PCR

**Principle:** PCR makes millions to billions of copies of a target DNA segment from a tiny starting sample. Amplification is exponential: about 2^n copies after n cycles.

**Components:** - Template DNA (the sample) - Two primers, forward and reverse, complementary to the sequences flanking the target - Heat-stable DNA polymerase (Taq polymerase, from *Thermus aquaticus*), which survives denaturation temperatures - Deoxynucleotide triphosphates (dNTPs) - Buffer with Mg2+

**Cycle steps (repeated about 25-40 times):** 1. **Denaturation (~94-95°C):** Heat separates the dsDNA into single strands. 2. **Annealing (~50-65°C):** Primers bind to complementary sequences on each strand. 3. **Extension/elongation (~72°C):** Taq polymerase synthesizes new DNA in the 5'→3' direction from the primers.

**Key variants:** - **RT-PCR (reverse transcriptase PCR):** Reverse transcriptase first converts RNA to cDNA, which is then amplified. It is used for RNA viruses (HIV, HCV, SARS-CoV-2, influenza). Don't confuse it with the "real-time" abbreviation. - **Quantitative/real-time PCR (qPCR):** Fluorescence is monitored each cycle, so the amount of product reflects the starting template. It is used for viral load (HIV, HBV, HCV, CMV, EBV) and gene expression. A lower Ct (cycle threshold) means more starting template. - **Multiplex PCR:** Several primer sets are used in one reaction to detect multiple targets, such as respiratory or GI pathogen panels. - **Nested PCR:** A second round with internal primers improves specificity.

**Clinical uses (high-yield):** - **Infectious disease:** - HSV encephalitis: CSF HSV PCR is the diagnostic test of choice. - Mycobacterium tuberculosis: Rapid NAAT (e.g., Xpert MTB/RIF) also detects rifampin resistance (*rpoB*). - Chlamydia/gonorrhea: NAAT on urine or swab. - HIV: Diagnosis in infants born to HIV+ mothers (antibodies are maternal IgG), acute/window-period infection, and viral load monitoring. - SARS-CoV-2, CMV, JC virus (PML), and many others. - **Genetics:** Detects specific mutations such as CFTR ΔF508, Factor V Leiden, and trinucleotide repeat expansions (Huntington, fragile X). Also used for prenatal diagnosis. - **Oncology:** Detects translocations such as BCR-ABL (CML, t(9;22)) and PML-RARA (APL, t(15;17)), and monitors minimal residual disease. - **Forensics/paternity:** Amplifies short tandem repeats (STRs) and VNTRs.

**Limitations/pitfalls:** - It requires known flanking sequences to design primers. - It is highly sensitive, so contamination causes false positives. - It detects nucleic acid, not necessarily viable organisms, so a positive result may reflect residual or nonviable genetic material. - Inhibitors in the sample (e.g., heme) can cause false negatives. - Taq has no proofreading (no 3'→5' exonuclease activity), so errors can occur. High-fidelity polymerases such as Pfu do proofread.

**Compare to other molecular techniques (commonly tested):** - **Southern blot:** DNA detection with a labeled DNA probe. - **Northern blot:** RNA detection. - **Western blot:** Protein detection with antibodies. - **ELISA:** Detects antigen or antibody. - **Microarray/FISH:** Detect expression or genomic changes, or localize sequences. - **Sanger sequencing:** Uses dideoxynucleotides (ddNTPs) as chain terminators.

**Naming note:** "PCR" also appears in other contexts. Examples are *protein:creatinine ratio* (urine, for proteinuria) and *C-reactive protein* (often written CRP or "PCR" in Spanish/Portuguese). On USMLE, PCR almost always means polymerase chain reaction.

**Mnemonic for cycle steps:** "Denature, Anneal, Extend" (heat-cool-warm: 95 → ~55 → 72°C).