Article Type: Feature Article

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

Mary Acosta Loyo, Gerson Caraballo and Howard Takiff
CeSAAN Lab, Venezuelan Institute for Scientific Research (IVIC)

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 (<0.5 ng) 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).

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.

Genotype of cadaver bones obtained using the PowerPlex(R) 16 System.

Figure 2. Genotyping of bones from a cadaver buried for approximately 3 months using the conventional PowerPlex^® 16 System.

Click on the image to view a larger version.

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(R) 16 HS System

Figure 3. Genotyping of bones from a cadaver buried for approximately 3 months using the PowerPlex^® 16 HS System.

Genotype of cadaver bones buried for 50 years obtained using the PowerPlex(R) 16 HS System

Figure 4. Genotyping of bones recovered from people who disappeared approximately 50 years ago using the PowerPlex^® 16 HS System.

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.

Reference

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 Research 16, 1215.

How to Cite This Article

Scientific Style and Format, 7th edition, 2006

Acosta Loyo, M., Caraballo, G. and Takiff, H. (2009) Successful multiplex genotyping of severely degraded DNA extracted from bone samples using the PowerPlex^® 16 HS System. Profiles in DNA 12(2); [Internet] 2009. [cited: year, month, date]; Available from: www.promega.com/profiles/1202/1202_03.htm

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