Polymerase Chain Reaction (PCR)

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Questions and Answers

In the context of recombinant DNA technology and utilizing Type II restriction endonucleases, what is the most critical biophysical consideration when designing a protocol for directional cloning of a 2.5 kb insert into a 7.0 kb plasmid vector, given both possess multiple recognition sites for EcoRI and BamHI?

Designing primers with flanking rare-cutter restriction sites (e.g., NotI, SfiI) for PCR amplification of the insert, coupled with compatible site insertion into a similarly digested vector, with careful consideration to translational reading frame maintenance. (correct)
Employing a combination of EcoRI and BamHI for digestion of both insert and vector, followed by phosphatase treatment of the vector to prevent recircularization, despite potential challenges in maintaining proper fragment orientation.
Ensuring complete digestion of both insert and vector with a single enzyme (either EcoRI or BamHI) followed by blunt-end ligation to minimize the risk of self-ligation.
Utilizing methylation-sensitive restriction enzymes to selectively digest the vector, preventing self-ligation, while using Dam-/Dcm- E. coli strains to propagate the insert, ensuring its protection from digestion.

Considering the challenges associated with amplifying highly structured RNA regions via RT-PCR, which of the following strategies would most effectively mitigate the formation of stable secondary structures that impede reverse transcriptase and polymerase activity, thereby ensuring accurate and efficient cDNA synthesis and subsequent amplification?

Employing a thermostable reverse transcriptase in conjunction with elevated reaction temperatures (e.g., 60-65°C) during the reverse transcription step, followed by a hot-start PCR to minimize primer dimers.
Utilizing modified nucleotides such as 7-deaza-GTP during cDNA synthesis to reduce secondary structure formation, in conjunction with a primer walking approach using sequence-specific primers designed at intervals along the RNA template. (correct)
Incorporating chemical denaturants such as DMSO or betaine at high concentrations (e.g., 10-15%) in both the reverse transcription and PCR mixtures, while gradually increasing annealing temperatures to compensate for the denaturing effect.
Designing multiple overlapping primer sets that target different regions of the transcript and performing several independent RT-PCRs, followed by pooling the amplified products to create a composite representation of the entire RNA sequence.

In the context of digital PCR (dPCR), which factor most significantly contributes to the enhanced precision and sensitivity of target quantification compared to traditional qPCR methods, particularly when analyzing samples with low target concentrations or complex backgrounds?

The exponential amplification phase in dPCR allows for more accurate quantification due to reduced competition between target and non-target sequences.
dPCR employs multiple rounds of amplification with nested primers to ensure that even the faintest signals are amplified above the detection threshold.
The use of intercalating dyes in dPCR provides a direct measure of the total DNA concentration in each partition, which normalizes for variations in amplification efficiency.
Sample partitioning into thousands of individual reactions in dPCR minimizes the impact of PCR inhibitors and stochastic amplification biases, enabling absolute quantification based on the fraction of positive partitions. (correct)

When constructing a genomic library using a lambda phage vector, which of the following considerations is most critical to ensure comprehensive and unbiased representation of the source genome, particularly when dealing with organisms possessing high AT or GC content?

Optimizing DNA fragmentation methods to minimize sequence-specific biases, such as using enzymatic methods (e.g., DNase I) to shear DNA randomly, followed by size selection to enrich for fragments within the optimal insert size range for the lambda phage vector. (C) Signup and view all the answers

In the context of allele-specific PCR, what is the most critical design parameter to ensure accurate and reliable discrimination between closely related alleles differing by a single nucleotide polymorphism (SNP), while minimizing the occurrence of false-positive amplification?

Designing allele-specific primers with the 3'-terminal nucleotide complementary to the target allele and incorporating a mismatch at the penultimate position to destabilize non-specific primer binding. (A) Signup and view all the answers

When employing inverse PCR to amplify unknown flanking regions adjacent to a known DNA sequence, what strategic optimization is most crucial to prevent self-ligation of the digested DNA fragments and ensure efficient circularization for subsequent PCR amplification?

Optimizing the restriction enzyme digestion to generate DNA fragments with compatible cohesive ends, followed by a low-concentration ligation reaction (e.g., 1-5 ng/µL) to favor intramolecular circularization. (C) Signup and view all the answers

What is the most critical factor when designing a multiplex PCR assay targeting multiple loci with varying amplicon sizes and GC content, while maintaining uniform amplification efficiency across all targets under stringent reaction conditions?

Using bioinformatics tools to identify primer sets with minimal cross-reactivity and similar thermodynamic properties, and incorporating a balanced mixture of dNTPs and additives to equalize amplification efficiency. (D) Signup and view all the answers

In assembly PCR, what is the most crucial parameter to optimize when synthesizing a long DNA sequence from overlapping oligonucleotides, in order to minimize the formation of misassembled products and ensure high-fidelity reconstruction of the target sequence?

Using a thermostable DNA polymerase with proofreading activity and optimizing the annealing temperature to promote specific hybridization of the overlapping oligonucleotides. (B) Signup and view all the answers

When using hot-start PCR, what is the underlying mechanism that prevents non-specific amplification during the initial stages of the PCR reaction, thereby enhancing the specificity and sensitivity of the assay?

The use of a chemically modified DNA polymerase that is only activated at high temperatures, preventing primer extension until the optimal reaction conditions are reached. (C) Signup and view all the answers

Considering the inherent limitations of traditional PCR in quantifying initial target concentrations, which modification would MOST accurately quantify the starting amount of a specific mRNA transcript in a complex sample?

Reverse transcription quantitative PCR (RT-qPCR), which combines reverse transcription of RNA into cDNA with real-time monitoring of PCR amplification, enabling accurate quantification of the initial mRNA transcript levels. (D) Signup and view all the answers

Flashcards

What is PCR?

A technique to amplify a single or few copies of DNA, generating thousands to millions of copies of a particular DNA sequence.

What are Restriction Enzymes?

Proteins that recognize and cut DNA at specific sequences, used in recombinant DNA technology.