Incorporating controls in STR DNA analysis is an essential and required part of the testing process.08, 09 Introducing controls at each step of the DNA process allows the analyst to identify and troubleshoot possible issues and ensure that the methods used produce accurate and reliable results.
An extraction blank is included in the extraction process to assist the analyst in determining if the reagents and/or techniques used may have introduced contamination. The extraction blank should be treated in exactly the same manner as the other samples.
During the amplification process two controls are used:
- A positive control is a known DNA sample that has been previously typed and is added to the sample set. The positive control verifies that the analysis processes are functioning properly. (Manufacturers provide positive control samples in STR kits)
- A negative control is included in the amplification process to assist the analyst in determining if the reagents and/or techniques used may have introduced contamination. The negative control should be treated in exactly the same manner as the other samples.
Samples are typed using capillary electrophoresis. Internal sizing standards (ISS) must be added to each sample.
Click here to read more about capillary electrophoresis in Subject 05, Module 02.
Internal size standards serve two purposes:
- Sizes of all fragments are established for a sample (relative fragment units). This is accomplished by using the ISS to establish a correlation coefficient, which is used to determine the size of sample fragments.
- The ISS serves as an effective control and provides information about instrument run conditions. For instance, if all of the peaks for the sizing standard are not present, it suggests a temperature, run time, or injection problem.
Laboratory temperatures can vary and fluctuations occurring during a run can affect electrophoresis. Including allelic ladders with each run corrects for this issue. Allelic ladders must be included in all instrument runs for the instrument’s software to properly type the sized fragments for each sample.
If a laboratory experiences relatively severe temperature variation within a run, it may be advisable to run several allelic ladders so that the run can be broken into smaller sizing projects.
< Previous Page :: Next Page >