公开(公告)号：US08945481B1

公开(公告)日：2015-02-03

申请号：US12145333

申请日：2008-06-24

申请人： Mark F. Oldham ,  Kenneth J. Livak ,  Jason E. Babcoke ,  H. Pin Kao ,  Stephen J. Gunstream ,  Kevin S. Bodner ,  Douglas P. Greiner ,  Nigel P. Beard ,  Dar Bahatt

发明人： Mark F. Oldham ,  Kenneth J. Livak ,  Jason E. Babcoke ,  H. Pin Kao ,  Stephen J. Gunstream ,  Kevin S. Bodner ,  Douglas P. Greiner ,  Nigel P. Beard ,  Dar Bahatt

IPC分类号： B01L3/00

CPC分类号： B01J19/0046 ,  B01L3/502715 ,  B01L3/502738 ,  B01L7/52 ,  B01L2200/027 ,  B01L2200/10 ,  B01L2300/0654 ,  B01L2300/0816 ,  B01L2300/0819 ,  B01L2300/0867 ,  B01L2300/185 ,  B01L2400/0655 ,  G01N21/17 ,  G01N2201/0407

摘要： Exemplary embodiments provide microfludic devices and methods for their use. The microfluidic device can include an array of M×N reaction sites formed by intersecting a first and second plurality of fluid channels of a flow layer. The flow layer can have a matrix design and/or a blind channel design to analyze a large number of samples under a limited number of conditions. The microfluidic device can also include a control layer including a valve system for regulating solution flow through fluid channels. In addition, by aligning the control layer with the fluid channels, the detection of the microfluidic devices, e.g., optical signal collection, can be improved by piping lights to/from the reaction sites. In an exemplary embodiment, guard channels can be included in the microfluidic device for thermal cycling and/or reducing evaporation from the reaction sites.

摘要翻译： 示例性实施例提供了用于它们的微流体装置和方法。 微流体装置可以包括通过与流动层的第一和第二多个流体通道相交形成的M×N反应位点的阵列。 流层可以具有矩阵设计和/或盲通道设计，以在有限数量的条件下分析大量样品。 微流体装置还可以包括控制层，其包括用于调节通过流体通道的溶液流动的阀门系统。 此外，通过使控制层与流体通道对准，可以通过向/从反应位置的管道灯来改善微流体装置的检测，例如光学信号收集。 在示例性实施例中，保护通道可以包括在微流体装置中用于热循环和/或减少从反应位置的蒸发。

公开(公告)号：US20110236984A1

公开(公告)日：2011-09-29

申请号：US12984269

申请日：2011-01-04

申请人： Hongye Sun ,  Eric S. Nordman ,  Mark F. Oldham ,  John R. O'Neill ,  Charles Connell ,  Umberto Ulmanella ,  Aldrich N.K. Lau ,  Theofilos Kotseroglou ,  Kenneth J. Livak

发明人： Hongye Sun ,  Eric S. Nordman ,  Mark F. Oldham ,  John R. O'Neill ,  Charles Connell ,  Umberto Ulmanella ,  Aldrich N.K. Lau ,  Theofilos Kotseroglou ,  Kenneth J. Livak

IPC分类号： G01N27/447 ,  G01Q60/24 ,  C08G69/42 ,  B82Y5/00

CPC分类号： C12Q1/6869 ,  B01L3/502761 ,  G01N27/3278 ,  G01N27/414 ,  G01N27/4145 ,  G01N27/4146 ,  G01N33/5438 ,  Y10S977/924 ,  Y10T436/143333 ,  C12Q2565/631

摘要： In some embodiments, an analyte detection system is provided that includes a nanochannel, an electrode arrangement, and a plurality of nanoFET devices disposed in the nanochannel. A plurality of nucleic acid base detection components can be used that include a plurality of nanopores, a plurality of nanochannels, a plurality of hybridization probes, combinations thereof, and the like. According to other embodiments of the present teachings, different coded molecules are hybridized to a target DNA molecule and used to detect the presence of various sequences along the target molecule. A kit including mixtures of coded molecules is also provided. In some embodiments, devices including nanochannels, nanopores, and the like, are used for manipulating movement of DNA molecules, for example, in preparation for a DNA sequencing detection. Nanopore structures and methods of making the same are also provided as are methods of nucleic acid sequencing using the nanopore structures. Surface-modified nanopores are provided as are methods of making them. In some embodiments, surfaced-modified nanopores for slowing the translocation of single stranded DNA (ssDNA) through the nanopore are provided, as are nanopores configured to detect each of a plurality of different bases on an ssDNA strand.

