Download Different Effects of PCR Inhibitors on Multiplex STR Assays

Different Effects of PCR Inhibitors on Multiplex STR Assays
Asilomar Conference
Dennis Y. Wang, Ph.D.*, Julio J. Mulero, Ph.D., and Lori K. Hennessy, Ph.D. Human Identification Group, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404
ABSTRACT
Figure 2. Low pH can affect PCR amplification
The effects of six common PCR inhibitors on the AmpFℓSTR® Identifiler® and AmpFℓSTR®
MiniFilerTM PCR Amplification Kits were investigated. For each PCR amplification kit, the impact on
changes in buffer acidity was first thoroughly evaluated, followed by determining the threshold
inhibitory concentration (concentration where allele dropout was first observed) for each PCR inhibitor.
In all studies, it was found that AmpFℓSTR® MiniFilerTM PCR Amplification Kit consistently
outperformed AmpFℓSTR® Identifiler® PCR Amplification Kit in the presence of PCR inhibitors.
Experiments were also conducted to identify methods for improving the quality of STR profiles under
inhibited PCR conditions.
1.75 mM Ca
50 ng/ul Collagen
= 0.0035N
= 0.0045N
Figure 5. In AmpFℓSTR® MiniFilerTM, low pH caused incomplete +A
addition, resulting in split-peak morphologies in FGA, D2, D7, and CSF
CSF
D2
D7
FGA
No acetic acid added
0.003N acetic acid
INTRODUCTION
In forensic human identification, DNA samples recovered from crime scenes are often commingled
with PCR inhibitors that present a great challenge for PCR amplification. Crime scene evidence such
as blood and semen which are often found on soil, sand, wood or leaf litter can contain substances
which co-extract with the perpetrator’s DNA and prevent PCR amplification. Textile dyes, leather and
wood from interior crime scenes can also contain inhibitors that interfere with the DNA polymerase’s
activity. The impact of these contaminants on the multiplex STR assays can vary from attenuation to
complete inhibition of the amplification process, resulting in partial STR profiles or profiles with unusual
peak morphology.
In the present study, a systematic approach was utilized to evaluate the effect of six PCR inhibitors
commonly found in forensic samples on two different multiplex PCR assays. Each multiplex PCR
assay has unique primer sequences and PCR buffer formulation. The six common PCR inhibitors
used in this study were:
● Hematin1 - the main oxygen carrier in blood.
● Indigo2 - the most common dye from denim.
● Melanin3 - a pigment present in skin and hair.
● Humic acid4 - the polyphenolic compounds found in soil and water samples.
● Collagen5 - a connective protein making up 90% of the organic fraction of bones.
● Calcium6 - another major component of bone samples.
The effects of these inhibitors were evaluated with the AmpFℓSTR® Identifiler® and AmpFℓSTR®
MiniFilerTM PCR Amplification Kits. For each multiplex PCR reaction, a range of inhibitor
concentrations was included during PCR amplification. The amplification results were evaluated based
on: 1) the ability of each multiplex PCR assay to generate full STR profiles, and 2) the quality of the
STR profiles obtained.
The results clearly illustrated an increase in tolerance to PCR inhibitors in the AmpFℓSTR®
MiniFilerTM PCR Amplification Kit, attributable to the robustness of the new PCR buffer formulation,
optimized PCR primer sequences and thermal cycling parameters. Furthermore, our investigation
revealed that the split peak tendency for certain loci in AmpFℓSTR® MiniFilerTM PCR Amplification Kit
under acidic PCR condition can be corrected by increasing the length of the final PCR extension step.
The performances of AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM in the presence of
increasing concentration of HCl or acetic acid were investigated. With AmpFℓSTR® Identifiler®, PCR
inhibition was observed when HCl and acetic acid is greater than 0.0035N and 0.0045N, respectively.
With AmpFℓSTR® MiniFilerTM, PCR inhibition was observed when HCl is greater than 0.0040N and full
STR profiles was generated even in the presence of 0.0055N acetic acid. The highest concentration
of the calcium and collagen used in this study are indicated on the respective graphs.
Figure 3. Effect of each PCR inhibitors on AmpFℓSTR® Identifiler® and
AmpFℓSTR® MiniFilerTM
0.004N acetic acid
0.005N acetic acid
Split-peak morphology in four STR loci was observed in AmpFℓSTR® MiniFilerTM under low pH PCR
condition. The degree of the split-peak morphology worsens with increasing concentration of acetic
acid. The remaining five STR loci were not affected.
