DNA Denaturation and Polymerase Chain Reaction

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What is the primary product of the process described in the text?

Reverse transcriptase

DNA copy

RNase H

RNA (correct)

What is the role of the primer in this process?

It is used to synthesize the double-stranded DNA from the cDNA.

It carries the binding site for RNA polymerase and is complementary to sequences in the target RNA. (correct)

It is used to degrade the RNA from the intermediate DNA-RNA hybrid.

It is used to amplify the target nucleic acid sequences.

What is the purpose of the heat used in the process?

To activate the reverse transcriptase

To denature the DNA-RNA hybrid (correct)

To separate the cDNA from the RNA target

To amplify the target nucleic acid sequences

What is the role of RNase H in this process?

It degrades the RNA from the intermediate DNA-RNA hybrid, eliminating the heating step. Signup and view all the answers

What is the purpose of the second primer in the process?

It binds to the cDNA and is extended by reverse transcriptase, producing double-stranded DNA. Signup and view all the answers

What is the purpose of the transcription of the cDNA by RNA polymerase?

To produce millions of copies of RNA Signup and view all the answers

What is the recommended concentration range of each nucleotide for PCR reactions?

0.1 to 0.5 mM Signup and view all the answers

Why is using a single solution containing a mixture of all four nucleotides recommended?

To reduce pipetting errors Signup and view all the answers

What can happen if dNTPs are stored in lower concentrations?

Hydrolysis to dNDP and dNMP Signup and view all the answers

What is the source of Taq Polymerase used in PCR?

Thermus aquaticus Signup and view all the answers

What unique feature does Tth Polymerase from Thermus thermophilus possess?

Reverse-transcriptase activity Signup and view all the answers

What can result from poor manufacturing conditions or contamination with heavy metals regarding dNTPs?

Nucleotide di- and monophosphates Signup and view all the answers

What is the primary purpose of the denaturation step in PCR?

To separate the double-stranded DNA into single strands Signup and view all the answers

What is the term used to describe the copies of DNA generated after the PCR process?

Amplicons Signup and view all the answers

Which of the following statements about primers in PCR is correct?

Primers determine the part of the template that will be amplified Signup and view all the answers

What is the primary advantage of the PCR technique?

It amplifies and effectively clones the target DNA sequences Signup and view all the answers

Which step of the PCR process is considered the most critical for specificity?

Annealing Signup and view all the answers

What is a typical annealing temperature range for primers in PCR?

50°C to 70°C Signup and view all the answers

What is the most effective method for decontamination and preparation of the workspace?

10% bleach (7 mM sodium hypochlorite) Signup and view all the answers

What is the primary purpose of the dUTP-UNG system?

To control contamination by enzymatic degradation of uracil-containing PCR products Signup and view all the answers

What is a necessary step for the UNG enzyme to function in the dUTP-UNG system?

A short incubation period at the beginning of the PCR amplification program, usually at 50°C for 2 to 15 minutes Signup and view all the answers

What is the purpose of wipe tests in the context of PCR?

To routinely check or confirm successful decontamination Signup and view all the answers

What should be avoided when using UV light for decontamination?

Both exposure to skin or eyes and damage to Plexiglas or some plastics Signup and view all the answers

What is the purpose of reverse transcriptase PCR (RT-PCR)?

To convert RNA to double-stranded DNA for better amplification Signup and view all the answers

What is the primary function of the Qβ replicase enzyme in this assay?

To amplify the reporter probes bound to the target nucleic acid Signup and view all the answers

What type of nucleic acid can be used as the target in this assay?

Both DNA and RNA Signup and view all the answers

What is the purpose of the polyG-tailed capture probes and magnetic beads covalently attached to polyC sequences?

To immobilize the target nucleic acid and reporter probes Signup and view all the answers

What is the purpose of the polyA capture probe (capture probe B)?

To release the target nucleic acid-reporter probe complexes from the magnetic beads Signup and view all the answers

What is the purpose of the polyT paramagnetic bead?

To further purify the target nucleic acid-reporter probe complexes Signup and view all the answers

What is the purpose of the colorimetric and real-time fluorogenic methods mentioned in the text?

To detect the amplified reporter probes Signup and view all the answers