Manoni M, Pergolizzi R, Luzzana M, De Bellis G
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie, Biomediche Avanzate, Milano, Italy.
Biotechniques. 1992 Jan;12(1):48-50, 52-3.
The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.
自动化荧光DNA测序系统和基于PCR的DNA测序方法的使用，在实际提高大规模DNA分析效率的工作中发挥着重要作用。在此，我们展示了使用单一荧光引物和双脱氧核苷酸终止剂的线性PCR在EMBL/Pharmacia荧光自动化DNA测序仪上的四个独立测序反应中的应用。我们利用双脱氧/脱氧核苷三磷酸比例和线性扩增循环条件，仅从400 ng双链DNA模板中获得了高达500个碱基及以上的准确测序反应，且无需化学变性。本文所述的测序方案有效地适用于提高自动化DNA测序系统的灵敏度和性能。
