Template Considerations for Extracted DNA
1. Use normal laboratory procedures to isolate DNA from your sample source. Assess quality and quantity of
extracted DNA to ensure there is sufficient template for PCR.
2. The 260/280 absorbance ratio should be greater than 1.80.
3. To expedite PCR setup, adjust the working template concentration to be approximately 10 ng/µL. Dilute the
template DNA in UV-treated molecular biology grade water or Low TE (10 mM TRIS pH 8; 0.1mM EDTA), when
required.
4. The amount of DNA added to an MX PCR pre-amplification assay should be at least 150 ng, for the detection
of low level mutations (≥0.01%). However, this may not always be possible. The DNA can still be added to
the MX PCR, but note the sensitivity for detecting mutations in the starting material is directly correlated to
genomic equivalents present in this sample. For most downstream sequence analysis platforms, there should be
approximately 5-10 mutant template copies present in the starting material for reproducible mutation detection.
5. The amount of water added to the pre-amplification Master Mix can be adjusted to account for variability in the
amount of input DNA used for the MX PCR portion of the assay. Note: If DNA contains contaminants (ethanol
carryover, EDTA etc.), increasing DNA volume may decrease MX PCR efficiency.
6. The formalin-fixation process used in preparing FFPE tumor biopsy samples may result in deamination of
cytosines. This deamination converts cytosine to uracil that becomes thymine in subsequent PCR reactions. There
are FFPE DNA isolation kits that use Uracil N-Glycosylase (UNG) treatment to break the DNA at uracils prior to
final isolation of DNA. Use of such a DNA isolation kit greatly reduces the potential for deaminated cytosines to
result in GC to AT transition mutations. Any residual uracils following UNG treatment would be exceedingly rare
events, but if copied early in the MX PCR cycling, they will “look” like mutations. These type of mutations do not
repeat upon repeating the MX PCR analysis, therefore independent, replicate analysis is recommended.
Mutations in Starting Material
1600
1500
1515
Intial Mutations (%)
0.01%
1%
1400
1200
0.10%
1000
0.001%
Column labels indicate the number of mutations
present at the initial mutation percentage
1000
800
600
455
400
200
0
303
152
15 2
500 ng
151,515
100
10 1
330 ng
100,000
152
45 5 0
150 ng
45,455
30 3 0
15 2 0
100 ng
30,303
50 ng
15,152
100
10 2 0
33 ng
10,000
30 3 0 0
10 ng
3,030
Starting DNA (ng)
Genomic Equivalents
Transgenomic®
Advancing Personalized Medicine
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Transgenomic is a registered trademark; ICEme, the helix and MX-ICP logos are trademarks of Transgenomic, Inc.
All other trademarks are trademarks of their respective holders.
©2015 Transgenomic, Inc. | All rights reserved | Document No. 602441-00 | 06.27.15
Template Considerations
1
MX-ICP