Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a method for exponentially amplifying a specific DNA sequence in vitro. It is foundational to nearly all molecular genetic testing, enabling detection of mutations, quantification of gene expression, pathogen identification, and sample preparation for downstream analyses like sequencing.

• Core components: (1) Template DNA, the sample containing the target sequence; (2) Primers, short oligonucleotides (~18–25 bp) flanking the target region; (3) Taq polymerase, a thermostable DNA polymerase from Thermus aquaticus that withstands repeated heating; (4) dNTPs, deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP); (5) Buffer with MgCl2, providing the cofactor required for polymerase activity.
• Thermal cycling: Three repeated steps. (1) Denaturation (~95 degrees C) separates double-stranded DNA; (2) Annealing (~50–65 degrees C) lets primers bind to complementary sequences; (3) Extension (~72 degrees C) lets Taq polymerase synthesize the new strand in the 5' to 3' direction. Typically 25–35 cycles.
• Exponential amplification: Each cycle doubles the target DNA. After n cycles, there are approximately 2^n copies of the target region.
• Qualitative PCR: Standard endpoint PCR detects presence or absence of a target sequence. Visualized by gel electrophoresis.
• Quantitative PCR (qPCR / real-time PCR): Measures DNA amplification in real time using fluorescent reporters (SYBR Green or TaqMan probes). Quantifies starting template amount. Used for gene expression analysis, viral load measurement, and copy number assessment.
• Reverse transcription PCR (RT-PCR): Converts RNA to complementary DNA (cDNA) using reverse transcriptase before PCR amplification. Used to study gene expression and detect RNA viruses. Not the same as real-time PCR (qPCR), despite confusing abbreviations.
• Methylation-specific PCR (MS-PCR): Uses bisulfite-treated DNA and primers specific to methylated vs unmethylated sequences. Used for disorders involving imprinting (e.g., Prader-Willi/Angelman syndromes) and tumor suppressor methylation.
• Allele-specific PCR: Primers designed to amplify only a specific allele. Used for targeted genotyping of known variants.

• Single gene testing: Amplification of specific exons before Sanger sequencing
• Trinucleotide repeat disorders: Specialized PCR (triplet-repeat primed PCR) for Huntington disease, fragile X, myotonic dystrophy
• Infectious disease: Detection and quantification of viral/bacterial DNA or RNA
• Pharmacogenomics: Genotyping of drug-metabolizing enzyme variants (e.g., CYP2D6)
• Carrier screening and prenatal diagnosis: Rapid detection of known familial variants
• Identity testing: STR (short tandem repeat) analysis for forensics and paternity

• Requires known target sequence: Primers must be designed for a specific region, so PCR cannot discover novel sequences
• Contamination risk: Extreme sensitivity means even trace contamination can produce false positives; requires strict laboratory protocols (separate pre- and post-PCR areas)
• Amplification bias: GC-rich regions, large targets, or repeat expansions may amplify poorly
• Size limitations: Standard PCR amplifies fragments up to ~5–10 kb; long-range PCR needed for larger targets
• Does not provide structural context: Amplifies a fragment in isolation; cannot determine chromosomal location or phase