摘要翻译： 在一些实施例中，提供了分析物检测系统，其包括纳米通道，电极布置以及设置在纳米通道中的多个纳米器件器件。 可以使用多个核酸碱基检测组分，其包括多个纳米孔，多个纳米通道，多个杂交探针及其组合等。 根据本教导的其他实施方案，将不同的编码分子与靶DNA分子杂交并用于检测沿着靶分子的各种序列的存在。 还提供了包含编码分子混合物的试剂盒。 在一些实施方案中，包括纳米通道，纳米孔等的装置用于操纵DNA分子的移动，例如用于DNA测序检测的准备。 还提供了制备其的纳米孔结构及其制备方法，使用纳米孔结构的核酸测序方法也是如此。 提供表面改性的纳米孔是制备它们的方法。 在一些实施方案中，提供了用于减缓通过纳米孔的单链DNA（ssDNA）易位的表面改性的纳米孔，以及配置成检测ssDNA链上的多个不同碱基中的每一个的纳米孔。

公开(公告)号：US20100317062A1

公开(公告)日：2010-12-16

申请号：US12504633

申请日：2009-07-16

申请人： Kai Qin LAO ,  Neil A. Straus ,  Kenneth J. Livak

发明人： Kai Qin LAO ,  Neil A. Straus ,  Kenneth J. Livak

IPC分类号： C12P19/34 ,  C12N9/12 ,  C07H21/00

CPC分类号： C12Q1/6876 ,  C07H21/02 ,  C07H21/04 ,  C12N9/00 ,  C12Q1/6844 ,  C12Q1/6848 ,  C12Q1/686 ,  C12Q2549/101 ,  C12Q2533/101 ,  C12Q2525/301

摘要： The present teachings provide methods, compositions, and kits for performing primer extension reactions. In some embodiments, a reverse transcription reaction is performed on a target polynucleotide with a hot start primer comprising a blunt-ended self-complementary stem, and a loop, and extension products form at high temperatures but reduce extension product formation at low temperatures.

摘要翻译： 本教导提供了用于进行引物延伸反应的方法，组合物和试剂盒。 在一些实施方案中，对靶多核苷酸进行逆转录反应，所述靶多核苷酸具有包含平端自补偿干杆的热起始引物，并且循环和延伸产物在高温下形成，但是在低温下减少延伸产物形成。

公开(公告)号：US20100203525A1

公开(公告)日：2010-08-12

申请号：US12614318

申请日：2009-11-06

申请人： Kenneth J. Livak ,  Junko Stevens ,  Katherine D. Lazaruk ,  Janet S. Ziegle ,  Lily Y. Wong

发明人： Kenneth J. Livak ,  Junko Stevens ,  Katherine D. Lazaruk ,  Janet S. Ziegle ,  Lily Y. Wong

IPC分类号： C12Q1/68 ,  G06F19/00

CPC分类号： G16B20/00 ,  C12Q1/6827 ,  C12Q1/6851 ,  C12Q2600/156 ,  C12Q2563/107 ,  C12Q2545/101 ,  C12Q2565/101 ,  C12Q2563/173 ,  C12Q2565/1015

摘要： Methods of detecting a candidate genetic anomaly such as a candidate duplication in a genome are disclosed. The methods comprise quantifying fluorogenic assays for alleles of a genetic locus from a plurality of individual genomes, identifying ranges of fluorescent intensities indicative of individual genomes homozygous for a first allele, homozygous for a second allele, or heterozygous for both alleles, and identifying individual genomes in which the fluorescence intensities are outside the range of intensities indicative of homozygosity or heterozygosity for the genetic locus.

