Integrated System for FFPE Sample Preparation

描述（由申请人提供）：从个人和动物身上采集的许多样品都是通过用化学品、福尔马林处理，然后用石蜡或蜡包裹来储存的；这些被称为福尔马林固定石蜡包封（FFPE）样品。通常这种储存方式非常适合保存它们以便在显微镜下观察，但在分析它们的DNA方面存在一些问题。遗传分析很重要，因为许多保存下来的样本都是癌症组织的活组织检查，而癌症是由DNA变化引起的疾病。通过了解这些样本的遗传成分，我们可以寻找可用于诊断癌症的特定DNA序列。我们还可以利用这些样本中的信息来确定哪种疗法可能效果最好，并衡量所应用的疗法的效果如何。有一些仪器和试剂试剂盒可用于提取FFPE样品中的DNA，但它们通常很复杂。他们经常使用危险的化学品。它们提取的DNA质量不一致，有时会被分解成小块或与蛋白质结合，这使得它们无法用于基因分析技术，比如测序，在测序中，DNA不同成分的顺序被读出。通常他们提取的DNA太少了。自适应聚焦声学（AFA）是一种将声能聚焦到小体积上的超声技术。它被用于许多过程，比如分解组织样本以提取蛋白质和DNA，以及将固体磨成均匀大小的颗粒。最近，AFA被应用于手工操作的FFPE样品中。这个过程去除石蜡，分解组织以提取DNA。它提供的DNA是其他方法的2-3倍，而且DNA质量更高。本项目将开发一个使用AFA对FFPE样品使用特殊仪器和特殊塑料盒的系统。仪器上的真空泵将移动药筒中的液体，AFA将去除蜡，提取DNA，并混合药筒中的液体和试剂。AFA从样品中去除的DNA也将通过与小磁珠结合来纯化，这些小磁珠由机器上的磁铁固定在适当的位置。这使得提取的DNA可以用于其他应用，比如DNA测序。该仪器和试剂盒将提供一种可靠、可重复、节省人力的方法来使用FFPE样品，这些样品非常有价值，有时是不可替代的，可用于研究或供医生诊断和治疗患者。我们将通过将整个过程分解成小块并单独开发它们来做到这一点。片段是用AFA提取的DNA；化学处理以去除蛋白质并破坏DNA与自身之间的化学键或剩余的蛋白质污染；净化DNA。每个步骤的性能将与手动方法进行比较。我们测量性能的方法包括通过添加荧光染料和测量荧光来确定有多少DNA；测量DNA分子的长度（这告诉我们它们是否太破碎而无法用于其他用途）；通过观察DNA溶液吸收了多少紫外线来测量残留蛋白的污染程度；最后使用一种叫做定量PCR的DNA扩增方法，这种方法只有在DNA完整且不与样本中的其他污染物结合的情况下才有效。我们还将对原型处理过的一些样品进行排序。当所有的部分都被单独开发出来后，它们将被组合在一起。在该项目中，完成这项工作的墨盒将使用计算机化的铣削或切割机制作，该机器可以制作高质量的结构模型，其尺寸小于人类头发的直径。当墨盒制成产品时，它将由模塑塑料制成。该仪器将由一台计算机运行，并在现有的科瓦利斯AFA仪器的基础上进行修改。如果这个项目成功，下一阶段将是解决制造问题，包括在墨盒上存储试剂，并将墨盒扩展到许多样品。

DESCRIPTION (provided by applicant): Many samples taken from individuals and animals are stored by treating them with chemical, formalin, and then encasing them in paraffin or wax; these are called formalin fixed paraffin encased (FFPE) samples. Usually this storage is very good for preserving them for observation under microscopes, but it presents some problems in terms of analysis of their DNA. Genetic analyses are important because many of these preserved samples are biopsies of cancer tissue, and cancers are diseases brought on by changes in the DNA. By understanding the genetic content of these samples, we can look for specific DNA sequences that can be used to diagnose cancer. We can also use the information in these samples to determine what therapies might work best, and to measure how well the therapies applied are working. There are some instruments and kits of reagents available for getting at the DNA in FFPE samples, but they are usually complicated. Often they use dangerous chemicals. They are inconsistent in the quality of DNA extracted, which can sometimes end up broken into small pieces or bound with proteins that make them unusable for genetic analysis techniques like sequencing, where the order of the different components of DNA are read out. Often they just extract too little of the DNA. Adaptive Focused Acoustics (AFA) is an ultrasonic technology that focuses sound energy onto small volumes. It is used for many processes like breaking up tissue samples to extract proteins and DNA and grinding solids into uniformly-sized particles. Recently AFA was applied to FFPE samples in a process performed manually. The process removes the paraffin and breaks the tissue down to extract the DNA. It provides 2-3 times as much DNA as other methods, and that DNA is of higher quality. This project will develop a system that uses AFA for FFPE samples with a special instrument and a special plastic cartridge. A vacuum pump on the instrument will move the liquids in the cartridge, and AFA will remove the wax, extract the DNA, and mix the liquids and reagents in the cartridge. The DNA that AFA removes from samples will also be purified by binding to little magnetic beads, which are held in place by magnets on the machine. This makes the extracted DNA ready for use in other applications, like DNA sequencing. This instrument and cartridge will provide a reliable, reproducible, and labor-saving way to use FFPE samples that are very valuable and sometimes irreplaceable and that can be used for research or by doctors in diagnosing and treating patients. We will do this by breaking down the whole process into small pieces and developing those individually. The pieces are the extraction of DNA using AFA; chemical treatment to remove proteins and to break any chemical bonds between the DNA and itself or remaining protein contamination; and purifying the DNA. The performance of each step will be compared to the manual method. The ways we will measure the performance include determining just how much DNA there is by adding a fluorescent dye and measuring the fluorescence; measuring the length of the DNA molecules (which tells us if they are too broken up to be used for other applications); measuring the contamination by residual protein by seeing how much UV light is absorbed by the DNA solutions; and finally by using a DNA amplification method called quantitative PCR that only works well if the DNA is intact and not bound to other contaminants in the sample. We will also sequence a few samples that have been processed by the prototype. When all of the pieces have been developed individually, they will be put together. For the project, the cartridge to do this work will be mae using a computerized milling or cutting machine that can make high quality models of structures with features of size less than that of the diameter of a human hair. When the cartridge is made into a product it will be made from molded plastic. The instrument will be run by a computer and modified from an existing Covaris AFA instrument. If this project is successful, the next phase will be to solve manufacturing problems, including storing reagents on the cartridge, and scaling the cartridge up to many samples.