat-stable DNA polymerase (Taq polymerase from Thermus aquaticus, or higher-fidelity alternatives — Phusion, Q5); dNTPs (building blocks); MgCl₂ (cofactor); and buffer.

PCR cycle (repeated 25–40 times): (1) Denaturation (~94–98°C, 30 sec): double-stranded DNA separates into single strands. (2) Annealing (~50–65°C, 30 sec): primers bind to complementary sequences on each single-stranded template; temperature depends on primer Tm. (3) Extension (~72°C, 1 min/kb): Taq extends from each primer in the 5'→3' direction. After n cycles, the target sequence is amplified 2ⁿ-fold (theoretical).

Variants: RT-PCR (reverse transcription PCR): converts mRNA to cDNA with reverse transcriptase, then amplifies; detects gene expression (requires mRNA isolation) or RNA viruses (SARS-CoV-2 COVID diagnosis). Quantitative PCR (qPCR/real-time PCR): fluorescent dyes (SYBR Green intercalates into dsDNA) or TaqMan probes (fluorophore + quencher on probe, hydrolysed by Taq during extension → fluorescence signal) quantify DNA/mRNA levels in real-time; used for gene expression analysis, viral load quantification (HIV, HBV, HCV), oncogene copy number. Multiplex PCR: multiple primer pairs in one reaction — STR profiling for forensics and paternity testing, pathogen panel (respiratory virus multiplex). Digital PCR: partition reaction into thousands of droplets, each containing 0 or 1 template → absolute quantification without standard curve; used for rare mutation detection (liquid biopsy circulating tumour DNA).

Gel Electrophoresis

Electrophoresis separates charged molecules in an electric field through a porous matrix. Agarose gel electrophoresis separates DNA and RNA by size. DNA is negatively charged (phosphate backbone) and migrates toward the anode (+) in an agarose gel (typically 0.5–3% agarose in TAE or TBE buffer). Smaller fragments migrate faster. DNA is visualised with ethidium bromide (EtBr, intercalating fluorescent dye — carcinogenic) or safer alternatives (SYBR Safe, GelRed) under UV light. A DNA ladder (molecular weight marker) allows size estimation. Applications: verifying PCR products, restriction digest analysis, Southern blotting (DNA transferred to membrane, hybridised with labelled probe — detects specific sequences).

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separates proteins by molecular weight. SDS denatures proteins and coats them uniformly with negative charge (proportional to mass) → migration in polyacrylamide gel is solely by size. Western blotting (immunoblotting): proteins transferred from gel to nitrocellulose/PVDF membrane, probed with primary antibody (specific to target protein) + HRP-conjugated secondary antibody → chemiluminescence detection. Detects protein expression, post-translational modifications, and protein-protein interactions.

Recombinant DNA Technology and Cloning

Recombinant DNA technology combines DNA sequences from different sources in vitro. Key tools: Restriction enzymes (restriction endonucleases): bacterial enzymes that cut double-stranded DNA at specific palindromic sequences (recognition sites, typically 4–8 bp). EcoRI cuts at 5'-GAATTC-3' → 5'-AATTC-3' and 5'-G-3' sticky (cohesive) ends; BamHI cuts 5'-GGATCC. Sticky ends facilitate directional cloning. DNA ligase: joins DNA ends (seals nicks) — used to insert foreign DNA into vectors. Cloning vectors: plasmids (circular, autonomously replicating DNA in bacteria; must have origin of replication — ori; antibiotic resistance gene for selection; multiple cloning site — MCS with restriction sites), bacteriophages (lambda), cosmids, BACs (bacterial artificial chromosomes — for large inserts), yeast artificial chromosomes (YACs — for very large inserts).

Expression vectors: designed for protein production; contain strong promoters (T7, CMV), ribosome-binding sites, and often His-tags or other affinity tags for purification. Used to produce recombinant proteins: insulin (in E. coli/yeast — replaced animal insulin), erythropoietin, G-CSF, hepatitis B surface antigen (vaccine), monoclonal antibodies (in CHO cells).

