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Commercial kits for short tandem repeats (STR) typing include allelic ladders and internal size standards (ISS) that are added to each sample.
Read more about Capillary Electrophoresis in course: Amplified DNA Product Separation.
Each sample from a run or gel should be assessed to determine if the ISS peaks have been called correctly. In general, the peaks or bands from an ISS are uniform in size or intensity. Lack of uniformity or miscalled peaks can indicate problems with the sample, injection, and/or run.
Above can be seen an example of a good internal size standard. Compare this to a miscalled ISS, below. Note that the first peak has not been called, and normal peak calls have begun with the second peak from the left.
Below is an example of a bad injection, as evidenced by the imbalanced peaks on the ISS.
The ISS is particularly useful in determining the precision of a capillary electrophoresis run. For example, using Applied Biosystems software, the 250 peak is not assigned in the ISS and the size of this peak (in each sample) can be used to determine the in-run precision. Temperature fluctuations can cause the in-run precision to exceed 1 base pair and evaluation of the ISS can assist analysts in identifying this issue.
- Allelic ladders should be assessed to ensure that all peaks have been called correctly. Ladder peaks that have not been called or have been miscalled can indicate a problem with the ladder sample, injection, and/or run.
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