A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP).
A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP).
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DOI：
10.1007/s00216-016-9479-y
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发表时间：
2016-12
影响因子：
4.3
通讯作者：
Ellington, Andrew D.
中科院分区：
文献类型：
--
作者：
Jung, Cheulhee;Ellington, Andrew D.
在核酸扩增反应中有多种引发方式。虽然大多数反应依赖于引物来启动扩增，但已经开发了DNA扩增的机制，其中单链寡核苷酸3'末端的发夹序列自身折叠以启动引发。不幸的是，这种方法对于诊断应用不太有用，因为自折叠效率低，并且仅在很窄的反应温度范围内起作用。为了使这种策略适用于分析应用，我们开发了一种变体，我们称之为硫代磷酸末端发夹形成和自引发延伸（PS-THSP）。在PS-THSP中，硫代磷酸酯（PS）修饰被掺入DNA骨架中，导致dsDNA的热稳定性降低和末端发夹的自折叠增加。通过优化初始模板中包含的PS键的数量，我们大大提高了自折叠效率和反应温度范围，最终实现了1 pM的检测限。这种改进的方法很容易适用于单核苷酸多态性的检测和非核酸分析物的检测，如碱性磷酸酶，其定量检测的限度为0.05 mU/mL，比商业测定约10倍。
There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3’ terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temperatures. In order to adapt this strategy for analytical applications we have developed a variant that we term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a reduction in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the number of PS linkages that are included in the initial template, we greatly increased self-folding efficiency and the range of reaction temperatures, ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alkaline phosphatase, which was quantitatively detected at a limit of 0.05 mU/mL, approximately 10-fold better than commercial assays.
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影响因子：
2.7
作者：
Bruse S;Moreau M;Azaro M;Zimmerman R;Brzustowicz L
通讯作者：
Brzustowicz L
影响因子：
14.9
作者：
LAPLANCHE, LA;JAMES, TL;ZON, G
通讯作者：
ZON, G
影响因子：
7.4
作者：
Jung, Cheulhee;Chung, Ji Won;Park, Hyun Gyu
通讯作者：
Park, Hyun Gyu
影响因子：
3.3
作者：
Nagamine, K;Hase, T;Notomi, T
通讯作者：
Notomi, T
DOI：
10.1101/sqb.1986.051.01.032
发表时间：
1986-01-01
期刊：
COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY
影响因子：
--
作者：
MULLIS, K;FALOONA, F;ERLICH, H
通讯作者：
ERLICH, H