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Real Time PCR
Introduction to Real Time PCR
As the name suggests, real time PCR is a technique used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. Real Time PCR is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. This occurs due to the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green ) or sequence specific probes (i.e. Molecular Beacons or TaqMan® Probes). Real time PCR facilitates the monitoring of the reaction as it progresses. One can start with minimal amounts of nucleic acid and quantify the end product accurately. Moreover, there is no need for the post PCR processing which saves the resources and the time. These advantages of the fluorescence based real time PCR technique have completely revolutionized the approach to PCR-based quantification of DNA and RNA. Real time PCR assays are now easy to perform, have high sensitivity, more specificity, and provide scope for automation. Real time PCR is also referred to as real time RT PCR which has the additional cycle of reverse transcription that leads to formation of a DNA molecule from a RNA molecule. This is done because RNA is less stable as compared to DNA
![AlleleID序列号](//l.b2b168.com/2019/07/18/10/201907181030407449034.jpg)
Taxa Specific/Cross Species identification: Probes with Mismatched Bases and Optimal Primers
Minimal Probes with Optimal Primers: When the homology among the aligned sequences is very low and both the designs mentioned above (a single taxa specific probe set or probe set with mismatch tolerance) are not feasible, AlleleID® designs the minimum number of probes possible to detect all the sequences in an alignment. The program designs probes for the majority consensus first and then for the minority consensus and remaining individual sequences. This design is very useful, especially for studying phylogenetic relationships among sequences obtained from different geographical locations.
![AlleleID序列号](//l.b2b168.com/2019/07/18/10/201907181030484134514.jpg)
Modern Techniques for Cross Species Detection
Real time PCR and microarrays offer a rapid, sensitive, specific and quantitative tool for the diagnosis of pathogens. Diseases are not readily distinguished based on symptoms when there is more than one pathogen in the sample. Microscopic identification requires culturing on specialized media for days to weeks. Recently, real time PCR assays were successfully used in the detection of fungal pathogens such as Rhizoctonia, Pythium, and Fusarium.
Designing a cross species assay involves the use of a multiple sequence alignment tool to identify conserved stretches of DNA and the design of a hybridization probe for detecting it. Recently there have been advances in micro-organism identification at the taxonomical level using molecular level identification techniques such as real time PCR and microarrays.
![AlleleID序列号](//l.b2b168.com/2019/07/18/10/201907181030459619184.jpg)
AlleleID 物种识别和分类鉴别的综合软件
的实验设计
AlleleID是一款旨在解决、病原体检测或物种的综合性桌面工具。以CulsSTW多序列比对为核心,AlleleID可以设计用于物种识别/交叉物种的基因芯片探针以及用于实时PCR的SYBR Green, TaqMan MGB, TaqMan探针, Molecular Beacons以及实时PCR引物。AlleleID还提供了支持可变剪切片段检测的微阵列实验设计。
物种分析/跨物种实验/等位基因分析
AlleleID使用ClustalW对序列进行排列,并分析保存和物种特定区域。您可以使用程序进行实时PCR引物设计(包含SYBR Green引物设计)和双标签探针设计(TaqMan probes,
TaqMan MGB and molecular beacons)。这些检测方法只用于进行菌株检测或从混合菌株中找到感兴趣的物种。为了进行交叉物种试验,AlleleID通过识别保守区域来设计通用探针。对于相关的生物体,当研究物种的基因组草图不可用时,可以使用AlleleID来研究其基因表达。这些强大的功能可以帮助解决了很多具有挑战性的难题,例如的检测、、定量以及污染物、环境等的监测。
广泛支持实时或定量PCR检测和SNP/表达微阵列
AlleleID可以设计优的SYBR Green引物、TaqMan探针、TaqMan Minor Groove Bingding
(MGB)探针、FRET探针或者分子信标,用于实时定量PCR差异基因表达SNP基因型分类试验。您也可以设计实时PCR引物和双标记探针多达一万个序列在一个单一的运行SNP检测或表达微阵列。
AlleleID®
Design qPCR and Microarray Assays for Related Organisms
AlleleID® is a comprehensive desktop tool designed to address the challenges of bacterial identification, pathogen detection or species identification. With ClustalW multiple sequence alignment at its core, AlleleID® can be used to design species identification/cross species probes for microarrays or real time PCR including SYBR® Green, TaqMan® MGB, TaqMan® probes, Molecular Beacons and real time PCR primers. AlleleID® also offers support for designing microarray experiments for detecting alternative splicing events.
Cross Species/Taxa Specific Assays
Species Identification Assays
Gene Splicing/Alternative Splicing using Microarrays
Exon Junction Primer Design
NEAT Assay Design
Database Support
Input & Output Formats
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