DNA chips: An array of possibilities
DNA chips: An array of possibilities
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DOI：
10.1038/nbt0198-27
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发表时间：
1998-01-01
影响因子：
46.9
通讯作者：
Hodgson, J
中科院分区：
文献类型：
--
作者：
Marshall, A;Hodgson, J
一年前，DNA芯片的非凡之处在于任何人都可以制造它们。现在，似乎每个人都在寻找制造它们的新方法。随着阵列制造的许多技术障碍被克服，许多公司和学术团体正在开发DNA样品制备、探针合成、靶标记和读出的新方法，使阵列设计更加灵活。这些进步应该提高这一技术的可及性和可负担性的板凳科学家。它们不仅有望提高阵列杂交方法的鲁棒性、准确性和重现性，还有望扩大其应用范围。分离很难：DNA样品的分离和“净化”绝非易事。根据阵列形式和应用，在标记和应用于阵列之前，通常需要扩增来自血液或组织活检的DNA。许多格式需要此扩增步骤，因为当前使用的读数通常对记录未扩增的标记样品不够敏感。根据分子动力学公司（桑尼维尔，加利福尼亚州）研究和业务副总裁大卫巴克的说法，这是诊断学中一个特别棘手的问题：“在肿瘤活检中检测杂合癌症基因与数千个”正常”基因的背景，显然需要有效和特异性的靶扩增”他说。但在溶液中通过PCR进行的“线性”扩增可能存在问题，因为低丰度信息通常难以通过凝胶分离检测到，并且不同靶标之间对引物的竞争意味着某些序列的扩增优于其他序列。几家公司现在正在研究解决这些问题的方法。Mosaic Technologies（Boston，MA）正在开发一种固相PCR系统，其中两组引物（靶DNA的每个方向各一个）排列在丙烯酰胺膜上，并与溶液中的DNA样品和PCR试剂混合。如果样品含有靶序列，则从引物的末端合成DNA，并且随着热循环的继续，扩增的双链DNA在引物之间形成环或“桥”。Mosaic的研究主管克里斯·博尔斯说：“通过在固相中进行反应，你可以避免竞争的问题。”
One year ago, the remarkable thingabout DNA chips was that anyone could make them at all. Now, it seems everyone is finding new ways of making them. With many of the technological hurdles to array fabrication overcome, numerous companies and academic groups are developing novel approaches to DNA sample preparation, probe synthesis, target labeling, and readout that make array design more flexible. These advances should improve both the accessibility and affordability of this technology for the bench scientist. They promise not only to increase the robustness, accuracy, and reproducibility of array hybridization methods, but also to extend their range of applications.Breaking up is hard to do: The sample Isolation and “cleanup" of a DNA sample is no easy task. Depending on the array format and application, amplification of DNA derived from blood or tissue biopsies is often necessary before labeling and application to the array. Many formats require this amplification step because readouts in current use are often insuf-ficiently sensitive to register unamplified labeled sample. According to Molecular Dynamics'(Sunnyvale, CA) David Barker, vice president of research and business, this is a particularly thorny problem in diagnostics:“Detection of a heterozygous cancer gene against a background ofthousands of" normal” genes in a tumor biopsy obviously requires efficient and specific amplification of the target" he says. But “linear” amplification by PCR in solution can be problematic because low-abundance messages are often difficult to detect by gel separation and competition between differ-ent targets for primers means that amplification of certain sequences is favored over others. Several companies are now working on ways to get around these problems. Mosaic Technologies (Boston, MA) is developing a solid-phase PCR system in which sets of two primers (one in each orientation of the target DNA) are arrayed onto an acrylamide film and mixed with DNA sample and PCR reagents in solution. If the sample contains the target sequence, DNA is synthesized from the ends of the primers and, as thermocy-cling continues, amplified double-stranded DNA forms loops or “bridges” between the primers.“By carrying out the reaction in solid phase, you avoid the problem of competition," says Chris Boles, Mosaic's director of research.