Fixing Fixed DNA

描述：(申请人提供) 从常规固定的人体组织中提取的DNA遭到破坏。模板 (碱基)损害可能比链条损害更大，因为福尔马林 与碱基反应，但不与糖或磷酸基团反应。固定组织中的DNA是 通常在琼脂糖凝胶上比通过聚合酶链式反应扩增的目标更长，甚至可以 用于Southern杂交分析。碱基损伤可能会阻止聚合酶链式反应 聚合酶停滞。最近发现的容易出错或绕过的DNA 聚合酶可以通过添加非模板碱基来桥接受损的模板。 用容易出错的聚合酶“修复”受损的DNA将导致“体外” 突变，但挽救了随后的聚合酶链式反应。然而，聚合酶链式反应产物可以保留 原始模板的信息，因为正确的碱基将出现在 大多数扩增的分子。例如，非常低的碱基水平 损坏可以显著减少完整模板的拉伸。3% 随机基础伤害，只有不到1%的模板会超过200 但97%的扩增分子会保留正确的碱基 每个站点。容易出错的聚合酶修复后的聚合酶链式反应应该产生更多的 更长的产品，并可能进一步释放这一无处不在的 临床资源。

DESCRIPTION: (provided by applicant) DNA extracted from conventionally fixed human tissues is damaged. Template (base) damage is likely to be greater than chain damage because formalin reacts with bases but not sugar or phosphate groups. DNA from fixed tissues is generally longer on agarose gels than targets amplified by PCR, and can even be used for Southern blot analysis. Base damage can stop PCR because of polymerases stalling. Recently discovered error-prone or by-pass DNA polymerases can bridge damaged templates by adding non-templated bases. "Repair" of damaged DNA by an error-prone polymerase will cause "in vitro" mutations but rescue subsequent PCR. However, PCR products can retain the information of original templates because the correct base would be present in the majority of amplified molecules. For example, very low levels of base damage can significantly reduce stretches of intact template. At 3 percent random base damage, less than 1 percent of templates would be longer than 200 bases but 97 percent of amplified molecules would retain the correct base at each site. PCR after "repair" by error prone polymerases should yield more and longer products, and may further unlock the utility of this ubiquitous clinical resource.