ThermoSleuth - PCR分析设计工具

ThermoSleuth原名为ThermoBLAST，针对基因组几何扫描多个引物以查找全部杂交和扩增子。ThermoSleuth发现全部错误杂交位点，消灭PCR中的假阳性。

解决引物异性

ThermoSleuth根据大型基因组数据库自动扫描寡核苷酸，以检测全部热力学稳定的命中。与BLAST不同，ThermoSleuth通过考虑以下因素来捕获关键的错误杂交命中：

• 适当的热力学评分，因为BLAST不会区分GC对和AT对

• 显示全部假扩增子

• 考虑3'的可扩展性和稳定的错配

• ThermoBLAST根据互补性而不是BLAST中的相似性进行评分

• ThermoBLAST允许溶液条件、盐、缓冲液、添加剂和其他实验因素

PCR假阳性问题

PCR中假阳性的常见原因是由于引物杂交导致假扩增子的形成，而BLAST未检测到这种情况，因为它使用序列相似性而不是使用匹配和错配互补性的正确规则对命中进行错误评分。此外，很多研究人员无法获得现代基于基因组的分析设计所需的计算能力或存储空间。

PCR假阳性的解决办法

ThermoBLAST CE和ThermoBLAST通过使用BLAST的速度扫描人类基因组或微生物组等全基因组数据库，同时将适当的热力学模型应用于错误杂交和交叉杂交结果，从而解决了引物和探针错误杂交的问题。ThermoBLAST CE的云集成使全部具有计算能力的研究人员能够捕获50倍于BLAST的热力学稳定和可扩展的命中。

预制和可定制的基因组集合

ThermoBLAST-CE包含一个庞大的精选和预格式化序列数据库存储库，可以进一步排列成定制的序列播放列表。下面显示了一些受欢迎的播放列表。客户现在可以在不限制内存、存储或计算能力的情况下构建所需大小的后台数据库，这对于那些本地计算能力有限的研究人员来说是一个巨大的痛苦来源。

【英文介绍】

Scans multiple primers against collections of genomes to find all hybridizations and amplicons.

ThermoBLAST finds all mishybridization sites, eliminates false positives in PCR.

Solving Primer Specificity

ThermoBLAST automatically scans oligos against large genome databases to detect all thermodynamically stable hits. Different from BLAST, ThermoBLAST captures the important mishybridization hits by considering the following:

• Appropriate thermodynamic scoring, as BLAST doesn't differentiate a GC pair from an AT pair

• Displays all false amplicons

• Consideration for 3' extensibility and stable mismatches

• ThermoBLAST will score based on complementarity as opposed to similarity in BLAST

• ThermoBLAST allows for solution conditions, salt, buffers, additives and other experimental factors

The problem of false positives in PCR

The most common cause of false positives in PCR is the formation of false amplicons due to primer mishybridization which is not detected by BLAST because it scores hits incorrectly using sequence similarity rather than using the proper rules for match and mismatch complementarity.  Additionally, many researchers do not have access to the computational capacity or storage required of modern genomic-based assay designs.

The solution to false positives in PCR

ThermoBLAST CE and ThermoBLAST solves the problem of primer and probe mishybridization by scanning against whole genome databases such as the human genome or microbiome using the speed of BLAST while applying the proper thermodynamic model to mishybridization and crosshybridization results. The cloud integration of ThermoBLAST CE enables all researchers with the computational power to capture 50X as many thermodynamically stable and extensible hits as BLAST.

Pre-made and customizable genome collections

ThermoBLAST-CE contains a huge repository of curated and pre-formatted sequence databases that can further be arranged into a customized sequence playlist. Some of the most popular playlists are shown below. Customers now have the capability to build a background database as large as they need without restricting memory, storage or computational capacity which is a huge source of pain for those researchers that have limited local computational capacity.