Pyrosequencing trade mark : A one-step method for high resolution HLA typing.
Pyrosequencing trade mark : A one-step method for high resolution HLA typing.
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DOI：
10.1186/1479-5876-1-9
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发表时间：
2003-11-26
影响因子：
7.4
通讯作者：
Wang, Lu
中科院分区：
文献类型：
--
作者：
Ramon, Daniel;Braden, Megan;Wang, Lu
虽然在人类白细胞抗原（HLA）的常规匹配中使用高分辨率分子分型有望改善非相关供体选择和移植结果，但HLA的遗传复杂性仍然使当前的方法受到限制和费力。焦磷酸测序是一种无凝胶的合成测序方法。在焦磷酸测序反应中，核苷酸结合沿着每个DNA模板按给定的核苷酸分配顺序（NDO）顺序进行，该顺序被编程到焦磷酸测序仪中。在这里，我们描述了一个NDO的设计，它可以为任何给定的等位基因或等位基因的组合产生一个独特的焦图。我们展示了从两个杂合HLA模板中产生的独特焦图的例子，否则使用基于标准测序的分型（SBT）方法将保持顺式/反式模糊。此外，我们还展示了长时间读取和线性信号生成的代表性数据。这些特性是实现高分辨率输入和自动化数据分析的先决条件。总之，焦磷酸测序是一种高分辨率DNA分型的一步法。
While the use of high-resolution molecular typing in routine matching of human leukocyte antigens (HLA) is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing trade mark is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given nucleotide dispensation order (NDO) that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard sequencing based typing (SBT) method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing.