Did you know that there are more bacteria in your body than there are human cells? That's right – not only do we have over 10 times more bacteria than other animals, but even the "human" cells are outnumbered by the bacterial ones.
Our bodies are teeming with tiny little organisms, and they all need a home. So what does this mean for PCR? It means that after 30 cycles of PCR, there are 230 copies of your DNA sequence! Each round of PCR doubles the number of copies (and remember: each cycle involves denaturing your DNA, adding primers to copy it again and again). So if you started with just one copy of your DNA sequence at the beginning of our example above, after 30 cycles you'd end up with 230 copies – enough to fill over 20 million swimming pools! That's pretty cool - but don't forget about those bacteria! After all...
You may have heard that PCR is like a photocopy machine for DNA. This analogy is accurate in that each round of PCR doubles the number of copies, just as a photocopier makes two copies from one original page. Here's how it works:
After 30 cycles of PCR, there are 230 copies of the DNA sequence per reaction tube (230 = 2 x 1 x 2 x 3...30). This exponential growth continues until you reach saturation—i.e., all available binding sites on all your primers are filled with DNA product and no new amplification can occur.
You may not be aware, but there are a lot more bacteria in your body than human cells. In fact, the average human being is home to around 100 trillion bacterial cells, while only 10 trillion of our own cells remain. So what makes bacteria so special? Well, they're extremely small and simple organisms that can live in a variety of environments. They also play an important role in maintaining our health by helping us digest food and protecting against harmful pathogens.
But despite their usefulness, there's no denying that some types of bacteria aren't beneficial at all—they can cause food poisoning or lead to deadly infections like anthrax or malaria!
So, after 30 cycles of PCR, there are 230 copies of the DNA sequence. That’s a lot of bacteria! In fact, it is more bacteria than there are humans on earth. This may sound alarming at first blush but don’t worry – this is what we were hoping for in order to make our DNA sample detectible by our sequencing machine.
After making these 230 copies, your sample will now be ready for analysis using next-generation sequencing technology.
To answer this question, you could run 30 cycles at a lower temperature. This would prevent the exponential growth of DNA and allow you to calculate a more accurate number of copies after 30 cycles.
For example, if we knew that 100 copies were generated in the first cycle (you find this by measuring fluorescence), then we would multiply that by 30 to get our final answer:
100 x 30 = 3000
As you can see, after every cycle of PCR, your copy numbers double. After 1 cycle of DNA replication by PCR, there is only one copy of the DNA template. After 2 cycles, there are two copies. This process continues until each reaction has reached its maximum amplification limit (about 30). At this point, a researcher would have 16 copies on average per reaction and could go back to their original sample (or another sample) to use as a new template for more PCR reactions.
After two cycles of PCR, you will have two copies of your DNA. After three cycles, you'll have four copies—two from the first cycle and one from each of the second and third. Four cycles gives us eight copies—two from each of the first three cycles' products. Eight is already more than we started with (one), so what happens at nine? Well, that's when things start getting interesting: 16!
Let's say you were trying to get 10 copies of your sample. After two cycles, you would have 10 copies. After 20 cycles, you would have 100 copies. And after 30 cycles, 1000 copies!
After 20 cycles of PCR, the number of copies is roughly 1.7 x 10^10 (1.7 billion).
This is a large number of copies and it's necessary to generate that many in order to achieve a high-fidelity sequencing output.
The amount of DNA amplified per cycle will continue to increase, but since this isn't as big an increase as from 5 to 10 cycles, we can assume that at some point there will be no further significant change in copy number (hence why I didn't bother showing you 15 or 20 cycles).
You have 30 cycles of PCR, so you would theoretically generate 230 copies.
However, if you were to use 1 µL of DNA and do 30 cycles of amplification, the final number would be closer to 187 copies. This is because once your sample gets diluted down over and over again throughout the amplification process, there are fewer DNA molecules available to produce new copies of themselves.
After 30 cycles of PCR, you would expect to have 230 copies. This means that the original DNA sequence with 5 copies of a particular segment has been amplified to 1 million copies of that segment. The answer is 230 or 1073741824. The number 231 is so large because there are four nucleotides in DNA (A,T,C,G), and 2^4 = 16 possible combinations or permutations for these letters to appear together at each site within a string of DNA (AAAAA...etc). If we multiply 16 by itself 30 times (30x30), then we get over 1 million different possibilities!
After 30 cycles, there are 230 copies – more bacteria than there are humans on earth!