In Part 1, we discussed using trypsin on bone specimens as a cleaning alternative to sanding in preparation for DNA extraction. In this second part of the series, we cover the details of attaining a higher yield of DNA using proteinase K.
Proteinase K for Higher DNA Yield from Bone Specimens
DNA in bone is located in the osteocytes (Hochmeister et al., 1991). Frost (1961) and Martin et al (1989) estimated that there are 20,000 to 26,000 osteocytes per cubic millimeter of calcified bone matrix. Hochmeister et al (1991) estimated that microgram quantities of DNA could potentially be extracted from a gram of bone. Thus, bone tissue should have enough DNA for analysis. However, skeletal fragments found in mass fatality incidents often have serious decomposition changes and may affect both chromosomal and mitochondrial DNA. High humidity and temperatures are factors that affect the degradation rate of DNA (Perry et al, 1988). Failure to obtain a DNA profile and identification of partial DNA profiles were reported in the World Trade Center cases (Shaler, 2002; Prinz, 2002).
Extract Trapped DNA with Proteinase K
Osteocytes are in a calcified matrix, which acts as a barrier to accessing the DNA when extracting. Thus, removing the matrix may improve the yield of DNA. The application of proteinase K by a number of groups is an approach to digesting the matrix barrier (Perry et al, 1988; Cattaneo et al, 1995 and 1997; Hochmeister et al, 1991). However, limited knowledge of the effect of proteinase K on the yield of DNA isolation from bone samples is available in literature.
Effect of proteinase K on DNA extraction yield remains to be characterized
To address the issue described above, we have conducted this study to characterize the effect of Proteinase K on the yield of DNA isolation from bone samples. It is known that the matrix protein network plays an important role in the structure of the bone. The matrix could be digested by a number of proteinases (Wang et al, 2001).
Proteinase K Helps Break DNA Barriers
It was hypothesized that proteinases would digest the matrix protein network by degrading the physical barrier around the osteocytes, thus facilitating DNA extraction. We are interested in identifying which proteinases can be used for digesting the matrix of bone tissue and optimizing proteinase treatment, particularly collagenases, which are known for playing a role in the degradation of the bone matrix proteins (Mallya et al, 1992).
Clostridiopeptidase A is one of the most potent collagenases and was used in a study by Li (2009) where they developed a modified DNA isolation method for bone tissue. The method increased the yield of isolated DNA. An increased yield of isolated DNA should provide more copies of DNA templates from bone samples. Therefore, it may increase the success rate of generating the genotype profiles for DNA analysis.
Image from (Li, 2009)
Proteinase K's ability to clean samples in a safer manner and increase DNA yield for analysis is all very important. Unfortunately, catastrophes and mass fatalities do occur, so when forensic specialists need to identify people through these means of bone sampling, at least scientists will have less hazardous working conditions and better chances of identifying loved ones who have been lost, by having more DNA material to work with. There is a solace in knowing the practice of science is able to do that for human and all life, especially when disastrous events happen. When they do, it's amazing to know scientists can help solve these big issues with the smallest tools.
- Li, R. (2009). Application of Proteinases for DNA Isolation of Bone Specimens.
- Proteinase K Solution (20 mg/mL) P-1266
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- Trypsin T-2763
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- Proteinase K Solution 20 mg/mL (Recombinant), Molecular/PCR Grade P-1264-SOL
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