DNA sequencing involves determining the precise order of nucleotides (A, T, C, G) within a DNA molecule. This process unlocks genetic information, fundamental to understanding biological functions and disease. Early methods used gel electrophoresis, a technique that visually separates DNA fragments, to decipher this sequence.
Visualizing the DNA Sequence on a Gel
A DNA sequencing gel is a long, thin slab, typically polyacrylamide or agarose, with multiple vertical lanes, each corresponding to one of the four nucleotide bases (A, T, C, G). DNA fragments, differing in length by a single nucleotide, separate by size as they migrate through the gel when an electric current is applied. Shorter fragments move faster, appearing closer to the bottom, while longer fragments remain higher up. To make these invisible DNA fragments detectable, they are tagged with radioactive labels (visualized on X-ray film) or fluorescent dyes (emitting light when excited by a laser). This labeling makes each separated DNA fragment appear as a distinct band.
Deciphering the Bands: The Underlying Logic
The distinct bands on the gel result from chain-termination sequencing, also known as Sanger sequencing. This method uses modified nucleotides called dideoxynucleotides (ddNTPs) to stop DNA synthesis. Unlike regular nucleotides, ddNTPs lack a hydroxyl group needed for extending the DNA chain. When a ddNTP is incorporated, DNA synthesis immediately terminates at that point.
In the sequencing reaction, four separate tubes are prepared. Each tube contains DNA polymerase, a DNA template, standard nucleotides, and a small amount of one specific ddNTP (ddATP, ddTTP, ddCTP, or ddGTP). This setup ensures DNA synthesis terminates randomly at every position where that particular base appears. For example, in the ddATP tube, DNA fragments are generated, each ending at an adenine nucleotide. When run on the gel, all fragments in the ‘A’ lane will share adenine as their terminal base.
Reading the Sequence: A Step-by-Step Guide
Reading a DNA sequence from a gel begins at the bottom, where the smallest DNA fragments have migrated fastest. Each band represents a DNA fragment that ended at a specific nucleotide, corresponding to its lane. To determine the sequence, one systematically moves upwards from the lowest band, identifying which lane each successive band appears in. For example, if the lowest band is in the ‘C’ lane, the first base is Cytosine.
Moving up the gel, the next band indicates the subsequent nucleotide. For instance, if the band directly above the ‘C’ is in the ‘G’ lane, the second base is Guanine. This process continues, identifying the base for each consecutive band as you ascend. The complete sequence is then read directly from bottom to top, compiling the bases in their order of appearance. The final sequence represents the complementary strand to the original template DNA.
Beyond the Gel: Why Methods Evolved
While revolutionary, manual gel electrophoresis for DNA sequencing had limitations that prompted more advanced methods. The technique was labor-intensive and time-consuming, requiring meticulous preparation and analysis. Manually pouring, running, and reading gels was impractical for high-throughput applications like sequencing large numbers of samples or entire genomes. Additionally, read lengths from a single gel were short, typically a few hundred bases at most.
These inefficiencies led to the evolution of sequencing technologies. Early advancements included automated capillary electrophoresis, which still used the Sanger method but automated fragment separation and detection. Later, next-generation sequencing (NGS) technologies enabled massively parallel sequencing. These newer methods eliminated gels, increasing sequencing speed, reducing costs, and expanding read lengths and throughput significantly.