摘要翻译： 公开了检测候选遗传异常（例如基因组中候选重复）的方法。 所述方法包括定量来自多个单个基因组的遗传基因座的等位基因的荧光测定，鉴定指示第一等位基因纯合的单个基因组的荧光强度的范围，对于第二等位基因纯合，或对于两个等位基因是杂合的，并鉴定个体基因组 其中荧光强度在表示遗传基因座的纯合性或杂合性的强度范围之外。

公开(公告)号：US06890718B2

公开(公告)日：2005-05-10

申请号：US10099738

申请日：2002-03-15

申请人： Christian A. Heid ,  Kenneth J. Livak

发明人： Christian A. Heid ,  Kenneth J. Livak

IPC分类号： G01N33/53 ,  C07H21/00 ,  C07H21/02 ,  C07H21/04 ,  C12N15/09 ,  C12P19/34 ,  C12Q1/68 ,  C12Q1/70

CPC分类号： C12Q1/686 ,  Y10S435/81 ,  C12Q2545/113 ,  C12Q2545/10

摘要： External control reagents for nucleic acid amplification are provided that verify the absence or presence of specific target sequences, and correct primers and probes. A single-stranded, external control polynucleotide is amplified with primers of the same sequence as target primers. Probes with detectable labels and sequences specific for target and external control polynucleotides allow for detection and measurement. The primers and the detectable probe are adjacent or substantially adjacent when hybridized to the external control polynucleotide. Target and control amplicons may be detected by increased fluorescence induced by polymerase-mediated 5′ nuclease cleavage or hybridization of a self-quenching probe complementary to both target and external control polynucleotides. A kit of PCR reagents can be dispensed into vessels for rapid and accurate nucleic acid amplification assay, with real-time or end-point measurements. The amplification control reagents, kits, and methods of the present invention provide positive and negative control tests which can be conducted concurrently with target amplification. Allelic differences at genetic loci can be detected, including single nucleotide polymorphisms (SNP).

摘要翻译： 提供用于核酸扩增的外部对照试剂，其验证特定靶序列的不存在或存在，以及校正引物和探针。 使用与靶引物相同序列的引物扩增单链外部对照多核苷酸。 具有可检测标记和具有针对和外部对照多核苷酸序列的序列的探针允许检测和测量。 当与外部对照多核苷酸杂交时，引物和可检测的探针相邻或基本相邻。 可以通过聚合酶介导的5'核酸酶切割诱导的增加的荧光或与靶和外部对照多核苷酸互补的自猝灭探针的杂交来检测靶和对照扩增子。 PCR试剂盒可以分配到容器中，用于快速准确的核酸扩增测定，具有实时或终点测量。 本发明的扩增控制试剂，试剂盒和方法提供可以与靶扩增同时进行的阳性和阴性对照试验。 可以检测遗传基因座中的等位基因差异，包括单核苷酸多态性（SNP）。

公开(公告)号：US06884583B2

公开(公告)日：2005-04-26

申请号：US10104774

申请日：2002-03-21

申请人： Kenneth J. Livak ,  Federico Goodsaid

发明人： Kenneth J. Livak ,  Federico Goodsaid

IPC分类号： G01N21/64 ,  C12M1/00 ,  C12Q1/68 ,  G01N33/53 ,  G01N33/566 ,  C07H21/02 ,  C12P19/34

CPC分类号： C12Q1/6858 ,  C12Q1/6818 ,  C12Q2561/101 ,  C12Q2537/143 ,  C12Q2563/107 ,  C12Q2531/119

摘要： A method is provided for genotyping a target sequence at at least two allelic sites by a 5′ nuclease amplification reaction. In one embodiment, the method includes performing a nucleic acid amplification on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5′→3′ nuclease activity and a primer capable of hybridizing to the target sequence in the presence of two or more sets of allelic oligonucleotide probes wherein: each set of allelic oligonucleotide probes is for detecting a different allelic site of the target sequence, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5′ relative to a sequence to which the primer hybridizes to the target sequence, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher postioned on the probe to quench the fluorescence of the fluorescer; detecting a fluorescence spectrum of the amplification; calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different allelic variants at the two or more different allelic sites based on the fluorescence contribution of each fluorescer to the combined fluorescence spectrum.

