Gel electrophoresis is a foundational method used in molecular biology to separate and analyze fragments of nucleic acids, such as DNA, based on their size. The standard unit of measurement used to describe the length of these fragments is the base pair, abbreviated as “bp.” This unit provides a universal language for reporting the size of the genetic material being studied.
Understanding the Base Pair Unit
A base pair (bp) is the fundamental unit of length for double-stranded DNA or RNA molecules. DNA exists as a double helix, where a base pair consists of two complementary nucleobases—adenine (A) paired with thymine (T), and guanine (G) paired with cytosine (C)—held together by hydrogen bonds. These paired bases form the “rungs” of the DNA molecule, and the number of base pairs directly corresponds to the physical length of the DNA sequence.
The designation “bp” is specifically used for double-stranded nucleic acids because it accounts for both strands of the helix. For single-stranded molecules, such as certain forms of RNA, the length is measured in bases (b) or nucleotides (nt), since there is no complementary pairing. A kilobase (kb or kbp) refers to one thousand base pairs, which is used to describe longer fragments.
How Gel Electrophoresis Separates DNA
Gel electrophoresis provides a separation mechanism that allows researchers to determine the size of a DNA fragment in base pairs. The process begins by placing DNA samples into small wells at one end of a porous gel matrix, typically made of agarose. Since the phosphate backbone gives DNA a strong negative charge, an electric current is applied across the gel, causing the fragments to migrate toward the positive electrode (anode).
The gel functions as a molecular sieve, impeding the movement of the DNA through its microscopic pores. Smaller DNA fragments, having fewer base pairs, navigate the pores more easily and quickly. Conversely, larger fragments encounter more resistance and move at a slower pace. This difference in migration speed separates the DNA mixture based solely on size, or length in base pairs.
The concentration of the agarose gel dictates the range of fragment sizes that can be optimally separated. After the electric current is run, the DNA fragments are visualized as distinct bands, with the shortest fragments migrating the farthest distance from the well. This arrangement allows scientists to assess the size distribution of the DNA in their sample.
Using Markers to Determine Fragment Length
The practical utility of the base pair unit is realized through the use of a standard known as a DNA ladder or molecular weight marker. This marker is loaded into one lane of the gel and is composed of DNA fragments with precisely known sizes, measured in base pairs. These fragments separate during the run, creating a visible “ladder” pattern of bands, each corresponding to a specific, predetermined length.
By running the unknown DNA sample alongside this DNA ladder, scientists compare the migration distance of the sample bands to the known bands of the marker. If an unknown band travels the same distance as a ladder band, it is assumed to have approximately the same length in base pairs. More precise size estimations are made by plotting the migration distance of the ladder fragments against their known base pair lengths to create a standard curve. The migration distance of any unknown fragment is then mapped onto this curve to accurately estimate its size in base pairs.