Site-directed mutagenesis: introduces specific mutations into a DNA sequence to study protein function — change codon, create or destroy restriction site, introduce stop codon.

DNA Sequencing

Sanger sequencing (dideoxy method, Frederick Sanger, Nobel Prize 1980): DNA is synthesised using a mix of dNTPs and a small amount of ddNTPs (dideoxynucleotides lacking 3'-OH → chain termination when incorporated). Different fluorescent dye-labelled ddNTPs (one per base) → four different chain lengths. Capillary electrophoresis separates fragments by size → fluorescence detector reads sequence. Reads ~600–1000 bp per reaction; used for single-gene sequencing, confirming cloned sequences, detecting known mutations.

Next-generation sequencing (NGS): massively parallel sequencing of millions of fragments simultaneously. Key methods: Illumina (sequencing by synthesis with reversible dye terminators — most widely used); Ion Torrent (detects H⁺ release during nucleotide incorporation); Nanopore (Oxford Nanopore — single molecule; detects changes in ionic current as DNA threads through a protein nanopore; long reads ~10–100 kb). Applications: whole genome sequencing (WGS); whole exome sequencing (WES — ~1.5% of genome but contains ~85% of disease-causing mutations); RNA-seq (quantitative transcriptome analysis); ChIP-seq (chromatin immunoprecipitation + sequencing, maps transcription factor binding sites and histone modifications); single-cell RNA-seq (scRNA-seq — gene expression of individual cells); liquid biopsy (circulating tumour DNA mutation detection, minimal residual disease).

CRISPR-Cas9 Genome Editing

CRISPR (clustered regularly interspaced short palindromic repeats) is a bacterial adaptive immune system repurposed as a genome editing tool (Jennifer Doudna and Emmanuelle Charpentier, Nobel Prize 2020). The system uses: Cas9 (a dual RNA-guided endonuclease from Streptococcus pyogenes); guide RNA (gRNA) — a single-guide RNA (sgRNA) comprising a 20-nt spacer sequence complementary to the target DNA + scaffold RNA. Cas9-sgRNA scans DNA, and when it finds a sequence complementary to the spacer adjacent to a PAM sequence (5'-NGG-3' for SpCas9), it unwinds the DNA and makes a blunt-ended double-strand break. Repair: (1) NHEJ (error-prone) → indels → frameshift → gene knockout; (2) HDR (homology-directed repair) using a donor template → precise substitution, insertion, or correction.

Applications: functional genomics screens (Cas9 + sgRNA library — identify gene function); disease modelling; gene therapy (BCR-ABL disruption; CCR5 knockout in HSCs to confer HIV resistance; γ-globin reactivation in β-thalassaemia/SCD); base editing (CBEs, ABEs — convert one base to another without DSB); prime editing (search and replace without DSB or donor template); epigenome editing (dCas9 fused to transcriptional activators or repressors). Challenges: off-target editing, delivery (AAV, LNPs), immunogenicity of Cas9, regulatory framework.

Other Key Techniques

ELISA (enzyme-linked immunosorbent assay): detects and quantifies antigens (or antibodies) using antibody-enzyme conjugates. Sandwich ELISA: capture antibody binds plate → antigen added → detection antibody (enzyme-conjugated) binds antigen → substrate added → colorimetric signal proportional to antigen concentration. Applications: diagnosis (HIV, hepatitis B, SARS-CoV-2 antigen/antibody tests), hormone quantification (hCG pregnancy test, TSH, troponin), cytokine measurement.

Flow cytometry: laser-based technique that measures physical and fluorescent characteristics of single cells as they flow through a laser beam. Cells labelled with fluorophore-conjugated antibodies to surface or intracellular markers. Detects cell size (forward scatter), granularity (side scatter), and up to 20+ fluorescent channels simultaneously. Applications: immunophenotyping (CD4 count in HIV — ART initiation threshold; leukaemia/lymphoma classification — e.g., CLL: CD5+/CD19+/CD23+); cell cycle analysis; apoptosis detection (Annexin V/PI staining); cell sorting (FACS — fluorescence-activated cell sorting).