Figure 6. Incomplete +A addition in AmpFℓSTR® MiniFilerTM under acidic
condition can be corrected by increasing the final PCR extension time
45 min
60 min
75 min
90 min
MATERIALS AND METHODS
PCR Inhibitor Preparation7
Stock solutions of 100 mM hematin and calcium were prepared in 0.1N NaOH and 0.1N HCl,
respectively. A 100 mM stock solution of indigo was prepared by dissolving the indigo powder in 0.2%
Triton X-100 in ddH2O. Stock solutions of 1 mg/ml melanin and collagen were prepared in 0.5N NaOH
and 0.1N acetic acid, respectively. Lastly, 1 mg/ml of humic acid was prepared in ddH2O. Dilution of
stock inhibitor solutions were made using ddH2O.
PCR Amplification, Data Collection and Data Analysis
PCR amplification was performed according to the protocols provided by the manufacturer. The
DNA input, PCR cycling condition and number of PCR cycles are shown in the table below.
The performances of AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM in the presence of
increasing concentration of PCR inhibitors were investigated. With all six PCR inhibitors tested,
AmpFℓSTR® MiniFilerTM consistently outperformed AmpFℓSTR® Identifiler® in overcoming PCR
inhibition. The threshold inhibitory concentration for each PCR inhibitor is listed in Table 1.
Table 1. Threshold inhibitory concentration of each PCR inhibitor for
AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM
Threshold Inhibitory Concentration =
the lowest concentration of inhibitor that
caused an allele dropout from at least one
locus from three replicate measurements
Data collection was performed on a 3130xl Genetic Analyzer and all collected data were analyzed
using GeneMapper® ID Software version 3.2.
RESULTS
150 uM Hematin
6 ug/ul Melanin
= 0.0003N
= 0.003N
CONCLUSIONS
The results of this study clearly demonstrated that different multiplex PCR assays can be
differentially affected by the presence of various PCR inhibitors. AmpFℓSTR® MiniFilerTM PCR
Amplification Kit, with enhanced PCR buffer formulation, optimized PCR primer sequences and
improved thermal cycling parameters, noticeably outperformed AmpFℓSTR® Identifiler® under all
inhibited PCR conditions.
Inhibition of multiplex PCR assays can result in reduced product yield, complete failure, or unusual
peak morphology (due to incomplete +A addition). During our investigation, we noticed the tendency
for certain loci in AmpFℓSTR® MiniFilerTM to exhibit split-peak morphology under low pH PCR
conditions. Subsequent analysis revealed that the split-peak morphology can be corrected by
increasing the length of the final PCR extension step, without affecting other STR loci.
REFERENCES
AmpFℓSTR®
Figure 1. High pH can affect PCR amplification
In the presence of 0.004N acetic acid, incomplete +A addition was observed in AmpFℓSTR®
MiniFilerTM PCR reactions. Significant reduction in incomplete +A addition was observed by
increasing the final PCR extension time from 45 minutes at 60°C to 60 minutes. Further increase
in final PCR extension time to 90 minutes effectively completed the +A addition.
Identifiler® amplification
Figure 4. Missing profiles in an inhibited
can be recovered by AmpFℓSTR® MiniFilerTM at the same inhibitor concentration
AmpFℓSTR®
Identifiler®
Missing D21, D7, CSF, D16, D2, D18, FGA
1.
2.
3.
4.
5.
6.
7.
Akane, A et al. (1994) J. Forensic Sci. 39, 362-72.
Shutler, G.G. et al. (1999) J. Forensic Sci. 44, 623-6.
Eckhart, L. et al. (2000) Biochem. Biophys. Res. Comm. 271, 726-30.
Tsai, Y.L. and Olson, B.H. (1992) Appl. Environ. Microbiol. 58, 2292-5.
Kim, C.H. et al. (2001) J. Dairy Sci. 84, 74-83.
Powell, H.A. et al. (1994) Lett. Appl. Microbiol. 18, 59-61.
Chung, D.T. (2004) Ph.D. Thesis, Ohio University.
ACKNOWLEDGEMENTS
AmpFℓSTR®
MiniFilerTM
The performances of AmpFℓSTR® Identifiler® and AmpFℓSTR® MiniFilerTM in the presence of
increasing concentration of NaOH were investigated. With AmpFℓSTR® Identifiler®, PCR inhibition
was observed when NaOH is greater than 0.003N. On the contrary, full STR profiles were generated
by AmpFℓSTR® MiniFilerTM even in the presence of 0.0055N NaOH. Hematin and melanin were
prepared using NaOH solution and the highest concentrations of the two inhibitors used in this study
are indicated on the graph.
CSF
D21
D18
D16
D2
D7
FGA
In the presence of 1.25 mM of calcium, several loci in AmpFℓSTR® Identifiler® failed to amplify
whereas AmpFℓSTR® MiniFilerTM generated a full STR profile. The STR profile generated by
AmpFℓSTR® MiniFilerTM was able to recover all the missing alleles that AmpFℓSTR® Identifiler® failed
to amplify.
The authors wish to acknowledge the Human Identification Group for helpful discussions during the
investigation.
© 2008 Applied Biosystems