How to generate your limit of detection and dilution curves



Author: Joe Whittaker
Source: Horizon Discovery
Category: Reference Standards

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 DNA100% 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 Frequency50% 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 Frequency50% 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 Frequency50% 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 Concentration50% Mutant (orange lid)Wild Type DNA (white 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


1. Westgard, J. O. 2008. Basic method validation, 3rd ed. Westgard QC, Inc., Madison, WI.
2. 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.

Back to top