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Published on: October 6, 2017
Polymerase resistance to polymerase chain reaction inhibitors in bone*
Kenneth D Eilert1, David R Foran
1Forensic Science Program, School of Criminal Justice, Michigan State University, East Lansing, MI 48824, USA.
Abstract:
Amplification of DNA from aged or degraded skeletal remains can be a challenging task, in part due to naturally occurring inhibitors of the polymerase chain reaction. PCR inhibitors may act by inactivating a polymerase itself, or compete with or bind other reaction components, although various polymerases may be differentially susceptible to such insult. In this study, ten thermostable polymerases from six bacterial species were examined for their ability to amplify DNA in the presence of bone-derived or individual PCR inhibitors. Two polymerases, one from Thermus aquaticus and one from Thermus thermophilus, showed lower susceptibility to inhibition from bone, while polymerases from Thermus flavus were highly susceptible. Addition of bovine serum albumin improved the activity of most of the enzymes. Taken together, the results indicate that thermostable DNA polymerases have different susceptibility to bone-derived PCR inhibitors, and that those most often used in forensic laboratories may not be optimal when working with DNA from skeletal remains.
Insights
Certain DNA polymerases are more resistant to inhibitors found in aged skeletal remains. This research tested ten polymerases, finding specific Thermus species enzymes performed better for DNA amplification from challenging samples.
Area of Science:
- Molecular Biology
- Forensic Science
- Biochemistry
Background:
- DNA amplification from aged or degraded skeletal remains is difficult due to PCR inhibitors.
- These inhibitors can inactivate polymerase or interfere with reaction components.
- Different polymerases exhibit varying susceptibility to these inhibitory substances.
Purpose of the Study:
- To evaluate the performance of ten thermostable DNA polymerases against bone-derived PCR inhibitors.
- To identify polymerases with enhanced resistance for improved DNA amplification from skeletal remains.
Main Methods:
- Tested ten thermostable polymerases from six bacterial species.
- Assessed DNA amplification in the presence of bone-derived and individual PCR inhibitors.
- Evaluated the effect of bovine serum albumin (BSA) on enzyme activity.
Main Results:
- Two polymerases, from Thermus aquaticus and Thermus thermophilus, showed reduced susceptibility to bone inhibitors.
- Polymerases from Thermus flavus were highly susceptible to inhibition.
- Bovine serum albumin improved the activity of most tested enzymes.
Conclusions:
- Thermostable DNA polymerases display differential susceptibility to bone-derived PCR inhibitors.
- Commonly used polymerases in forensic labs may not be optimal for skeletal remains DNA.
- Enzyme selection is critical for successful DNA amplification from challenging forensic samples.
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