Solid-phase PCR in microwells:: Effects of linker length and composition on tethering hybridization, and extension
Solid-phase PCR in microwells:: Effects of linker length and composition on tethering hybridization, and extension
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DOI：
10.2144/02322rr03
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发表时间：
2002-02-01
期刊：
影响因子：
2.7
通讯作者：
Kresovich, S
中科院分区：
文献类型：
--
作者：
Carmon, A;Vision, TJ;Kresovich, S
在固相PCR（SP-PCR）过程中，原位延伸与可溶性模板互补并固定在表面上的DNA寡核苷酸。虽然主要用于病原体检测，SP-PCR具有更广泛的应用潜力，包括疾病诊断，基因分型和表达研究。目前在微孔中进行SP-PCR的方案适用于固定化产物的酶促检测，但产量通常不足以使用常规荧光探针直接检测产物。在这里，我们定量测量的拴系，杂交和固相延伸的结果，并检查在拴系的寡核苷酸的5'端的间隔区的组成和长度的影响。我们的研究结果表明，空间位阻主要影响聚合酶的活性，而不是模板和拴系的寡核苷酸之间的杂交效率。SP-PCR产率对于五单元六乙二醇（HEG）间隔区比对于更常用的10-残基脱氧胸苷间隔区显著更高。相对于先前报道的玻璃表面上SP-PCR的值，最佳的5' HEG间隔区导致延伸效率增加60倍。因此，优化的间隔区应该允许SP-PCR产物的直接定量，为各种应用提供简单、定量和成本有效的样品分析手段。
During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells, are suitable for enzymatic detection of Immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.