Question

Place the steps of real time DNA synthesis detection using a probe in the correct order starting at the top with cooling the reaction in the annealing step. Which of the following is the correct order of the steps?

1) 3, 1, 4, 5, 2
2) 3, 1, 5, 4, 2
3) 3, 1, 2, 4, 5
4) 3, 1, 2, 5, 4

Answer 1

The correct sequence of events for real-time DNA synthesis detection using a probe, starting with the annealing step, is represented by option 3 (3, 1, 2, 4, 5), which corresponds to annealing, extension, and denaturation. The correct option is 3.

Step-by-step explanation:

The question pertains to the process of real-time DNA synthesis detection, specifically looking at the incorporation of fluorescent dideoxynucleotides (ddNTPs) in DNA sequencing. The steps of this process, particularly in the context of Polymerase Chain Reaction (PCR), start with the cooling of the reaction in the annealing step. PCR consists of three main stages:

1. Annealing (cooling the reaction to allow the primers to attach to their complementary sequences on the DNA strands)
2. Extension (where the DNA polymerase synthesizes the new DNA strand by adding nucleotides)
3. Denaturation (heating the reaction to separate the newly synthesized DNA strands)

For the given options in the question, the correct order starting with cooling in the annealing step would be: annealing, extension, denaturation.

This translates to option 3: 3, 1, 2, 4, 5. It's important to note that in DNA sequencing using Sanger method, which includes the use of ddNTPs, the steps after PCR amplification include chain elongation and eventually gel electrophoresis for analysis of the DNA fragments. The correct option is 3.