Question 1

Microbial community analysis — In PCR-based cloning/sequencing of mixed rRNA gene amplicons, why is a cloning step included before sequencing?

Accepted Answer

Introduction / Context: Environmental microbiology often amplifies rRNA genes from complex communities to profile diversity. Before high-throughput sequencing was widespread, cloning PCR products into plasmids and sequencing individual clones was the standard approach. Understanding the rationale for the cloning step clarifies how heterogeneous mixtures are resolved into individual sequences. Given Data / Assumptions: We have a mixed PCR product containing rRNA gene fragments from many organisms. Sequencing directly would produce overlapping chromatograms (in Sanger methods) that cannot be interpreted. Cloning into vectors allows isolation of single inserts for clean reads. Concept / Approach: Cloning distributes heterogeneous amplicons among individual plasmids, each carried by a separate colony. Picking single colonies ensures each sequencing reaction reads one unique insert, thereby separating the mixture into resolvable components. This step is not primarily to “improve amplification” or to “prevent contamination,” although good technique reduces contamination. Modern amplicon sequencing (e.g., Illumina) uses barcodes rather than cloning, but the underlying goal remains the same: deconvolute sequence mixtures. Step-by-Step Solution: Amplify a community rRNA gene region using broad-range primers.Clone the pooled amplicons into a plasmid vector and transform competent cells.Pick individual colonies; each colony harbors one amplicon type for clean, interpretable sequencing. Verification / Alternative check: Sanger chromatograms from mixed templates show overlapping peaks; after cloning, chromatograms are clear and single-peaked, confirming separation. Why Other Options Are Wrong: The amplification process: cloning does not amplify; PCR already does. Preventing contamination: sterile technique and controls address contamination, not cloning per se. None of these: incorrect because separation of mixed sequences is the point. Common Pitfalls: Assuming cloning is required for PCR success. It is required for resolving mixtures into single sequences when using Sanger sequencing of complex samples. Final Answer: separating the different rRNA gene sequences in the mixture

Question 2

PCR fundamentals — The thermostable DNA polymerase originally popularized for routine PCR was obtained from which source enzyme?

Accepted Answer

Introduction / Context: Polymerase chain reaction (PCR) relies on repeated high-temperature denaturation steps. Early PCR protocols required adding fresh polymerase after each cycle because mesophilic enzymes were heat-inactivated. The breakthrough came with thermostable polymerases that survive denaturation temperatures, enabling automated cycling and revolutionizing molecular biology. Given Data / Assumptions: “Originally popularized” refers to the first widely used thermostable DNA polymerase in PCR workflow. Thermostability is required to withstand ~94–98°C during denaturation. We distinguish DNA polymerases from RNA polymerases and mesophilic sources. Concept / Approach: Taq polymerase, isolated from Thermus aquaticus, is thermostable and became the standard enzyme for PCR in the late 1980s and 1990s. While E. coli DNA polymerase I (Klenow fragment) was used in very early manual protocols, it lacked thermostability. E. coli RNA polymerase transcribes RNA and is not used for DNA-dependent DNA synthesis in PCR. Step-by-Step Solution: Identify the property needed: thermostability at denaturation temperatures.Recall the canonical enzyme: Taq polymerase from thermophilic bacteria.Choose “Taq polymerase” as the historically correct and widely accepted answer. Verification / Alternative check: Thermocyclers paired with Taq enabled exponential amplification without manual enzyme addition, demonstrating its practicality and popularity. Why Other Options Are Wrong: E. coli DNA polymerase: heat-labile; impractical for automated cycling. E. coli RNA polymerase: not a DNA-dependent DNA polymerase. All of these: incorrect because only Taq satisfies the thermostable PCR requirement. Common Pitfalls: Confusing later high-fidelity polymerases (e.g., Pfu, Phusion) with the original widely used enzyme; the question asks about the original popularized choice. Final Answer: Taq polymerase

Question 3

Template requirements in PCR — Under standard DNA PCR (not RT-PCR), which nucleic acid template is used for amplification?

