Double stranded DNA sequencing as a choice for DNA sequencing
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A step-by-step protocol for the alkaline denaturation of template DNA, and recommendations for the sequencing reaction.Abstract:
Double stranded DNA sequencing (1) is favored because of its simplicity, and convenice. However it is only recently that the quality of this method has become comparable with single stranded DNA sequencing. We believe that two factors have limited the popularity of double stranded DNA sequencing: 1, the quality of the template DNA; 2, the inherent propertv.,of the DNA polymerase. The modified T7 DNA polymerase (Sequenase ) has several properties which make it more suitable for sequencing (2). We replaced the Klenow Polymerase I with Sequenase in our double stranded DNA sequencing, and by paying careful attention to the conditions used to denature and recover the template DNA, we are now routinely producing sequencing results which are as good as single stranded DNA sequencing (see figure). Here, we present a step-by-step protocol for the alkaline denaturation of template DNA, and our recommendations for the sequencing reaction.read more
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