John M. S. Bartlett.pdf - Bio-Nica.info

268 Case-Green, Pritchard, and Southern
10. Instead of performing the assays in two steps, a hybridization followed by a ligation
or polymerization, an all-in-one reaction can be used. There are several advantages to
conducting all-in-one reactions, such as the saving time and effort and the potential to
cycle the temperature allowing the target DNA to be used as a template for more than one
ligation or polymerization. Conducting all-in-one reactions puts additional constraints on
the conditions because the majority of enzymes work in only a small range of temperatures
and salt concentrations. The target single-stranded DNA generally needs to be purified.
11. The methods described use Thermus Thermophilus DNA ligase for ligation reactions
and Sequenase in extension reactions. Other enzyme types can also be used and have
advantages if particular temperatures are required, particularly in all in one reactions.
Therefore, for example, Taq and Thermosequenase have all been used successfully.
12. The extension method described adds a dideoxynucleotidetriphosphate (ddNTP). For most
applications, appropriate deoxynucleotidetriphosphate (dNTP) can be used.
References
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