A size standard is a mixture of DNA fragments with known fragment lengths. It is used to calibrate
the fragments of samples where the fragment lengths are unknown.
Prerequisites for the calibration process
The size standard calibration process requires the following information:
1. the peak locations in the size standard channel/lane (a peak detection prior to the calibration
2. knowledge of the base sizes of each fragment.
Although each electrophoresis run is unique it would be very time consuming to assign the bands
in the size standard lane for each run manually. Therefore, a size standard template is defined
and can be applied to any sample containing this size standard.
Size standard templates
A size standard template contains the mobility and the base size for each peak of an exemplary
electrophoresis run. Of course, a 'good' run will be used to define a size standard template.
Table 1 shows how a size standard template for the 'ABI GeneScan 50-500' size standard could
look like. This size standard contains 16 fragments. The fragment with a length of 248 bases
often runs atypically and, therefore, is usually not included into the size standard template.
Size standard calibration process
The size standard calibration process applies a size standard template to the detected peaks in a
size standard lane of a sample. In fact the mobility data of both data sets are compared and the
best path using some kind of error function is found. Then the base sizes are assigned to the
peaks in the sample.
The task is to assign the right base size labels to these peaks using the values of Tab.1.
Figs. 2 and 3 show two trace curves before and after applying a size standard template.
Tab. 1. Peak data of a size standard
template (ABI GeneScan 35-500).
Each row contains a data point value
(the midpoint of a peak) and its
corresponding base size.
Tab. 2. Data point values of peaks
as detected in the size standard
channel of a sample run.
Comparison between the peak
mobilities in the size standard
channel of a sample and the pre-defined mobilities of a size standard
template. The values were taken
from Tab. 1. (ABI GeneScan 35-500
size standard). The green dots
indicate the optimal path through the
two data sets.
Fig. 2. This image shows two
sample runs with the same size
The data is scaled by data points,
i.e. according to the temporal
appearance. In the second sample,
the sample peaks appear earlier and
in a shorter time interval. The whole
sample appears 'compressed' if
compared with the first one.
Fig. 3. The same data as in Fig. 2
but after a calibration using a size
Each peak obtained a base size
label and the samples can now be
scaled by base sizes, i.e. the real