There are two common methods in which to construct a DNA molecular-weight size marker. One such method employs the technique of partial ligation. DNA ligation is the process by which linear DNA pieces are connected to each other via covalent bonds; more specifically, these bonds are phosphodiester bonds. Here, a 100bp duplex DNA piece is partially ligated. The consequence of this is that dimers of 200bp, trimers of 300bp, tetramers of 400bp, pentamers of 500bp, etc. will form. Additionally, a portion of the 100bp dsDNA will remain. As a result, a DNA "ladder" composed of DNA pieces of known molecular mass is created on the gel.
The second method employs the use of restriction enzymes and a recognized DNA sequence. The DNA is digested by a particular restriction enzyme, resulting in DNA pieces of varying molecular masses. One of the advantages of this method is that more marker can readily be created simply by digesting more of the known DNA. On the other hand, the size of the DNA pieces are based on the sites where the restriction enzyme cuts. This makes it more difficult to control the size of the fragments in the marker.
More recently, another method for constructing DNA molecular-weight size markers is being employed by laboratories. This strategy involves the use of Polymerase Chain Reaction (PCR). This is achieved one or two ways: 1) a DNA target is amplified at the same time via primer sets, or 2) different DNA targets are amplified independently via particular primers.