Accepted Answer

Introduction / Context: PCR amplifies specific DNA sequences using cycles of denaturation, primer annealing, and extension. Clarifying what nucleic acid serves as the template in standard PCR (as opposed to reverse-transcription PCR) helps avoid common laboratory errors and misinterpretations. Given Data / Assumptions: The question refers to conventional DNA PCR, not RT-PCR. Primers are DNA oligonucleotides that anneal to complementary DNA sequences. Thermocycling includes an initial denaturation step to separate strands. Concept / Approach: Standard PCR uses double-stranded DNA (dsDNA) as the starting template. The first denaturation step separates the two strands, producing single-stranded regions for primer binding. Although PCR can, in principle, amplify from single-stranded DNA if a primer can anneal, the canonical and routinely used template is dsDNA. RNA templates require an additional reverse transcription step to generate complementary DNA (cDNA) for amplification (RT-PCR), and therefore are not the direct template in standard PCR. Step-by-Step Solution: Recognize the workflow: denature dsDNA → anneal primers → extend with DNA polymerase.Exclude RNA because standard PCR polymerases are DNA-dependent DNA polymerases.Select “double stranded DNA” as the default and most accurate answer. Verification / Alternative check: Protocol manuals specify an initial denaturation of dsDNA templates prior to cycling, confirming the routine use of dsDNA. Why Other Options Are Wrong: RNA: requires reverse transcription first (RT-PCR). Single-stranded DNA: possible but not the standard template described in basic PCR methods. None of these: incorrect because dsDNA is correct. Common Pitfalls: Confusing RT-PCR (RNA → cDNA → PCR) with DNA PCR; always verify the nature of the input nucleic acid. Final Answer: double stranded DNA

Question 4

PCR efficiency — In later cycles, the amplification efficiency of PCR often declines primarily due to which combined reasons?

Accepted Answer

Introduction / Context: While PCR is exponential in early cycles, yields eventually plateau. Understanding why the reaction slows helps optimize protocols, troubleshoot poor yields, and interpret quantitative data. Multiple factors converge to limit efficiency as the reaction proceeds. Given Data / Assumptions: dNTPs and primers are finite and become limiting over time. Polymerase activity can decline; extension time may be insufficient as products lengthen. Accumulated amplicons can re-anneal to themselves or form secondary structures. Concept / Approach: The “plateau effect” reflects resource depletion (substrates like primers and dNTPs), enzyme performance limits (partial denaturation/inactivation, limited extension time), and product inhibition (amplicon-amplicon reannealing competing with primer binding). These factors collectively reduce the effective amplification per cycle, making “all of these” the correct integrative choice. Step-by-Step Solution: Consider substrate depletion: as primers/dNTPs are consumed, extension cannot proceed efficiently.Consider enzyme/time: polymerase may be partly inactivated; longer products need more extension time.Consider product buildup: high amplicon concentration promotes product reannealing, reducing primer access. Verification / Alternative check: qPCR curves show sigmoidal kinetics: early exponential phase gives way to a plateau despite further cycling, reflecting these constraints. Why Other Options Are Wrong: Single-cause answers miss the multifactorial nature of the efficiency decline. Common Pitfalls: Attempting to “fix” plateau phase by merely adding more cycles; without addressing limiting factors, extra cycles add little product. Final Answer: all of these

Question 5

Diagnostic applications — Which of the following infections can be identified using polymerase chain reaction (PCR)-based assays?

Accepted Answer

Introduction / Context: PCR and related nucleic acid amplification tests (NAATs) have transformed diagnostics by detecting pathogen-specific DNA or RNA with high sensitivity and specificity. The technology applies across viruses, bacteria, and parasites, enabling detection even when culture is slow or hazardous. Given Data / Assumptions: PCR can target DNA directly or, via reverse transcription, RNA genomes. Clinical assays exist for multiple pathogens in blood, tissue, and respiratory samples. The listed pathogens include both viruses and bacteria of major clinical importance. Concept / Approach: HIV-1/HIV-2 (RNA retroviruses) are detected by RT-PCR to quantify viral load or confirm infection. Hepatitis B virus (DNA virus) is detected with PCR for diagnosis and monitoring. Mycobacterium tuberculosis (bacterium) is identified using PCR-based tests (e.g., MTB complex detection), which are faster than culture and can include resistance markers. Therefore, all listed infections are amenable to PCR-based detection. Step-by-Step Solution: Match virus/bacterium nucleic acid type to PCR or RT-PCR workflow.Note widespread clinical adoption of NAATs for HIV, HBV, and MTB.Conclude that each option is correct; therefore choose “All of these.” Verification / Alternative check: Clinical guidelines recommend NAATs for early HIV detection and viral load, HBV DNA monitoring, and rapid MTB diagnostics, validating PCR utility. Why Other Options Are Wrong: Selecting a subset ignores established PCR assays for the others. Common Pitfalls: Assuming PCR is only for DNA viruses; RT-PCR extends detection to RNA viruses as routine practice. Final Answer: All of these

Question 6

rRNA gene sequencing — The PCR/sequencing approach targeting rRNA genes from an unknown bacterium is most informative for determining:

