Gene Sequencing with the Sanger Method
If you’ve sat down in a classroom during the last decade, you’ve already heard that DNA is kind of like blueprint your body uses to build all the stuff that make you unique. And the whole blueprint is written with only four letters! (A, T, G, and C.) Each of these letters represent one of the four nucleotides, or bases, that form the fundamental building blocks of DNA itself — adenine, thiamine, guanine, and cytosine.
But the body is far too complex to have one measly running the show alone. If a single piece of DNA is like one blueprint, then a genome is like an instruction manual containing every single individual blueprint. It’s the most complete set of instructions your body has to offer. In humans, this can be over 20,000 genes long! Scientists are already working on ways to edit this instruction manual and engineer life for a variety of reasons too numerous to list.
But before the twenty-first century, scientists didn’t even know what that book said. It was thanks to the Sanger method that we finally decoded the mystery of the human genome and paved the way for gene-editing technologies like CRISPR. The Sanger method was named by its creator, Carl Sanger, who developed it during the mid-1970s, and is the most tried-and-true DNA sequencing method in the world.
First, the sample must be prepared to be sequenced. This involves isolating the target DNA from the rest of the organism, but can be incredibly taxing. This pain-staking preparation process does not typically yield enough DNA to do any meaningful work. To obtain as many samples as possible, companies use a process called PCR amplification (or polymerase chain amplification). This involves using a heat-resistant enzyme to make copies of the DNA out of broken pieces of the original sample, and is done in three steps:
- Denaturation. Samples are placed in a test tube which is heated to 96 degrees Celsius to denature the DNA. The newly single-stranded DNA now has only half of its genetic information.
- Annealation. The DNA is cooled to 55 degrees Celsius. A specialized batch of primer is added to the mixture, which anneals to a section of DNA just before the region of interest.
- Extension. The sample is reheated to 72 degrees Celsius. A heat-resistant, polymerase, a cutting enzyme, and free-floating nucleotides are added to the mixture. The polymerase begins at the primer and traverses the strand of DNA, binding specific nucleotides to the corresponding bases along the strand. The process will eventually fizzle out, either because the polymerase has run out of nucleotides, or because it has reached the end of the sample, or any other number of biological variables that could end the process.
After this cycle is finished the amount of DNA samples doubles. Lab technicians can undergo as many of these cycles as their project requires, but most times, they’ll repeat this process about thirty-five times. This results in 2³⁵ copies of the initial amount of DNA being made.
The samples are then purified to clean up excess biological materials like leftover nucleotides, incomplete DNA strands, spent polymerase, or any other number of other unwanted materials.
Next, the samples must be prepared to be sequenced. This preparation process puts the DNA through the same denaturation, annealation, and extension steps as they did during PCR amplification, with an additional batch of terminator nucleotides being added during the extension phase. These terminator nucleotides are almost identical to regular nucleotides, but with one important distinction: they’re modified to prevent the polymerase from attaching any other nucleotides after it. In other words, they’ll randomly stop the extension sequence at any point in the chain.
They’re also given a fluorescent identification marker depending on their base. For example, adenine terminators might be colored blue, thiamine might be green, adenine might be orange, and guanine might be pink. One of these terminator nucleotides will eventually bind to every single complementary nucleotide from the sample.
Now the DNA can finally be sequenced! Typically, this is done through capillary gel electrophoresis, which is a complicated way to sort the samples by size. First the samples are loaded into a tube filled with a porous capillary gel, where an electrical current will pass through.
The negatively-charged nucleotides will pass through the capillary gel towards the positive end. Here the gel acts as a filter, where smaller segments will end up closer to the positive end than larger segments, since they can pass through more pores. A laser detector then passes along the tube, starting at the end with the smallest nucleotides, and reads the fluorescent color of the terminator bases. The information is passed through a chromatogram and scientists translate the data into the letter representations of the bases.
Congratulations! You’ve just sequenced a piece of DNA.
