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Successful Multiplex Genotyping of Severely Degraded DNA Extracted From Bone Samples...

Successful Multiplex Genotyping of Severely Degraded DNA Extracted From Bone Samples Using the PowerPlex® 16 HS System

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Mary Acosta Loyo, Gerson Caraballo and Howard Takiff

CeSAAN Lab, Venezuelan Institute for Scientific Research (IVIC)

Publication Date: 2009

Introduction

Short tandem repeat (STR) marker multiplex kits are commonly used tools for parentage testing and forensic casework genotyping, but results can be unreliable when the DNA is insufficient (<0.5ng) or severely degraded or contains polymerase inhibitors. In March 2009, we tested a new multiplex kit manufactured by Promega, the PowerPlex ® 16 HS System, which includes an efficient hot-start Taq DNA polymerase that is resistant to common inhibitors and is capable of amplifying very small quantities of DNA. Using the new Promega kit, we were able to obtain successful results from some samples that could not be analyzed using other kits.

“The PowerPlex® 16 HS System can generate informative and interpretable results even when samples contain only small amounts of degraded DNA that could not analyzed with other kits.”

Materials and Methods

A total of 11 human bone samples, corresponding to three forensic cases, were pulverized in a micromill system. All bone fragments showed varying degrees of damage and degradation. Five were bones recovered from an airplane disaster (Figure 1), three were from exhumed remains thought to be from politically motivated disappearances approximately 50 years ago, and three were from cadavers buried for approximately 3 months. The DNA was extracted by a salting out method(1) and purified with an in-house-adapted protocol using MagneSil® Green (Promega). DNA concentrations were estimated by visualization in a 1% agarose gel. Amplified products were detected and separated by capillary electrophoresis on an Applied Biosystems 3130xl Genetic Analyzer. Fragment sizes were determined using the Genescan® Analysis Software, version 3.1, and allele designation using GeneMapper® ID software, version 3.1 (Applied Biosystems).

Photographs of bone samples.Figure 1. Bone samples recovered from an airplane disaster.

Results

In the first attempt, all samples were amplified using the conventional PowerPlex® 16 System, and additionally, some were amplified with the Y-filer® kit, but results could not be analyzed for any of the 11 samples (Figure 2 and data not shown). These samples then were processed using the new PowerPlex® 16 HS System.

Representative electropherograms for direct amplification of DNA from buccal and blood samples preserved on various card types.Figure 2. Genotyping of bones from a cadaver buried for approximately 3 months using the conventional PowerPlex® 16 System.

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For five of eleven samples, full profiles were obtained, and interpretable results were obtained for nearly half of the remaining samples. We even obtained partial but interpretable results from sample "A", which appeared to be completely carbonized (Figure 1). Representative results are shown in Figures 3 and 4.

Genotype of cadaver bones buried for 3 months obtained using the PowerPlex 16 HS System.Figure 3. Genotype of cadaver bones buried for 3 months obtained using the PowerPlex® 16 HS System.

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Genotype of cadaver bones buried for 50 years obtained using the PowerPlex 16 HS System.Figure 4. Genotyping of bones recovered from people who disappeared approximately 50 years ago using the PowerPlex® 16 HS System.

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Conclusion

The PowerPlex® 16 HS System System can generate informative and interpretable results even when samples contain only small amounts of degraded DNA not analyzable with other kits. It may prove to be a valuable tool for forensic casework.

References

  1. Miller, S.A., Dykes, D.D. and Polesky H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215.

How to Cite This Article

Loyo, M., Caraballo, G. and Takiff, H. Successful Multiplex Genotyping of Severely Degraded DNA Extracted From Bone Samples Using the PowerPlex® 16 HS System. [Internet] 2009. [cited: year, month, date]. Available from: http://france.promega.com/resources/profiles-in-dna/2009/successful-genotyping-of-dna-from-bone-using-the-powerplex-16-hs-system/

Loyo, M., Caraballo, G. and Takiff, H. Successful Multiplex Genotyping of Severely Degraded DNA Extracted From Bone Samples Using the PowerPlex® 16 HS System. Promega Corporation Web site. http://france.promega.com/resources/profiles-in-dna/2009/successful-genotyping-of-dna-from-bone-using-the-powerplex-16-hs-system/ Updated 2009. Accessed Month Day, Year.

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Figures

Photographs of bone samples.Figure 1. Bone samples recovered from an airplane disaster.
Representative electropherograms for direct amplification of DNA from buccal and blood samples preserved on various card types.Figure 2. Genotyping of bones from a cadaver buried for approximately 3 months using the conventional PowerPlex® 16 System.

/~/media/images/resources/figures/profiles in dna/1202/acostafigure02_large2.jpg?la=fr-FR
Genotype of cadaver bones buried for 3 months obtained using the PowerPlex 16 HS System.Figure 3. Genotype of cadaver bones buried for 3 months obtained using the PowerPlex® 16 HS System.

/~/media/images/resources/figures/profiles in dna/1202/acostafigure03_large2.jpg?la=fr-FR
Genotype of cadaver bones buried for 50 years obtained using the PowerPlex 16 HS System.Figure 4. Genotyping of bones recovered from people who disappeared approximately 50 years ago using the PowerPlex® 16 HS System.

/~/media/images/resources/figures/profiles in dna/1202/acostafigure04_large2.jpg?la=fr-FR

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