Author: Joe Whittaker
Definition: Limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value (3xSD).
When validating molecular tests, the number of data points (and therefore confidence) may be limited by sample availability and budget concerns. Often in literature, 20 measurements at, above, and below the probable LOD as determined by preliminary dilution studies is recommended to establish the analytical sensitivity of an analyte.1,2
To establish your LOD, HDx™ Reference Standards can be used to determine the sensitivity and specificity of your assay. Available as mutant DNA and matched Wild Type DNA, precise dilutions can be performed to create your desired allelic frequencies.
If your assay requires a different concentration to that supplied in HDx Reference Standards (50ng/µl), please view the concentration dilution table.
To generate your LOD dilution curve, you will require the following:
| 50% Mutant DNA | 100% Matched Wild Type DNA | ||||
|---|---|---|---|---|---|
|
Total DNA Amount |
1µg |
Total DNA Amount |
5µg |
||
|
Concentration |
50ng/µl |
Concentration |
50ng/µl |
||
|
Volume |
20µl |
Volume |
10µl |
||
|
Allelic Frequency |
50% |
Allelic Frequency |
100% |
||
Next Generation Sequencing
The following table is an example dilution table, assuming a theoretical LOD ranging between 1-10%.
| Target Allelic Frequency | 50% Mutant DNA (orange lid) | Wild Type DNA (white lid) |
|---|---|---|
|
20% |
4µl |
6µl |
|
10% |
2µl |
8µl |
|
5% |
1µl |
9µl |
|
1% |
1µl of prepared 10% dilution |
9µl |
|
0% |
0µl |
10µl |
|
Total Volume |
7µl |
42µl |
Eg. For a 10% allelic frequency - take 2µl of 50% mutant DNA (orange lid) and add to 8µl of 100% Wild Type (white lid)
qPCR Sequencing
The following table is an example dilution table, assuming a theoretical LOD ranging between 1-2%.
| Target Allelic Frequency | 50% Mutant DNA (orange lid) | Wild Type DNA (white lid) |
|---|---|---|
|
10% |
4µl |
16µl |
|
5% |
1µl |
9µl |
|
2% |
1µl of prepared 10% dilution |
4µl |
|
1% |
1µl of prepared 10% dilution |
9µl |
|
0% |
0µl |
10µl |
|
Total Volume |
5µl |
48µl |
eg. For a 5% allelic frequency - take 1µl of 50% mutant DNA (orange lid) and add to 9µl of 100% Wild Type (white lid)
Sanger Sequencing
The following table is an example dilution table, assuming a theoretical LOD ranging between 15-25%.
| Target Allelic Frequency | 50% Mutant DNA (orange lid) | Wild Type DNA (white lid) |
|---|---|---|
|
50% |
10µl |
0µl |
|
20% |
4µl |
6µl |
|
10% |
2µl |
8µl |
|
0% |
0µl |
10µl |
|
Total Volume |
16µl |
24µl |
Eg. For a 20% allelic frequency - take 4µl of 50% mutant DNA (orange lid) and add 6µl of 100% Wild Type (white lid)
We provide Genomic DNA HDx Reference Standards at a concentration of 50ng/µl (1µg).
DNA Concentration Dilution Table
For use with Sanger Sequencing, qPCR and NGS applications, a concentration of between 5-50ng/µl is required. The provided 50ng/µl DNA can be diluted with Tris-EDTA (TE) buffer (Tris-HCl 10mM, 1mM EDTA, pH 8.0) to produce a more dilute DNA concentration.
| Target DNA Concentration | 50% Mutant (orange lid) | TE Buffer | Total Volume |
|---|---|---|---|
|
5ng/µl |
2µl |
18µl |
20µl |
|
10ng/µl |
4µl |
16µl |
20µl |
|
25ng/µl |
10µl |
10µl |
20µl |
Eg. For a 10ng/µl - take 4µl of Mutant DNA (orange lid) and add 16µl of TE Buffer
References
- Westgard, J. O. 2008. Basic method validation, 3rd ed. Westgard QC, Inc., Madison, WI.
- CLSI/NCCLS. 2004. Protocols for determination for limits of detection and limits of quantitation. Approved guideline. CLSI document EP17-A. Clinical and Laboratory Standards Institute, Wayne, PA.