The human genome is 3×10⁹ bp. You wish to design a primer to ampl...

Question

The human genome is 3×10⁹ bp. You wish to design a primer to amplify a specific gene in the genome. In general, what length of oligonucleotide would be sufficient to amplify a single unique sequence? To simplify your calculation, assume that all bases occur with an equal frequency.

Accepted Answer

Hi, everyone. Welcome back. Let's look at our next problem. It says we need to calculate the minimum length of a primer for amplifying a specific gene in the P P Saum plant. Assuming a desired melting temperature of 60 degrees Celsius A G C content of 50% and an equal frequency of all bases in the plant's genome, which is about 4.45 billion base pairs, which of the following formulas to calculate the minimum primer length is correct. Well, when we look at our answer choices, we see that they're all very similar. So we don't need to think about competing formulas in terms of wildly different things. Just need to know the, all the correct parts of the formula we're going to use. In this case, we're calculating the minimum length of a primer. We're given the desired melting temperature in the G C content. So the rule we're going to be looking at is the Wallace rule of thumb, which is for calculating the minimum length of a primer. And it takes into account the desired melting point and the G C content because the desired melting point is affected by or the length of the primer is affected by G C content to have the desired melting point. Because G C pears have more hydrogen bonds than a tea pairs. You can recall they have three while A T pairs just have two and therefore more G C content in a given DNA sequence means a higher melting point, it takes more kinetic energy to break the greater number of hydrogen bonds. So if you're trying to calculate, how long does that primer have to be to be at the correct melting point, I need to take into account uh how many, how, what is my G C content of my given DNA sequence? So Wallace rule of thumb is what I want. And my four answer choices are presented all in, as I said, a very similar format. Just there are two constants in the formula, one in the numerator and one in the denominator. And each of these has different values for these contents. So we need to pick the one with the correct values. So let me just list the general format. Then we'll go through answer choices and see how they differ. So the general format of this formula is in the numerator, the desired melting temperature, I'll put melting tea minus sine 64.9 degrees Celsius multiplied by and will write constant one. So that's our con in the numerator that is all divided by. And then, oh, let me put parentheses around my subtraction problem here. So in parentheses, desired melting temperature minus 64.9 degrees Celsius closed parentheses multiplied by constant one. Then all that divided by. And in my denominator, I have G C content sign A T content minus sine constant two. So a constant in the numerator, a constant in the denominator. So in each of these cases, my answer choice just differs by that constant one or constant two. So choice, a constant one is 4.1 in the numerator and constant two is 16.4. On the denominator, choice B constant one in the numerator is five constant two in the nominator, 16.4. Choice C constant one in the numerator is constant two. And the denominator is 16.4. And choice D constant one in the numerator is five constant two in the num denominator is 20.4. So the correct formula of the Wallace rule of thumb is choice B in parentheses, desired melting temperature minus 64.9 degrees Celsius closed parentheses multiplied by five, all divided by. And then in the denominator G C content plus sign A T content minus sine 16.4. So when we look at choice A, we see that the constant in the numerator is incorrect. It's 4.1 instead of five. When we look at choice C, the constant in the numerator is also incorrect. It's 10 instead of five. And when we look at choice D the constant in the denominator is incorrect. It's 20.4 instead of 16.4. So once again, our answer choice is choice B our correct formula has five as constant one in the numerator and 16.4 as constant 12 in the denominator. See you in the next video.

The human genome is 3×10⁹ bp. You wish to design a primer to amplify a specific gene in the genome. In general, what length of oligonucleotide would be sufficient to amplify a single unique sequence? To simplify your calculation, assume that all bases occur with an equal frequency.

Key Concepts

Oligonucleotide Length

Unique Sequence Probability

Base Composition and Frequency

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