Dna chain termination sequencing

[Puchinita5]

Homework Statement

A DNA 5-[32P] CCT TCG T TCG TTG TTC CCT A GGC TGT ATA GCC CCT ACC TTT TTG GTA GGG GCT ATA CAG CC was elongated by DNA polymerase in four reaction mixtures in the presence of dATP, dTTP, dGTP (dCTP was omitted due to an experimental mistake) and one ddNTPs. The four reaction mixtures contained different ddNTPs. Predict the band pattern after separation of the reaction mixtures ion gel and autoradiography.I thought I was starting to understand until I saw a classmate talking about the primer ending at the lone A in the middle.

I thought that you would start sequencing at the 3 prime end, so you would get GGC ..etc.
but since there are no C;s since dCTP was omitted, it would just stop there, so you would just get a band patter of two lines in the ddGTP column.

But now I don't get this whole primer thing. Is that entire chunk from that single A in the middle all the way to the end just a primer?

So then would I sequence,
AGGGAAC and then stop at that C?

Is this right? And if so, how do you know that the primer ends at that middle A?

Homework Equations

The Attempt at a Solution

 

[nate808]

First of all, great job on understanding the concept so far! It's important to have a good grasp on the basics before diving into more complex experiments like this.

To answer your question, yes, the entire chunk from the single A in the middle to the end is indeed the primer. The primer is a short piece of DNA that serves as a starting point for DNA synthesis by DNA polymerase. In this experiment, the primer was labeled with 32P, making it visible on the gel and autoradiogram.

Now, for the band pattern prediction, let's start with the primer itself. Since it is labeled with 32P, it will show up as a band on the gel. This band will be at the bottom of the gel, as it is the smallest fragment.

Next, let's look at the elongation products. In the presence of dATP, dTTP, and dGTP, the DNA polymerase will add these nucleotides to the end of the primer, creating longer fragments. However, since dCTP was omitted, the polymerase will not be able to add a C to the end of the primer. This means that the elongation products will stop at the C in the primer sequence, which is the fifth nucleotide from the 3' end. So, the bands for these elongation products will be at the fifth position from the bottom of the gel.

Now, for the ddNTPs. These are chain-terminating nucleotides, meaning that when they are incorporated by the polymerase, they stop the synthesis of the DNA chain. In this experiment, there are four different ddNTPs, each labeled with a different fluorescent dye. This means that there will be four bands on the gel, each representing the end of the elongation products.

To summarize, the band pattern on the gel and autoradiogram will look like this:

1. Primer (labeled with 32P) at the bottom of the gel
2. Elongation products (labeled with dATP, dTTP, and dGTP) at the fifth position from the bottom of the gel
3. Four bands representing the ddNTPs at different positions on the gel

So, to answer your question, the sequencing will start at the 3' end of the primer and stop at the C in the primer sequence. This is because the primer serves as a starting point for DNA synthesis,