Arlinghaus H F, Kwoka M N, Jacobson K B
Atom Sciences, Inc., Oak Ridge, Tennessee 37830, USA.
Anal Chem. 1997 Sep 15;69(18):3747-53. doi: 10.1021/ac970267p.
Two novel DNA-sequencing methods are described that use DNA hybridization biosensor chips. These two techniques involve either labeling the free nucleic acid with enriched stable isotopes or hybridizing DNA without labels to immobilized peptide nucleic acid (PNA) and detecting the phosphorus present in the DNA but not in the PNA. Sputter-initiated resonance ionization microprobe analysis was used to detect the presence of enriched tin isotope-labeled DNA and of phosphorus in natural DNA as a means to identify the presence of DNA after hybridization to oligodeoxynucleotides (ODNs) or PNAs, respectively, immobilized on a biosensor chip. The data clearly demonstrate that excellent discrimination between complementary and noncomplementary sequences can be obtained during hybridization of DNA to either ODNs or PNAs. The capability to detect different enriched stable isotope-labeled DNAs simultaneously allows high degrees of multiplexing which may be very advantageous for hybridization kinetics studies in complex systems, as well as significantly increasing the speed of analysis. Alternatively, by using natural DNA with PNA biosensor chips, discrimination for single-point mutation could be increased because of improved hybridization kinetics and direct analysis of genomic DNA may become possible without amplification. Both methods have the potential to provide a rapid method for DNA/RNA sequencing, diagnostics, and mapping.
本文描述了两种使用DNA杂交生物传感器芯片的新型DNA测序方法。这两种技术要么用富集的稳定同位素标记游离核酸，要么将未标记的DNA与固定化的肽核酸（PNA）杂交，并检测DNA中而非PNA中存在的磷。采用溅射引发共振电离微探针分析来检测富集锡同位素标记的DNA以及天然DNA中的磷，以此分别鉴定与固定在生物传感器芯片上的寡脱氧核苷酸（ODN）或PNA杂交后DNA的存在情况。数据清楚地表明，在DNA与ODN或PNA杂交过程中，能够很好地区分互补和非互补序列。同时检测不同富集稳定同位素标记的DNA的能力允许高度多重化，这对于复杂系统中的杂交动力学研究可能非常有利，并且还能显著提高分析速度。或者，通过将天然DNA与PNA生物传感器芯片结合使用，由于杂交动力学得到改善，对单点突变的区分能力可能会提高，并且无需扩增就有可能直接分析基因组DNA。这两种方法都有可能为DNA/RNA测序、诊断和作图提供一种快速方法。