Accepted Answer

Introduction / Context: Ribosomal RNA (rRNA) genes, especially 16S rRNA in bacteria, are highly conserved with variable regions that allow phylogenetic placement. Sequencing these genes is a cornerstone of bacterial taxonomy and culture-independent community analysis. Given Data / Assumptions: Amplified rRNA gene sequences are compared to databases for identification. rRNA genes contain conserved and variable regions suitable for phylogeny. Phenotypic traits like morphology or motility are not directly encoded in rRNA sequence patterns. Concept / Approach: By aligning the rRNA gene sequence against reference sequences, we infer evolutionary relationships and likely taxonomic identities (from phylum down to genus or species, depending on resolution). While certain lineages may correlate with metabolisms or morphologies, rRNA sequences do not directly specify those traits; they primarily inform relatedness. Step-by-Step Solution: Sequence the rRNA gene amplicon from the bacterium.Align against curated databases to calculate similarity and phylogenetic trees.Infer the closest related taxa based on sequence similarity and clustering. Verification / Alternative check: Consistent phylogenetic placement across multiple variable regions (e.g., V3–V4, V4–V5) strengthens identification, confirming relatedness. Why Other Options Are Wrong: Morphology, metabolism, motility: phenotypic traits require microscopy or functional assays; rRNA sequencing alone does not define them. Common Pitfalls: Assuming 16S rRNA can always resolve to species; sometimes only genus-level identification is reliable, depending on divergence. Final Answer: the species to which the bacteria are most closely related

Question 7

Thermal cycling design — A standard PCR cycle is composed of how many fundamental steps repeated each cycle?

Accepted Answer

Introduction / Context: PCR relies on repeating temperature-driven steps that denature template DNA, allow primers to hybridize, and extend new strands. Recognizing the canonical step count helps when programming thermocyclers and troubleshooting amplification problems. Given Data / Assumptions: We consider the classic, three-step PCR cycle. Some protocols merge steps (e.g., two-step PCR for high-Tm primers), but the canonical model still has three distinct phases. Optional initial denaturation and final extension are not counted per-cycle steps. Concept / Approach: The classic PCR cycle consists of: (1) denaturation (typically ~94–98°C), (2) annealing (primer binding at a temperature based on primer Tm), and (3) extension (DNA synthesis at polymerase optimum, often 68–72°C). These three are repeated for 25–40 cycles depending on the application. Step-by-Step Solution: Identify the distinct thermal phases per cycle.Confirm that initial and final holds are outside the per-cycle count.Select “three steps” as the canonical description. Verification / Alternative check: Manufacturer protocols for Taq polymerase describe three steps per cycle, validating the conventional model. Why Other Options Are Wrong: Two steps: used in specialized protocols (e.g., long primers) but not the standard. Four or five steps: overcounts by splitting phases or adding non-cycle holds. Common Pitfalls: Confusing initial denaturation or final extension as additional per-cycle steps; they are single events outside the cycle loop. Final Answer: three steps

Question 8

From RNA to PCR template — Bulk RNA can be analyzed by PCR after reverse transcription into which nucleic acid form?

Accepted Answer

Introduction / Context: Standard PCR requires DNA as a template. To analyze RNA transcripts, the RNA must first be converted into complementary DNA (cDNA) by reverse transcription, creating a DNA template suitable for amplification. This combined method is called reverse-transcription PCR (RT-PCR) or, in quantitative formats, RT-qPCR. Given Data / Assumptions: Input is RNA (e.g., total RNA or mRNA). Reverse transcriptase synthesizes a DNA strand complementary to the RNA template. Subsequent PCR uses DNA polymerase on this cDNA. Concept / Approach: Reverse transcription yields cDNA, a DNA copy of the RNA sequence. This cDNA is then amplified using PCR primers complementary to sequences of interest. tRNA and rRNA are specific RNA classes, not DNA forms; “m DNA” is not a recognized term. Therefore, the correct product enabling PCR from RNA is cDNA. Step-by-Step Solution: Isolate RNA from the sample (e.g., using spin columns).Use reverse transcriptase with primers (random hexamers, oligo-dT, or gene-specific) to generate cDNA.Perform PCR on cDNA with DNA polymerase to amplify targets. Verification / Alternative check: Successful RT-PCR shows amplicons even when no genomic DNA is present; minus-RT controls confirm the amplification derives from cDNA, not contaminating DNA. Why Other Options Are Wrong: tRNA / rRNA: RNA species, not DNA; cannot serve as DNA templates for standard PCR. m DNA: not a standard term in molecular biology. Common Pitfalls: Forgetting DNase treatment of RNA to remove genomic DNA, which can otherwise confound RT-PCR results. Final Answer: c DNA

Polymerase Chain Reaction Questions