Polymerase Chain Reaction (PCR) is a widely used technique i…
Polymerase Chain Reaction (PCR) is a widely used technique in molecular biology that enables the amplification of specific DNA sequences. The process consists of three main steps: denaturation, annealing, and extension. Each cycle approximately doubles the amount of DNA, leading to exponential amplification. The reaction requires a DNA polymerase enzyme, typically Taq polymerase, which remains stable at high temperatures. The specificity of PCR is determined by the primers, which are short DNA sequences that bind to the target region. Several modifications of PCR have been developed, including quantitative PCR (qPCR), which allows real-time monitoring of amplification using fluorescent dyes or probes. Reverse transcription PCR (RT-PCR) converts RNA into complementary DNA (cDNA) before amplification, enabling the detection of RNA viruses such as SARS-CoV-2. Nested PCR enhances specificity by using two sets of primers in sequential reactions. In contrast, multiplex PCR allows the simultaneous amplification of multiple targets within a single reaction by using different sets of primers. PCR efficiency can be affected by several factors, including primer design, template quality, and inhibitors such as heparin or excess salts. The technique’s sensitivity makes it prone to contamination, necessitating stringent laboratory practices. Advances in PCR technology have led to digital PCR (dPCR), which partitions the sample into numerous micro-reactions to allow absolute quantification of nucleic acids. During a qPCR experiment, a researcher uses SYBR Green dye to quantify gene expression. However, the amplification curve for one of the samples shows a late threshold cycle (Ct), suggesting low DNA yield. Which of the following factors is LEAST likely to be responsible for this observation?