为了准备引物,从NCBI数据库中获取了这些基因的序列,这些序列来自1171个完整的大肠杆菌基因组。使用MAFFT进行多重序列比对,选择保守片段,并基于这些片段构建了用于新型多重PCR测试的引物。 然后,我们在一个由47370个不同肠杆菌科基因组组成的大型数据集上使用体外PCR(in silicoPCR, isPCR)对这些引物进行了测试。结果显示...
National Center for Biotechnology Information, National Library of Medicine, Building 38A, Bethesda, MD, 20894.http://blast.ncbi.nlm.nih.gov/blastcgihelp.shtml Nomenclature for incompletely specified bases in nucleic acid sequences (1984)http://www.chem.qmul.ac.uk/iubmb/misc/naseq.html ...
为了确保特异性,引物设计时应使用专门设计的引物设计工具,如NCBI Primer-BLAST和UCSC In-Silico PCR等。这些软件可以帮助在基因组中引物,检测与其他序列的杂交,从而提高引物的特异性。 4.避免自身或互相杂交:引物之间和引物自身之间的互补序列会导致偶联和二次结构的形成,从而干扰PCR扩增。因此,在设计引物时应避免引物...
OligoPicker - OligoPicker picks specific oligos by skipping regions with contiguous bases common in other sequences. In addition, oligo specificity is double-checked by NCBI BLAST. Sequence regions similar to non-coding RNAs are avoided because total RNA is often used for array hybridization. Low-...
采用BeaconDesigner8.0软件,设计特异性引物及探针,在NCBI数据库进行blastn比对分析,再通过在线生物信息工具“insilico”模拟待选引物的PCR扩增结果,最后经过菌株基因组DNA的普通PCR扩增验证引物特异性,确认荧光定量PCR引物的序列如下: TQM-f:5’-cagaccgttatagtgaattc-3’。(SEQIDNO.1) TQM-r:5’-ctgcattcttacctg...
In in silico test using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), the designed primer sets for latipes and sakaizumii were found to amplified only the target medaka species. In real-time single PCR with extracted genomic DNA, both of the developed primer probe sets...
NCBI BLAST+ blastn Biopythonunderpython3 pandoc Rversion 3.4.2 or higher with the following packages installed: readr dplyr tidyr stringr knitr Installation ... Command line options Main script usage % clermonTyping.sh Script usage : -h : print this message and exit --fasta : fasta contigs...
In-silico two state melting hybridizations were performed to understand the Tm variations using the DINAmelt application. The final list of primers and probes used in this study are listed in Table 1S. Due to the presence of mutations in the primer binding region of the Omicron variant and ...
采用beacondesigner8.0软件,设计特异性引物及探针,在ncbi数据库进行blastn比对分析,再通过在线生物信息工具“insilico”模拟待选引物的pcr扩增结果,最后经过菌株基因组dna的普通pcr扩增验证引物特异性,确认荧光定量pcr引物的序列如下: tqm-f:5’-cagaccgttatagtgaattc-3’。(seqidno.1)...
在本次实验中,除使用iPCRess软件和In-silicoPCR软件来预测候选引物是否非特异性扩增外还使用NCBIPrimerBlast和MFEPrimer3两款网页工具作为对比,预测的结果请参见表5。 表5:非特异性扩增的预测结果 从表5中能够看出,ATMe59_F7的PCR实验的电泳结果有多条带或无扩增;KLKB1e6_F12的PCR实验的电泳结果有多条带;L_...