Sanger sequencing stands as a foundational method for determining the precise order of nucleotides within a DNA molecule. This technique has been instrumental in advancing our understanding of genetics. A fundamental step involves the accurate separation of DNA fragments according to their size. This separation is paramount for resolving DNA sequences and is achieved through a specialized gel material.
The Specific Gel Material
Polyacrylamide gel is the specific material used to separate DNA fragments in Sanger sequencing. It forms from a polymer of acrylamide and N,N’-methylenebisacrylamide. Acrylamide monomers link together to form long chains, cross-linked by bis-acrylamide into a dense, three-dimensional mesh. This network forms a matrix with uniform, tunable pores.
Polyacrylamide is chosen for its ability to create exceptionally fine and consistent pores. Unlike agarose gels, polyacrylamide gels can separate DNA fragments that differ in length by just a single nucleotide. This precision is necessary for accurately reading a DNA sequence. The concentration of these components can be adjusted to optimize pore size for the range of DNA fragment sizes being analyzed.
Mechanism of Separation
DNA fragments separate in polyacrylamide gel through gel electrophoresis. An electric field is applied, causing negatively charged DNA molecules to migrate towards the positive electrode. As DNA moves through the dense polymer network, smaller fragments navigate the pores more easily and quickly than larger ones. This differential migration rate separates fragments by size. The gel’s uniform pore size ensures that even fragments differing by a single base pair migrate at observably different speeds, leading to their distinct separation.
The Gel’s Role in Reading DNA
The precise separation achieved by the polyacrylamide gel is fundamental to interpreting the DNA sequence in Sanger sequencing. In this method, DNA synthesis reactions produce a series of fragments, each terminated at a specific nucleotide by modified dideoxynucleotides. These fragments, ranging in length from a few to hundreds or thousands of bases, effectively form a “ladder” of DNA molecules. When these fragments are separated by the polyacrylamide gel, they arrange themselves by size, with the smallest fragments at the bottom and progressively larger ones above them.
To make these separated fragments visible and allow for sequence determination, they are typically labeled with either radioactive isotopes or fluorescent dyes. In modern automated Sanger sequencing, each of the four dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP) is tagged with a unique fluorescent color. As the fragments migrate through the gel and pass a detection point, a laser excites these fluorescent tags, and a detector records the color emitted. The order of colors detected as fragments pass by reveals the sequence of nucleotides, effectively “reading” the DNA sequence from the smallest to the largest fragment. This precise resolution by the gel is what translates the physical separation into a decipherable genetic code.