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針對(duì)基因組幾何掃描多個(gè)引物以查找全部雜交和擴(kuò)增子。ThermoBLAST發(fā)現(xiàn)全部錯(cuò)誤雜交位點(diǎn)，消滅PCR中的假陽(yáng)性。
解決引物異性
ThermoBLAST根據(jù)大型基因組數(shù)據(jù)庫(kù)自動(dòng)掃描寡核苷酸，以檢測(cè)全部熱力學(xué)穩(wěn)定的命中。與BLAST不同，ThermoBLAST通過(guò)考慮以下因素來(lái)捕獲關(guān)鍵的錯(cuò)誤雜交命中：
適當(dāng)?shù)臒崃W(xué)評(píng)分，因?yàn)锽LAST不會(huì)區(qū)分GC對(duì)和AT對(duì)
顯示全部假擴(kuò)增子
考慮3'的可擴(kuò)展性和穩(wěn)定的錯(cuò)配
ThermoBLAST根據(jù)互補(bǔ)性而不是BLAST中的相似性進(jìn)行評(píng)分
ThermoBLAST允許溶液條件、鹽、緩沖液、添加劑和其他實(shí)驗(yàn)因素
PCR假陽(yáng)性問(wèn)題
PCR中假陽(yáng)性的常見(jiàn)原因是由于引物雜交導(dǎo)致假擴(kuò)增子的形成，而BLAST未檢測(cè)到這種情況，因?yàn)樗褂眯蛄邢嗨菩远皇鞘褂闷ヅ浜湾e(cuò)配互補(bǔ)性的正確規(guī)則對(duì)命中進(jìn)行錯(cuò)誤評(píng)分。此外，很多研究人員無(wú)法獲得現(xiàn)代基于基因組的分析設(shè)計(jì)所需的計(jì)算能力或存儲(chǔ)空間。
PCR假陽(yáng)性的解決辦法
ThermoBLAST CE和ThermoBLAST通過(guò)使用BLAST的速度掃描人類基因組或微生物組等全基因組數(shù)據(jù)庫(kù)，同時(shí)將適當(dāng)?shù)臒崃W(xué)模型應(yīng)用于錯(cuò)誤雜交和交叉雜交結(jié)果，從而解決了引物和探針錯(cuò)誤雜交的問(wèn)題。ThermoBLAST CE的云集成使全部具有計(jì)算能力的研究人員能夠捕獲50倍于BLAST的熱力學(xué)穩(wěn)定和可擴(kuò)展的命中。
預(yù)制和可定制的基因組集合
ThermoBLAST-CE包含一個(gè)龐大的精選和預(yù)格式化序列數(shù)據(jù)庫(kù)存儲(chǔ)庫(kù)，可以進(jìn)一步排列成定制的序列播放列表。下面顯示了一些受歡迎的播放列表?？蛻衄F(xiàn)在可以在不限制內(nèi)存、存儲(chǔ)或計(jì)算能力的情況下構(gòu)建所需大小的后臺(tái)數(shù)據(jù)庫(kù)，這對(duì)于那些本地計(jì)算能力有限的研究人員來(lái)說(shuō)是一個(gè)巨大的痛苦來(lái)源。
【英文介紹】
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.
