Primers of all genes in Bacillus subtilis strain 168 without recognition sequence of NdeI and BamHI

Primer design is a critical part of the molecular cloning workflow for amplifying target genes from a template. Serving as a molecular marker for the region in which polymerase chain reaction should amplify a particular gene segment, primers should have perfect complementary base pairing with the target strand of the DNA duplex in which the gene resides. Beyond the binding sequence that anneals a primer to the target DNA strand at the correct location, recognition and flanking sequences are also typically appended to primer sequence to aid downstream restriction enzyme digestion for molecular cloning purposes. Specifically, recognition sequence appended to the primer facilitates recognition by specific restriction enzyme for digestion and excision of the gene fragment that is subsequently inserted into an expression vector. However, one critical requirement for the choice of restriction enzyme is that the gene of interest should not contain recognition sequence of the restriction enzyme. In this contribution, a primer database for all genes in <i>Bacillus subtilis</i> strain 168 (Genbank ID number: NC_000964.3) that do not contain recognition sequence of restriction enzymes, NdeI and BamHI was obtained using an in-house MATLAB software processing the annotated genome sequence of <i>B. subtilis</i> strain 168 from Genbank. The database comprises the gene identities together with the forward and reverse primers necessary for amplifying and inserting the gene of interest into an expression vector. NdeI recognition sequence was appended to the forward primer, while that of BamHI was appended to the reverse primer. Melting temperature of the primer was designed to be 55 ^oC. Two nucleotide flanking sequence of ‘AT’ was appended to the start of the forward and reverse primers to help anchor the RNA polymerase during transcription. Altogether, the database comprises 3202 genes that are without recognition sequence of NdeI and BamHI. Collectively, a primer database comprising genes in <i>B. subtilis</i> strain 168 that are without recognition sequence of NdeI and BamHI restriction enzymes was created using a MATLAB algorithm. Both forward and reverse primers sequence are available in the database and should find use in workflows attempting to clone specific genes from the <i>B. subtilis</i> strain 168 genome using NdeI and BamHI for the forward and reverse primers respectively.<br><br>

引物设计是从模板扩增靶基因的分子克隆流程中的关键环节。作为聚合酶链式反应（PCR）扩增特定基因片段区域的分子标记，引物需与该基因所在DNA双链的靶链实现完美的碱基互补配对。除了能在正确位置将引物与靶DNA链退火结合的结合序列外，通常还会在引物序列中添加识别序列与侧翼序列，以辅助后续分子克隆所需的限制性内切酶酶切操作。具体而言，添加至引物的识别序列可帮助特定限制性内切酶识别并酶切、切除基因片段，以便后续将其插入表达载体。但选择限制性内切酶时有一项关键要求：目的基因中不得包含该内切酶的识别序列。本研究中，我们利用自研MATLAB软件处理从GenBank获取的枯草芽孢杆菌（Bacillus subtilis）168菌株的注释基因组序列，构建了针对该菌株所有不含限制性内切酶NdeI与BamHI识别序列的基因的引物数据库。该数据库包含基因标识，以及用于扩增目的基因并将其插入表达载体所需的正向引物与反向引物序列。正向引物末端添加了NdeI识别序列，反向引物末端则添加了BamHI识别序列。引物的解链温度（Tm）设计为55℃。在正向与反向引物的起始端各添加了两个碱基的侧翼序列“AT”，以辅助转录过程中RNA聚合酶的锚定。最终该数据库共收录了3202个不含NdeI与BamHI识别序列的基因。综上，本研究利用MATLAB算法构建了针对枯草芽孢杆菌168菌株中不含NdeI与BamHI限制性内切酶识别序列的基因的引物数据库。数据库中收录了所有正向与反向引物序列，可用于以NdeI（正向引物）、BamHI（反向引物）分别进行酶切，从枯草芽孢杆菌168菌株基因组中克隆特定基因的相关实验流程。