PCR & DNA Sequencing
PCR (Polymerase Chain Reaction) and DNA sequencing are the two most transformative technologies in molecular biology. PCR amplifies specific DNA sequences and sequencing determines the exact nucleotide order.
PCR — Principles
Exponential amplification of a target DNA sequence in vitro. Requires: specific primers (18–25 nt, flanking target), Taq polymerase (thermostable), dNTPs, MgCl₂ (cofactor), buffer, and thermal cycler. 30 cycles → ~10⁹-fold amplification.
- Denaturation (94–95°C, 30s): DNA strands separate
- Annealing (50–65°C, 30s): Primers bind to complementary strand (Tm of primer determines temperature)
- Extension (72°C, 1min/kb): Taq extends from primer 5'→3'
PCR Variants
- RT-PCR: RNA → cDNA (Reverse Transcriptase) → PCR. Detects RNA viruses (HIV, SARS-CoV-2), measures gene expression.
- Real-time PCR (qPCR): PCR with fluorescent dye (SYBR Green) or probe (TaqMan). Quantifies DNA/RNA in real-time. Used for viral load (HIV, HBV), gene expression quantification.
- Multiplex PCR: Multiple primer sets in one reaction → detect multiple targets simultaneously.
- LAMP (Loop-mediated Isothermal Amplification): Amplification at constant temperature; used in POC diagnostics.
- Digital PCR (dPCR): Partition sample into thousands of droplets; absolute quantification without calibration curve.
Sanger Sequencing
Chain-termination method (Frederick Sanger, Nobel Prize 1980). Uses dideoxynucleotides (ddNTPs — lack 3'-OH → terminate chain). Four reactions (one per ddNTP) produce fragments of all possible lengths → gel electrophoresis → read sequence. Modern automated Sanger uses fluorescent ddNTPs + capillary electrophoresis. Reads ~800 bp/run; gold standard for accuracy. Used for: Mutation confirmation, clinical genetics, sequencing individual genes.
Next-Generation Sequencing (NGS)
Massively parallel sequencing — millions of short reads (100–300 bp) simultaneously. Key platforms: Illumina (sequencing by reversible termination), Oxford Nanopore (long reads), PacBio (single molecule, real-time). Applications:
- Whole Genome Sequencing (WGS) — full 3 billion bp in <24h
- Whole Exome Sequencing (WES) — coding regions only; rare disease diagnosis
- RNA-Seq — transcriptome, gene expression
- ChIP-Seq — protein-DNA interactions
- Cancer genomics (somatic mutations, tumor heterogeneity)
- Pathogen identification (metagenomic sequencing)
CRISPR-Cas9 Gene Editing
Guide RNA (gRNA, 20 nt) directs Cas9 endonuclease to specific genomic site → DSB → repaired by NHEJ (indels, gene disruption) or HDR (precise gene correction). Applications: Sickle cell disease cure (HbF induction), Duchenne muscular dystrophy, CAR-T cell engineering, functional genomics screens, agricultural biotechnology. Ethical concerns: germline editing.
DNA Fingerprinting (RFLP/STR Analysis)
- RFLP (Restriction Fragment Length Polymorphism): Restriction digest → Southern blot → probe with minisatellite sequences → unique pattern. Largely replaced by:
- STR (Short Tandem Repeat) analysis: PCR amplify polymorphic microsatellite loci → capillary electrophoresis → fragment sizing. More sensitive, requires minimal DNA. Standard for forensics, paternity testing, ID of disaster victims.