Novel Two-Dimensional LIF Detector for DNA Sequencing

描述（申请人摘要）：该第一阶段SBIR项目引入了 新型激光诱导荧光（LIF）检测器，将提高DNA测序 提升到一个新的水平所有商业测序仪都从一维序列中进行碱基比对。 荧光数据，即，四个波长的强度。荧光 另一个碱基判定选项是发育期。我们的理念独一无二 生成完全二维的数据阵列作为完整的荧光衰减 在几个波长下同时测量分布。的方法 依靠高重复率脉冲激光，没有移动部件，并使用 用一个简单的光电倍增管代替CCD摄像机。在众多好处中， 最值得注意的是，二维数据格式提供了乘法 碱基判定准确性的提高。如果一个不准确的 光谱数据的basecall为5%，寿命数据为10%， 组合数据将不准确性降低到0.5%。此外， 分辨重叠峰并区分背景荧光 大大提高，允许更长的片段测序， 更短的时间。到第二阶段结束时的预计业绩包括： 在16个毛细管上平行测量，读取长度大于1000个碱基， 碱基判定准确率高于99.5%，运行时间少于90分钟 时间没有任何一种商业仪器能接近这种速度和 精度 拟定商业应用： 自动DNA测序仪彻底改变了遗传研究和诊断程序。 年自动DNA测序设备的销售额估计为2亿美元， 1999年，预计市场规模将在未来五年内急剧增长。 需要一种能够提供更准确碱基判定的自动DNA测序仪， 更长的真实的长度和更短的运行时间将通过所提出的研究来满足，其中， 将开发出第一个用于测序的二维荧光检测器。

DESCRIPTION (Applicant's abstract): This Phase I SBIR project introduces a novel laser-induced fluorescence (LIF) detector that will raise DNA sequencing to a new level. All commercial sequencers make basecalls from one-dimensional fluorescence data, viz., intensities at four wavelengths. Fluorescence lifetime, another basecalling option, is developmental. Our concept uniquely generates fully two-dimensional data arrays as complete fluorescence decay profiles are measured at several wavelengths simultaneously. The approach relies on a high repetition rate pulsed laser, has no moving parts, and uses a simple photomultiplier tube instead of a CCD camera. Of the many benefits, the most notable is that the two-dimensional data format offers a multiplicative improvement in basecalling accuracy. If the probability of an inaccurate basecall is 5 percent for spectral data and 10 percent for lifetime data, the combined data reduces the inaccuracy to 0.5 percent. Moreover, the ability to resolve overlapping peaks and discriminate against background fluorescence improves dramatically, allowing far longer fragments to be sequenced in much shorter time. Projected performance by the end of Phase II includes ability to measure in parallel on 16 capillaries, read length greater than 1000 bases with better than 99.5 percent basecalling accuracy, and less than 90-minute run time. No commercial instrument comes close to this combination of speed and accuracy. PROPOSED COMMERCIAL APPLICATION: Automated DNA sequencers have revolutionized genetic research and diagnostic procedures. Annual sales of $200 million were estimated for automated DNA sequencing equipment in 1999 and the size of the market is expected to increase sharply during the next five years. The need for an automated DNA sequencer that can provide more accurate basecalling, longer real lengths, and shorter run times will be met by the proposed research, in which the first two-dimensional fluorescence detector for sequencing will be developed.