A virology laboratory switches from a SYBR Green based real-time PCR assay to a probe-based (TaqMan) assay for enterovirus detection. What is the principal limitation of SYBR Green chemistry that the new assay overcomes?
- A SYBR Green cannot detect RNA targets even after reverse transcription
- B SYBR Green requires a separate standard curve for every run
- C SYBR Green binds any double-stranded DNA, so non-specific products and primer dimers also generate signal ✓
- D SYBR Green fluorescence cannot be read in real time during amplification
Correct answer: C. SYBR Green binds any double-stranded DNA, so non-specific products and primer dimers also generate signal
Explanation
SYBR Green is an intercalating dye that fluoresces upon binding any double-stranded DNA present in the tube, including primer dimers and off-target amplicons, which lowers specificity. Sequence-specific hydrolysis probes such as TaqMan fluoresce only when the probe bound to the correct target is cleaved by polymerase 5' nuclease activity. SYBR Green works fine on cDNA from RNA templates, reads in real time, and needs no special curve beyond normal quantification standards.
Reference: Murray's Basic and Medical Microbiology, 9th ed.
High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP
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