摘要翻译： 提供了一种通过5'核酸酶扩增反应在至少两个等位基因位点对靶序列进行基因分型的方法。 在一个实施方案中，该方法包括使用具有5'→3'核酸酶活性的核酸聚合酶和能够在目标序列中与靶序列杂交的引物对具有至少两个不同等位基因位点的靶序列进行核酸扩增 两组或更多组等位基因寡核苷酸探针，其中：每组等位基因寡核苷酸探针用于检测靶序列的不同等位基因位点，每组等位基因寡核苷酸探针包括与等位基因上的不同等位基因变体互补的两个或多个探针 通过该组探针检测位点，所述等位基因位点相对于引物与靶序列杂交的序列为5'，并且至少所有等位基因寡核苷酸探针中的所有等位基因寡核苷酸探针包括与其它探针不同的荧光剂，和 探针上的猝灭剂用于淬灭荧光剂的荧光; 检测扩增的荧光光谱; 计算每个荧光剂对荧光光谱的荧光贡献; 并且基于每个荧光剂对组合的荧光光谱的荧光贡献，确定两个或更多个不同等位基因位点处的不同等位基因变体的存在或不存在。

公开(公告)号：US06821727B1

公开(公告)日：2004-11-23

申请号：US09627753

申请日：2000-07-28

申请人： Kenneth J. Livak ,  Susan J. A. Flood ,  Jeffrey Mamoro ,  Khairuzzaman Bashar Mullah

发明人： Kenneth J. Livak ,  Susan J. A. Flood ,  Jeffrey Mamoro ,  Khairuzzaman Bashar Mullah

IPC分类号： C12Q168

CPC分类号： C12Q1/6818 ,  C12Q1/6834 ,  Y10S977/924 ,  C12Q2525/301 ,  C12Q2565/1015 ,  C12Q2565/107 ,  C12Q2561/101

摘要： A hybridization assay is provided which uses an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded confirmation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibits different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.

摘要翻译： 提供了使用寡核苷酸探针的杂交测定法，其包括荧光报告分子和能够猝灭报告分子荧光的猝灭剂分子。 构建寡核苷酸探针，使得探针在未杂交的情况下以至少一种单链确认存在，其中淬灭剂分子足够接近报道分子以淬灭报道分子的荧光。 当寡核苷酸探针与靶多核苷酸杂交时，寡核苷酸探针也存在至少一个构象，其中猝灭剂分子不能与报道分子紧密接近以猝灭报道分子的荧光。 通过采用这些杂交和非杂交构象，当探针杂交和未杂交时，探针上的报道分子和猝灭剂分子表现出不同的荧光信号强度。 结果，可以基于报道分子，猝灭剂分子或其组合的荧光强度的变化来确定探针是杂交还是未杂交。 此外，因为探针可以被设计成使得当探针不杂交时，猝灭剂分子淬灭报告分子，所以探针可被设计成使得报告分子在荧光探针杂交或消化之前显示有限的荧光。

公开(公告)号：US06309829B1

公开(公告)日：2001-10-30

申请号：US09205114

申请日：1998-12-03

申请人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

发明人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

IPC分类号： C12Q168

CPC分类号： C12Q1/6827 ,  C12Q1/6837 ,  C12Q1/6858 ,  C12Q1/6874 ,  C12Q2565/514 ,  C12Q2533/101 ,  C12Q2525/151 ,  C12Q2525/131 ,  C12Q2565/515 ,  C12Q2525/161 ,  C12Q2535/125 ,  C12Q2525/197 ,  C12Q2563/107

摘要： Disclosed is a method for determining the number of repeat units in a repeat region of a target nucleic acid. In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle. In a second aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer-extension reagent; separating the target-primer hybrid from unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent and unreacted primer termination reagent; measuring a signal produced by the label; and repeating the above steps until a signal is detected indicating incorporation of the nucleotide terminator. The invention further includes kits useful for practicing the above methods.

摘要翻译： 公开了确定靶核酸重复区域中重复单元数目的方法。 在第一方面，本发明的方法包括将引物退火至靶核酸的步骤; 使用第一引物延伸试剂进行第一引物延伸反应; 分离靶 - 引物杂交体和未反应的第一引物延伸试剂; 使用第二引物延伸试剂进行第二引物延伸反应，其中第一或第二引物延伸试剂中的至少一个包括具有附着标记的标记的可延伸核苷酸; 将靶引物杂交体与未反应的第二引物延伸试剂分离; 测量标签产生的信号; 处理标签以使标签不可检测; 并重复上述步骤，直到信号基本上小于在先前循环中检测到的信号。 在第二方面，本发明的方法包括将引物退火至靶核酸的步骤; 使用第一引物延伸试剂进行第一引物延伸反应; 将靶引物杂交体与未反应的第一引物延伸试剂分离; 使用第二引物延伸试剂和引物终止试剂进行第二引物延伸反应，所述引物终止试剂包括具有附着标记的核苷酸终止子; 从未反应的第二引物延伸试剂和未反应的引物终止试剂中分离靶引物杂交体; 测量标签产生的信号; 并重复上述步骤，直到检测到指示掺入核苷酸终止子的信号。 本发明还包括可用于实践上述方法的试剂盒。

