Getting Rid of Contaminating DNA and the DNase Used to Destroy it
Because virtually all RNA samples have trace amounts of contaminating DNA, most protocols specify DNase treatment for RT-PCR applications. DNase I treatment is clearly the best way to rid an RNA sample of contaminating DNA. However, some preparations of DNase may be contaminated with RNases, and the DNase must be completely inactivated prior to RT-PCR so that it doesn't degrade newly synthesized DNA. Unfortunately, removal or inactivation of this enzyme is problematic; DNase removal methods can be inconvenient, ineffective and even detrimental to RNA integrity.
Commonly used methods for removal or inactivation of DNase after digestion include: heat inactivation, proteinase K treatment followed by phenol:chloroform extraction, chelation of essential ions with EDTA, and purification using a glass-filter binding method such as RNAqueous® (see the sidebar at right, "RNA Isolation for RT-PCR). Each of these inactivation or removal methods has its drawbacks.
Heat inactivation: Probably the most common method of DNase inactivation is heat treatment, typically for 5 minutes at 75°C. Although this method appears straightforward, the divalent cations in the DNase digestion buffer can cause (chemically-induced) strand scission of RNA when heated. Studies at Ambion have shown that much of an RNA sample is destroyed when heated to 80°C for 5 minutes in the presence of 2.5 mM MgCl2 and 0.1 mM CaCl2 (salts typically found in DNase I digestion buffer).
Proteinase K treatment and organic extraction: Proteinase K treatment followed by phenol:chloroform extraction is probably the most rigorous method for DNase inactivation and removal, but it is time-consuming, and organic extractions often cause some sample loss. Sample loss can be minimized by back extraction of the phenol:chloroform phase, but this adds another step to an already time-consuming procedure. Additionally, many people prefer to avoid working with hazardous phenol.
EDTA chelation of cations: The addition of EDTA to DNase digestion reactions chelates ions in the digestion buffer, that are required for DNase I activity. The DNase I can then be safely heat inactivated without loss of RNA. However, Mg2+ is needed for enzymatic activity of both the reverse transcriptase and the thermostable DNA polymerase. Thus the chelation capacity of the EDTA must be saturated with additional ions prior to subsequent enzymatic reactions. This can make the assembly of a simple reaction quite complicated.
RNA purification: Some filter-based RNA isolation methods treat with DNase directly on the filter after binding of the lysate. This treatment may not completely eliminate contaminating DNA because the DNase will not be in an optimal environment for digestion (traces of lysis solution and other contaminants may interfere with optimal digestion). Alternatively, RNA preparations that have been treated with DNase in solution, can be purified away from DNase over such columns. Although this technique adequately removes DNase from the prep, it requires both an extra step, and expensive materials.
Commonly used methods for removal or inactivation of DNase after digestion include: heat inactivation, proteinase K treatment followed by phenol:chloroform extraction, chelation of essential ions with EDTA, and purification using a glass-filter binding method such as RNAqueous® (see the sidebar at right, "RNA Isolation for RT-PCR). Each of these inactivation or removal methods has its drawbacks.
Heat inactivation: Probably the most common method of DNase inactivation is heat treatment, typically for 5 minutes at 75°C. Although this method appears straightforward, the divalent cations in the DNase digestion buffer can cause (chemically-induced) strand scission of RNA when heated. Studies at Ambion have shown that much of an RNA sample is destroyed when heated to 80°C for 5 minutes in the presence of 2.5 mM MgCl2 and 0.1 mM CaCl2 (salts typically found in DNase I digestion buffer).
Proteinase K treatment and organic extraction: Proteinase K treatment followed by phenol:chloroform extraction is probably the most rigorous method for DNase inactivation and removal, but it is time-consuming, and organic extractions often cause some sample loss. Sample loss can be minimized by back extraction of the phenol:chloroform phase, but this adds another step to an already time-consuming procedure. Additionally, many people prefer to avoid working with hazardous phenol.
EDTA chelation of cations: The addition of EDTA to DNase digestion reactions chelates ions in the digestion buffer, that are required for DNase I activity. The DNase I can then be safely heat inactivated without loss of RNA. However, Mg2+ is needed for enzymatic activity of both the reverse transcriptase and the thermostable DNA polymerase. Thus the chelation capacity of the EDTA must be saturated with additional ions prior to subsequent enzymatic reactions. This can make the assembly of a simple reaction quite complicated.
RNA purification: Some filter-based RNA isolation methods treat with DNase directly on the filter after binding of the lysate. This treatment may not completely eliminate contaminating DNA because the DNase will not be in an optimal environment for digestion (traces of lysis solution and other contaminants may interfere with optimal digestion). Alternatively, RNA preparations that have been treated with DNase in solution, can be purified away from DNase over such columns. Although this technique adequately removes DNase from the prep, it requires both an extra step, and expensive materials.
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