Rapid non-invasive prenatal Down syndrome detection using a DNA-molecule counter

摘要唐氏综合症（DS）是最常见的可存活的遗传疾病，在美国每年影响691人中有1人，或约6000名活产婴儿。目前基于超声和血液样本测量的非侵入性产前筛查是不准确的，而绒毛膜绒毛取样和羊膜穿刺术等确认性检查是可能导致流产的侵入性程序。最近，一种新的无创大规模平行测序检测方法可以检测母体血浆中胎儿DNA中的21三体。虽然这种方法可以对非整倍体做出明确的诊断，而不会给胎儿带来风险，但这种检测很昂贵，而且准父母通常要等上10天或更长时间。为了改进目前的方法，我们的目标是开发一种更快、更便宜的单分子计数系统，从母体血浆DNA中检测胎儿非整倍体。我们最近展示了一种计算DNA单分子的新方法。一组序列条形码用于随机标记单个DNA分子，扩增后，可以很容易地检测和计数不同条形码的数量，以揭示最初存在的相同分子的拷贝数量。因此，我们将计算相同分子的单个副本的困难任务转变为检测存在的不同条形码数量的简单任务。在我们发表的工作中，我们使用微阵列检测器对条形码进行计数，并从非常低的亚纳克样品输入开始，显示了染色体拷贝的精确和准确的数字绝对定量。在这项初步研究中，我们将进一步确定条形码和扩增反应条件，并构建一个合适的微阵列条形码计数器，以检测21号染色体的额外拷贝的非常小的增加。我们的检测方法的测量精度和统计置信度将被彻底调查，以证明后续商业产品开发阶段的可行性。我们的公司由该领域的主要领导者组成，包括Stephen Fodor，他是第一个发明和开发微阵列技术的人，Stephen Quake，他是第一个展示单分子DNA测序和唐氏综合症无创产前检测的人。两位创始人都将在这项新技术的开发和商业化中发挥关键作用。此外，我们还与斯坦福大学基因组技术中心的Ronald Davis建立了合作关系，使我们能够获得这个世界级研究机构提供的仪器和专业知识。

DESCRIPTION (provided by applicant): Rapid non-invasive prenatal Down syndrome detection using a DNA-molecule counter Abstract Down syndrome (DS) is the most common survivable genetic disorder affecting 1 in 691, or approximately 6,000 live births in the United States annually. Current non-invasive prenatal screening tests based on ultrasound and blood sample measurements are inaccurate, and confirmatory tests such as chorionic villus sampling and amniocentesis are invasive procedures that can cause miscarriage. Recently, a new non-invasive massively parallel sequencing test to detect trisomy 21 in the fetal DNA present in maternal plasma has become available. Although the approach produces a definitive diagnosis for aneuploidy without risk to the fetus, the test is expensive, and expectant parents usually have a long wait time of ten days or more. To improve the current method, our objective is to develop a faster and cheaper single molecule counting system to detect fetal aneuploidy from maternal plasma DNA. We recently demonstrated a novel approach to count single molecules of DNA. A collection of sequence barcodes is used to randomly label individual DNA molecules, and after amplification, the number of different barcodes can be easily detected and counted to reveal the number of copies of identical molecules originally present. Thus, we transform the difficult task of counting individual copies of identical molecules into a simple one of detecting the number of different barcodes present. In our published work, we used a microarray detector to count barcodes, and showed precise and accurate digital, absolute quantitation of chromosomal copies starting with very low, sub-nanograms of sample input. For this proposed pilot study, we will further define the barcoding and amplification reaction conditions, and construct a suitable microarray barcode counter to enable the detection of very small increases of additional copies of chromosome 21. The measurement accuracy and statistical confidence of our detection method will be thoroughly investigated to demonstrate feasibility for a subsequent commercial product development phase. Our company is composed of key leaders in the field including Stephen Fodor who was the first to invent and develop microarray technology, and Stephen Quake who was the first to demonstrate single molecule DNA sequencing and a non-invasive prenatal test for Down syndrome. Both founders will play critical roles in the development and commercialization of this novel technology. Additionally, we have established collaborations with Ronald Davis at the Stanford University Genome Technology Center, giving us access to instruments and expertise available at this world class research facility.