![]() ![]() “Staden”, containing a graphic user interface, provides a free and powerful package for analyzing giant genome sequencing by combining the “SPIN” and “EMBOSS” ( Staden, Judge & Bonfield, 2003), again, this package is complicated for some users. “Cap3” is a free and effective program for merging DNA fragments, however, the command-line is complicated for users who are not familiar with commands of “Cap3” ( Huang & Madan, 1999). Nevertheless, the web tool relies on web access and server status. In terms of free software, there is an online tool that is able to merge overlapping long sequence fragments based on the program merger in the “EMBOSS” suite ( Bell & Kramvis, 2013 Rice, Longden & Bleasby, 2000). Thereby, some functions of these commercial software are not essential, and the price of these software might be too high for the routine work of molecular cloning. For protein science and molecular biology, sequencing is mostly used to confirm whether the subcloning or mutagenesis is correct. To merge the multiple walking results, several commercial software can be applied, such as DNASTAR (DNASTAR, Inc., Madison, WI, USA), DNAMAN (Lynnon Biosoft, San Ramon, CA, USA), Vector NTI (Thermo Fisher Scientific Inc., Denver, CO, USA), and SnapGene (GSL Biotech LLC., San Diego, CA, USA), which are robust, however, expensive. It is preferred to merge all the walking results before alignment with the correct gene file, rather than aligning each walking result with the target gene file separately ( Tang et al., 2020a, 2020b), especially for those large genes, which may be over 10,000 bps in length. These results have to be aligned to the correct gene file for confirmation. To sequence a large gene, several reactions might be required in both forward and reverse directions. To get the full-length sequence of a target gene, it is necessary to carry out DNA walking sequencing using a new primer based on the previous sequencing result. However, in other cases, the lengths of the genes exceed one thousand nucleotides, beyond the scope of one single Sanger sequencing reaction. For those genes with lengths of only several hundred nucleotides, it is possible to go through each one of the whole sequences with one single Sanger sequencing reaction, and the results can be directly aligned to the correct gene files. However, the number of nucleotides determined by one Sanger sequencing reaction is around 1,000. Though the next-generation sequencing has started a new era, Sanger sequencing is still one of the most popular methods due to its reliability, affordability, and feasibility ( Schuster, 2008). Sanger sequencing has been widely applied to determine the DNA sequence since it was reported first time by Sanger et al. ![]() Sanger sequencing is a DNA sequencing technology by incorporating chain-terminating dideoxynucleotides, which are fluorescently labeled and can be read-out by automated sequencing machinery. To ensure that the gene sequence is correct within the constructed plasmid, the Sanger sequencing is utilized to sequence the gene, and the results are aligned and checked ( Zimmermann et al., 1988). For the most part, the gene of concern is ligated to a vector with a canonical method of restriction enzyme cutting and ligation, or the new-fashioned technique of seamless ligation ( Okegawa & Motohashi, 2015). Gene cloning is customarily required to study the gene functions in vivo and in vitro. ![]()
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