Attaining High DNA Yield from Sperm Cells using Proteinase K

February 27, 2018 Categories AG Scientific Blog, AG Scientific Products, Proteomics
proteinase k

Attaining High DNA Yield from Sperm Cells using Proteinase K

There are many techniques that can be used to isolate high quality and high molecular weight DNA from mammalian somatic cells. However, these techniques are ineffective for mammalian sperm. Unlike somatic cells, nearly all histones in sperm cells are replaced by protamines held together by disulfide bonds, which compacts the sperm nucleus, rendering it resistant to conventional lysis procedures.

Sperm DNA Extraction

It has been a gradual process for the development of efficient methods for isolating DNA from mammalian sperm. Bahnak et al. reported a protocol using guanidine thiocyanate in a lysis buffer made with sodium citrate, sodium lauroyl sarcosinate (Sarkosyl), and β- mercaptoethanol (reducing agent) to isolate high quality mammalian sperm DNA. The DNA extracted was then successfully visualized in Southern blot analysis. The procedure required lengthy steps such as CsCl ultracentrifugation for 20 hours and dialyzing the banded DNA for 24 hours against Tris-HCl and ethylene diaminetetra acetic acid (EDTA).

Proteinase K Shortened Procedure Time

The inclusion of proteinase K in subsequent methods to enhance the activity of chaotropic agents such as guanidine thiocyanate by digesting nucleoproteins eliminated the need for lengthy ultracentrifugation and dialyzing steps. In a method developed by Pacheco et al., sperm pellets were lysed for 16 hours in a solution containing Tris-HCl, dithiothreitol (DTT; another commonly used reducing agent), sodium chloride, EDTA, sodium dodecyl sulfate (SDS), proteinase K, and β-mercaptoethanol. DNA was then extracted using a phenol/chloroform protocol, and DNA was ethanol precipitated.
 
While the authors were able to extract DNA used for subsequent DNA methylation analyses, the method still required at least an overnight incubation. The method also used harmful organic solvents (phenol and chloroform) that are undesirable for simple laboratory procedures. A method by Hossain et al. was one of the first to eliminate these overnight procedures from the sperm DNA extraction protocol.

Proteinase K

Proteinase K Eliminated Overnight Procedures

Hossain et al. modified the original protocol by Bahnak et al. for the preparation of human sperm DNA by including proteinase K in the lysis buffer (containing guanidinium thiocyanate) to digest nucleoproteins, and isopropanol to precipitate DNA. This modification eliminated the need to mechanically homogenize the cells, the use organic solvents for extraction, and the use ultracentrifugation for DNA precipitation. The degradation of DNA through mechanical homogenization and organic solvents was minimized, and the overall procedure could be performed in ordinary laboratory facilities in a reduced amount of time with only an incubation period of 3 hours for lysis needed. However, incomplete protein digestion and removal of chaotropic salts persisted limited the quality of the DNA yield.

Griffin’s DNA Extraction Method

Modifications were made to Hossain’s protocol by Griffin to increase the quality and yield of mammalian sperm DNA by eliminating incomplete protein digestion and removal of chaotropic salts that may coprecipitate with DNA. Descriptions of the lysis and extraction components as well as the steps of and modifications to Hossain’s protocol were given in detail.

Guanidine thiocynate Enhances Proteinase K Activity

Guanidine thiocyanate is one variety of chaotropic agent employed during DNA extractions. Its functions include:
 

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