Basic protocols and the theory behind them

The goal of this blog is to provide the basic overview of how and why we process samples the way we do and to provide insight into the molecular biology techniques that are being used. At the highest level we are going to extract DNA from an organism, amplify a small section of the DNA whose sequence can tell us about its identity, we will check to see if we amplified the DNA and if we did we will send our amplified DNA out to be sequenced. When we get the sequence back we will use databases to see if it matches known species and we will publish our data to the global database for DNA sequences. 

DNA Extraction

DNA (deoxyribonucleic acid) in most organisms is buried deep inside cells away from where it can't get harmed by the outside world. We want to look at its code of As, Ts, Cs, and Gs, so we need to get it out of the cell to do this. Different cell types provide different challenges to getting the DNA out and this will often influence the procedure that is chosen to extract DNA. 

 Generalized examples of cell structure for a Bacteria, Animal and Plant cell. Bacteria and Plants often have cell walls that make their cells very difficult to access in comparison to animal cells. Images from wikicomons and made by  Mariana Ruiz Villarreal.  

Generalized examples of cell structure for a Bacteria, Animal and Plant cell. Bacteria and Plants often have cell walls that make their cells very difficult to access in comparison to animal cells. Images from wikicomons and made by Mariana Ruiz Villarreal. 

Different procedures can also get you DNA of different qualities; broken up into small pieces, intact long strands, clean, or in solution with a bunch of the other cellular junk. The more intact you want the DNA and the cleaner you want it the more time and money will go into getting it. Since we are on a relatively tight budget and only looking a a very small section of the DNA we will use a quick and dirty method to extract DNA as described below.  

 Generalized workflow for extracting DNA with whats going on at human scale and at the micro scale. 

Generalized workflow for extracting DNA with whats going on at human scale and at the micro scale. 

A small portion of the organism we are interested, about the size of half a pea, will be added to a microcentrifuge tube. The material will then be crushed using a sterile piece of plastic and an equal volume of distilled water will be added to aid with the crushing. This physical crushing can open cells up letting their contents spill out into the water, but it also serves to separate cells from each other which can make them more vulnerable to heat. Heating the sample causes more of the cells to rupture and frees their DNA into the water. Spinning of the tube causes the heavier organelles, cell walls, etc to pellet in the bottom of the tub leaving a layer of liquid on the top that is free of larger particales called the supernate. The supernate contains the DNA we are interested in. It probably also contains some of the other lighter components of the cell, but it should be clean enough for our uses. 

DNA Amplification (Polymerase Chain Reaction)

Once we have our somewhat clean DNA isolated we want to amplify just a small section of it whose sequence can help us determine what species we have. We need to amplify it because we want to be able to look at just one little section of all the DNA that we have and we need to make enough of it that we can see it and manipulate it. We are going to amplify less then 0.0005% of the DNA we have and from one copy we are going to make about 100 million copies. The process were are going to use is called the Polymerase Chain Reaction. It derives its name from the enzyme that copies DNA, called polymerase, and from the multiple rounds or chains or reactions that are used to copy the DNA. The technique was developed in Emeryville in the 1980s and is a foundational technology in most contemporary biological labs.

pcr.jpg

The illustration above can help you visualize whats going on in a PCR reaction, but an animation is really needed to get a solid understanding of how the system works and this is the best one I've found:

Just like in the animation we are going to take our extracted DNA and mix it with nucleotides (individual building blocks of DNA), primers (DNA fragments that tell the polymerase where to start copying), and polymerase. Once its well mixed we will place it on our little thermal cycler, which is a tool for quickly heating and cooling our reaction tube. The PCR reaction will take anywhere from 1 hour to 4 hours to complete. We will attempt to use an optimzed protocol to minimize how long it runs. 

QC of PCR using Gel Electrophoresis

When we remove our reaction from the thermal cycler it looks like nothing has happened as the solution in our tube is still the same clear color it was when we put it in and that is because any DNA that has amplified is still to small for us to see unaided even though there are now potentially hundreds of millions of copies. Our Quality Control (QC) check of our PCR will require the use of gel electrophoresis for us to visualize any DNA that was amplified. Gel electrophoresis is a form of chromatography similar to how you can get colors from a pen to separate on a piece of paper by letting water wick up the paper. The different colors have different properties that cause them to move faster or slower as the water wicks through the paper. Gel electrophoresis is similar as it uses the size of DNA molecules and their charge to separate them in a gel. 

 Negatively charged DNA fragments migrate towards the positive end of the gel and as they do that they separate out based upon their size. A stain is used to visualize the DNA

Negatively charged DNA fragments migrate towards the positive end of the gel and as they do that they separate out based upon their size. A stain is used to visualize the DNA

This is another process where an animation can be much more helpful at fully conceptualizing how gel electrophoresis works and this is one of the better animations I've found:

After our gel has run and we've added the stain to see the DNA with our naked eye we will be looking for the presence of a band of DNA between 400 and 800 bases in the lane we loaded our sample in. If we see a band we know that our PCR was successful and we have a lot of amplified DNA that can be sent out for sequencing.