公开(公告)号：US5945284A

公开(公告)日：1999-08-31

申请号：US863437

申请日：1997-05-27

申请人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

发明人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

IPC分类号： C12N15/09 ,  C12Q1/68 ,  C07H21/02 ,  C07H21/04

CPC分类号： C12Q1/6869

摘要： In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle. In a second aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer-extension reagent; separating the target-primer hybrid from unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent and unreacted primer termination reagent; measuring a signal produced by the label; and repeating the above steps until a signal is detected indicating incorporation of the nucleotide terminator. The invention further includes kits useful for practicing the above methods.

摘要翻译： 在第一方面，本发明的方法包括将引物退火至靶核酸的步骤; 使用第一引物延伸试剂进行第一引物延伸反应; 分离靶 - 引物杂交体和未反应的第一引物延伸试剂; 使用第二引物延伸试剂进行第二引物延伸反应，其中第一或第二引物延伸试剂中的至少一个包括具有附着标记的标记的可延伸核苷酸; 将靶引物杂交体与未反应的第二引物延伸试剂分离; 测量标签产生的信号; 处理标签以使标签不可检测; 并重复上述步骤，直到信号基本上小于在先前循环中检测到的信号。 在第二方面，本发明的方法包括将引物退火至靶核酸的步骤; 使用第一引物延伸试剂进行第一引物延伸反应; 将靶引物杂交体与未反应的第一引物延伸试剂分离; 使用第二引物延伸试剂和引物终止试剂进行第二引物延伸反应，所述引物终止试剂包括具有附着标记的核苷酸终止子; 从未反应的第二引物延伸试剂和未反应的引物终止试剂中分离靶引物杂交体; 测量标签产生的信号; 并重复上述步骤直到检测到指示掺入核苷酸终止子的信号。 本发明还包括可用于实践上述方法的试剂盒。

公开(公告)号：US5723591A

公开(公告)日：1998-03-03

申请号：US559405

申请日：1995-11-15

申请人： Kenneth J. Livak ,  Susan J.A. Flood ,  Jeffrey Marmaro ,  Khairuzzaman Bashar Mullah

发明人： Kenneth J. Livak ,  Susan J.A. Flood ,  Jeffrey Marmaro ,  Khairuzzaman Bashar Mullah

IPC分类号： C12N15/09 ,  C07H21/00 ,  C12N15/00 ,  C12Q1/68 ,  C07H19/00 ,  C07H21/02 ,  C07H21/04

CPC分类号： C12Q1/6818 ,  C12Q1/6834 ,  C12Q1/6837

摘要： An oligonucleotide probe is provided which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.

摘要翻译： 提供了一种寡核苷酸探针，其包括荧光报道分子和能够猝灭报道分子荧光的猝灭剂分子。 构建寡核苷酸探针，使得探针在未杂交的情况下以至少一个单链构象存在，其中猝灭剂分子足够接近报道分子以淬灭报道分子的荧光。 当寡核苷酸探针与靶多核苷酸杂交时，寡核苷酸探针也存在至少一个构象，其中猝灭剂分子不能与报道分子紧密接近以猝灭报道分子的荧光。 通过采用这些杂交和非杂交构象，当探针杂交和未杂交时，探针上的报道分子和猝灭剂分子表现出不同的荧光信号强度。 结果，可以基于报道分子，猝灭剂分子或其组合的荧光强度的变化来确定探针是杂交还是未杂交。 此外，因为探针可以被设计成使得当探针不杂交时，猝灭剂分子淬灭报告分子，所以探针可被设计成使得报告分子在荧光探针杂交或消化之前显示有限的荧光。