PCR variants, sequencing, hybridization, FISH, microarray, nucleic acid extraction, contamination control, and common molecular assays in infectious disease and oncology.
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- What does real-time PCR (qPCR) measure during amplification?
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Fluorescence signal as DNA is amplified, allowing quantification in real time.- What is the purpose of reverse transcription in RT-PCR?
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Converts RNA into complementary DNA (cDNA) so it can be amplified by PCR.- What is nested PCR designed to improve?
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Specificity and sensitivity, by using two sequential rounds of PCR with an inner primer set.- What defines a multiplex PCR reaction?
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Multiple primer sets are used in one reaction to amplify several target sequences simultaneously.- What does digital PCR (dPCR) provide that conventional PCR does not?
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Absolute quantification of target DNA without needing a standard curve.- What problem does hot-start PCR prevent?
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Nonspecific amplification and primer-dimer formation from polymerase activity before thermal cycling begins.- What does the Ct (cycle threshold) value represent in real-time PCR?
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The PCR cycle number at which fluorescence first rises above background, inversely related to starting target amount.- How does a TaqMan probe generate a fluorescent signal?
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Taq polymerase's 5' exonuclease activity cleaves the probe during extension, separating the fluorophore from the quencher.- How does a molecular beacon probe signal target binding?
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Its hairpin structure opens upon hybridization to the target, separating the fluorophore from the quencher.- How does SYBR Green detect PCR product?
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It intercalates into double stranded DNA and fluoresces, with signal increasing as more product accumulates.- What is melting curve analysis used to assess after real-time PCR?
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Product specificity, by measuring the temperature at which double stranded DNA denatures.- What does allele-specific PCR (ARMS-PCR) detect?
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Known point mutations or SNPs, using primers that only extend when perfectly matched to the target allele.- What is the chemical basis of Sanger sequencing?
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Chain termination sequencing using dideoxynucleotides that stop DNA synthesis when incorporated.- Why do dideoxynucleotides (ddNTPs) terminate DNA synthesis in Sanger sequencing?
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They lack the 3' OH group needed to form a bond with the next nucleotide.- What distinguishes next-generation sequencing (NGS) from Sanger sequencing?
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NGS sequences millions of DNA fragments simultaneously (massively parallel) rather than one fragment at a time.
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