Blöcker H, Lincoln D N
GBF-Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
Comput Appl Biosci. 1994 Apr;10(2):193-7. doi: 10.1093/bioinformatics/10.2.193.
In principle it is most economical to sequence large DNA fragments consecutively ('primer walking'), provided there is an immediate supply of sequencing primers. To solve the problem of primer supply we previously suggested generating a bank of short oligonucleotide primers ('shortmers'). In every sequencing reaction shortmers would have to be selected from this bank that are suitable to hybridize adjacently on the sequencing template. After their ligation the shortmers would form a long, and hence more specific, primer in the subsequent sequencing reaction. In the present study a computer simulation of large sequencing projects revealed a reduced set of approximately 12,000 selected octanucleotides (out of all 65,536) retaining maximum priming flexibility and minimum redundant information on the simulated sequence analyses. Establishing routine protocols for nucleic acid sequencing following the shortmer approach will abolish the tightest bottleneck of the consecutive sequencing route (primer supply) and hence may render this general scheme more attractive than the shotgun sequencing scheme. A twofold (or more) speed-up of genome sequencing projects by the shortmer approach may be assumed.
原则上，如果有现成的测序引物供应，连续对大的DNA片段进行测序（“引物步移法”）是最经济的。为了解决引物供应问题，我们之前建议构建一个短寡核苷酸引物库（“短引物”）。在每次测序反应中，都必须从这个库中选择适合在测序模板上相邻杂交的短引物。短引物连接后，会在后续测序反应中形成一个长的、因而更特异的引物。在本研究中，对大型测序项目的计算机模拟显示，从所有65536个八聚体中选出约12000个八聚体，在模拟序列分析中保留了最大的引物灵活性和最小的冗余信息。按照短引物方法建立核酸测序的常规方案，将消除连续测序途径中最严重的瓶颈（引物供应），因此可能使这种总体方案比鸟枪法测序方案更具吸引力。可以设想，短引物方法可使基因组测序项目的速度提高两倍（或更多）。
