Standard curve
Based on the discussion in the previous section, you should understand that when we run the restriction digests from our PCR amplification on a gel, the smaller fragments will separate from the larger ones because the smaller fragments will move faster through the gel matrix. This alone may be enough to allow us to determine the genotypes of our samples. However, it is also possible to obtain a quantitative estimate of the mass of a fragment by measuring the distance it traveled and comparing it with the distances traveled by fragments of known lengths, using a method called a standard curve.
A standard curve is necessary when there is a linear (or other predictable) relationship between a desired quantity and a measurable characteristic, but when the measurable characteristic varies from trial to trial. A standard curve is used to establish the exact relationship for a particular trial. For example, although bands in a gel move in a predictable way, their exact position at the end of the gel run depends on the voltage at which you run the gel and when you turn the power off. So the particular relationship varies from gel to gel and must be established by comparing known masses of bands with the distance they traveled from the origin.
Molecular mass standards
For this reason, it is standard practice to include molecular mass standards (also known as size standards or a DNA ladder) as one lane on a gel. Size standards are a mixture of DNA fragments that have masses spanning a certain range. Frequently the fragments are produced by digestion of a large molecule with restriction enzymes.
Fig. 3. Mass standards used in our experiments (Invitrogen 1 kb Plus DNA Ladder No. 10787-018).
In our experiments, we will use a commercially available set of mass standards. Fig. 3 shows the banding pattern that is produced when the mass standards are run on an agarose gel. Notice that the 2000 and 1650 bands are closer to each other than the other bands. You can use this trait to orient the relative positions of the other bands. The masses of the heavier bands are not labeled on this image but are at intervals of 1000 kb. The complete fragment list is: 100, 200, 300, 400, 500, 650, 850, 1000, 1650, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000.
Creating a standard curve
Fig. 4. Relationship between distance traveled on a gel and fragment mass (note: this example is an SDS-PAGE protein gel)
It is known that the distance traveled by a linear DNA fragment is nearly linearly proportional to the log of its molecular mass. By plotting the logarithm of molecular mass vs. distance from the origin to the band, the relationship between these two quantities can be established (Fig. 4). The equation describing this relationship (derived from a linear regression) can then be used to estimate the masses of unknown fragments. Because the masses of DNA's four nucleotides are similar, basepairs (bp) can be used as a unit of mass. It does not matter whether natural log (ln) or base 10 log (log10) is used.
