3
$\begingroup$

I am learning about biotechnology. I have no education in biology or chemistry and am simply interested in the subject of biotechnology.

I am wondering why we need to have multiple copies of a piece of DNA in order to do something with it (ie. PCR)? Why is one piece of DNA not enough? Is machinery not precise enough to work with one piece of a DNA sequence in order to do something with it?

I can understand that an experiment may fail. In that case, make 8-16 copies for assurance. But why an extraordinary (billions) amount of copies? What practical use does it have? What applications require there to be an extraordinary amount of DNA to be succesful? Do the billions of copies just increase the speed of the experiment by being able to quickly "fish out" a piece of DNA? I just can't quite grasp the idea.

$\endgroup$
  • 1
    $\begingroup$ Although your question about DNA has been answered, you need to be aware of the general limits of detection of molecules in the lab. If you thought you could detect one molecule of DNA, think about detecting one molecule of glucose. How much would that weigh? You can find the answer by reflecting on Avagadro's number — the number of atoms in a mole of a compound. This has a value of about 6 x 10^23, so there would be that many molecules in 180 g glucose! Catch one! $\endgroup$ – David Oct 7 '19 at 22:34
5
$\begingroup$

This is a question I also remember wondering about when I was younger in school. Now as a professional it's way too obvious to even explain. But i think it's an important and common question, which warrants an example or two from common daily lab practice.

Preface

You have to understand that DNA is a molecule. It's really tiny. It's not trivial to work precisely with it, nor detect it or discriminate two different pieces of DNA from each other, let along sequencing them. We make a lot of DNA through amplification so that we can prepare a sufficient amount to make reactions with.

Visualization

We usually make DNA visible by using chemical dyes that emit light (fluorophores) or incorporated radioactive pieces to make the DNA visible.

Here's a common case in the lab: we need to get enough DNA to use in subsequent reactions. When we work with DNA it is very rare that we obtain exactly what we want 100% of the time, or perfect yield. For example, we want to cut a piece of DNA once. But we have scissors (i.e. restriction enzymes) that will chop up the DNA multiple times, especially when we leave the reaction going too long. What we do is take 100 units of DNA, subject them to the reaction, and then view the results using electrophoresis using an agarose gel, for instance. We can then see 40 units of uncut DNA, 30 units of single cut DNA, and some 30 units of DNA cut multiple times (2+ times, so they are small fragments). We need enough single cut DNA to proceed with the next reaction, with full knowledge that there are no contaminating chunks of DNA. Typically there are many reactions in a row that we do to achieve anything in the lab. Cloning, in vitro amplification, sequencing, gene editing, preparing genes for transgenesis, creating RNAi probes, what have you.

More is better

Another case: if you want to know the sequence of DNA, you must sequence it. However, sequencing is imperfect and errors occur. To be sure, we re-sequence the same DNA many times to get a confident picture of what the sequence is like, beyond reasonable doubt. Sequencing DNA ten times is really more reliable than sequencing it once. This could be because of how the molecules behave in solution, or because of degradation, or tens of other things. You must understand this is also not specific to sequencing; a lot of reactions follow the same logic.

Amplification as a specification tool

By using primers in PCR, we can make sure we are obtaining what we want. In principle, this means that we can make a gajillion copies of something we are interested in (e.g. a gene), even from a single strand of DNA which may contain things we are uninterested in (e.g. the rest of the genome).

Some thoughts

This answer is certainly not extensive, but I hope it's a primer for you to understand that DNA work is just really smart nanotechnology, and we are not yet at the level of performing nanotechnology on single molecules, with full confidence, precision or efficacy. On the daily, we need enough to see it with our eyes so we amplify and react it with intercalating dyes to make them visible to the naked eye!

$\endgroup$
  • $\begingroup$ Thank you for the succinct answer. I was failing to understand the precision of current nanotechnology, and your practical examples (which sound really fun) really brought the point home. $\endgroup$ – ErikTang Oct 7 '19 at 16:56

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.