When starting your journey in learning synthetic biology, you will inevitably run into something in your reading called PCR (polymerase chain reaction).
I found it a little confusing at first because in some contexts, PCR refers to the process of amplifying DNA, while at other times, it refers to the physical hardware used to perform the amplification.
Now that I’ve gained some experience in running PCR, I thought I’d share a little more about how I learned it using internet resources alone.
What is PCR?
The short-version is that PCR is a technique that is used to make a lot of copies of DNA. Why? DNA is incredibly small and so we must make a lot of copies of it in order to study it in more detail. Sequencing, for example, requires a certain concentration of DNA to provide a reliable reading of nucleotide sequences.
How PCR works is more involved but the short version is that you heat and cool a sample of DNA in a series of cycles. In each cycle, a special enzyme (DNA polymerase) mixed into your tube of extracted templated DNA works to double the amount of DNA with each heat / cool cycle.
The heat / cool cycles are also referred to as denaturation and annealing in the literature.
In days of old, temperature cycling was achieved by moving the tube of DNA back and forth between a hot and cold bath of specific temperatures for specific periods of time. As you might imagine, this process required much attention and energy over several hours and is prone to error.
Nowadays, the process is automated via the use of a thermocycler machine.
How I Learned PCR Fundamentals
I spent some time watching the videos, reading the articles and making notes – really making sure I understood what was being said to the extent that I could explain it by heart to someone else.
In addition, I found it helpful to search for images, like the one below, that could help me develop a mental model of what was happening at the molecular level.
Personally, I learn my saturating myself with information to the point that certain terms and methods begin to repeat themselves across sources and eventually come into focus. To saturate myself with knowledge and develop a clear understanding, I have to be exposed to the same information in a variety of formats and contexts. This is quite different than how most folks are taught in school where most people are given a single source (usually a textbook) and the learning typically revolves around memorizing, not internalizing, the information.
diyBio Thermocycler Options
There are a few options available to diybio folks who want to run PCR reactions at home. Keeping in mind that all thermocyclers really do is heat and cool DNA samples over a series of cycles, the difference in options comes down to price, size and degree of automation.
Here are some of the options.
MiniPCR mini8 Cost: $650, Samples: 8
PocketPCR Cost: ~$120 Samples: 5
Bento Lab Cost: $1600 Samples 32
There was also OpenPCR but it looks like the project has been discontinued and replaced with a qPCR machine that starts at around $5000. I’m going to skip that one since that is out of the range for most diy biologists.
So which thermocycler did I end up choosing?
I weighed all of the options and went in another direction: buying a used thermocycler on eBay!
Buying on eBay is inherently risky, but it can be done. You absolutely need to make sure the listing states the the unit has been tested and works. You absolutely need to make sure it comes with the heat block (the big chunk of metal that the tubes sit in); otherwise, buying a new block can cost more than the machine itself.
I’ve heard some folks get lucky with repairing broken units, but I wouldn’t recommend this approach if you are new to diybio as you’re already going to be overwhelmed with things to do and learn.
I ended up buying an Applied Biosystems GeneAmp PCR 9700 for about $300 ($150 for the unit + $150 for shipping).
This option was the cheapest for me out of all the options mentioned above, even with the high shipping costs. Although it’s not portable and it cannot be controlled via an app, it works. It has a 96-well heat block which is more than I’ll ever need for my home lab. It allows me to easily program and store my PCR routines. And surprisingly, many of the disposables and peripherals are still sold by ThermoFisher.
Additional Components Necessary for Running PCR
A thermocycler is only one piece of the puzzle in amplifying DNA. There are other procedures and required tools + reagents you’ll need to successfully run a PCR reaction.
My experience is in extracting and amplifying the ITS gene from fungal samples, but the same general requirements will be needed for any gene(s) you want to amplify:
- DNA Extraction – you’ll need a sample, protocol for extracting the dna, reagents to break the DNA out of your cultures’ cells, centrifuge, and 1.5ml or .2ml Eppendorf tubes.
- Oligonucleotides (Primers) – primers are little strands of DNA that prefix and suffix the gene(s) you wish to amplify. Once you extract your DNA, you’ll mix in the primers per your protocol prior to running PCR. During PCR, the primers will attach to the gene of interest where the next reagent below will work to double the gene of interest.
- TAQ Polymerase – this reagent is added with the primers and it’s a special heat-resistant enzyme that facilitates the doubling of the gene(s) of interest between each denaturation / annealing cycle.
- Buffer – buffer is usually just water and Tris-HCL pH 8.0 (sometimes with EDTA) but it’s role it’s to balance and stabilize the extracted DNA and/or create primer dilutions.
After running PCR, gel electrophoresis can be used to test whether or not your PCR reaction was successful. This is an important step because it ensures you’ve amplified the correct gene before proceeding with costly next steps like sequencing.
In closing, I’ll say that learning PCR is an incredibly useful and versatile tool. Many of the folks I’ve spoken with reflect on their time spent learning and running PCR reactions fondly. Much of modern-day genetic work would not be possible without PCR. So go forth and learn it! It’ll be worth your time.