Homologous sequences, either long or short, always accompany our DNA recombination trials in laboratory. We must also admit that the principles underlining our DNA recombination methods exist in nature, and we have been learning from nature in developing these methods. The first successful DNA recombination trial was achieved with the aid of enzymatically added “A” and “T” polymers (Jackson et al., 1972), to which a Nobel Prize in Chemistry was awarded in 1980. The second earliest trial was the restriction enzymes (RE)-based plasmid construction (Cohen et al., 1973), which pioneered the later DNA recombination. RE-based DNA recombination joins a DNA fragment and a linearized vector. When the construct is transferred into a bacterial cell, it replicates there and simultaneously the DNA fragment is cloned. In RE-based cloning, the DNA fragment can be oriented when the differential cohesive ends after RE digestion are available. The RE-based recombination generally involves cycles of tedious digestion and ligation and introduces unwanted scars into the construct, and the fragments must be free of restriction sites used to retrieve or generate them. These limitations become almost insurmountable when the number and the length of the DNA fragments increase. The ways of DNA recombination towards the high efficiency and precise directionality have been continuously paving for the DNA recombination. Sets of recombination techniques have been developed or are developing nowadays, which are receiving endless modifications, improvements and combinations.

It seems that the word “recombination” is not fully appropriate any more for describing the process of joining DNA fragments together; the number, the length and the directional preciseness of the joined fragments have increased significantly. In contrast, “DNA assembly” has evolved as an alternative of DNA recombination. Both recombination and assembly document the process of joining DNA fragments while assembly implies large number and size and precise directionality. Accordingly, the “DNA assembly” has been adopting by researchers to document the process of DNA recombination. The phrase has also been using by genomic and transcriptomic sequencers to document in silico DNA assembly in which the clean reads generated on sequencing platforms are assembled into contigs (continuous sequences) according to the sequence overlapping information alone or overlapping and linkage information in combination. The avalanching genomic and transcriptomic data have featured in silico DNA assembly. Assembling DNA fragments is also a hidden step in the chemical synthesis of DNA. At present, the chemical synthesis of DNA sequences longer than 200 bp remains unaffordable. To overcome such limitation, the technologies detouring complete chemical synthesis have been developed for long DNA chemical synthesis, which are like DNA assembling methods to some extent. In addition, the phrase “DNA assembly” has been used to describe the making processes of DNA nanostructures. An early synonym of assembling, DNA splicing, compared the DNA assembly to the splicing of messenger RNA precursors (Zarghampoor et al., 2020). Splicing implies the possibility of multiple products while assembling assures the unique product. Currently, DNA assembly is widely used as the alternative of DNA recombination.

Ahead of the word “cloning”, we always use the words like “molecular”, “DNA” and “gene” to restrict its connotation. To my understanding, gene cloning must isolate gene sequence and clarify its function simultaneously while DNA cloning just take to amplify the DNA sequence itself. Unless otherwise stated, DNA assembling refers to DNA recombining, DNA cloning, DNA joining among others in this review. We used “cloning” when the assembly is aimed to amplify DNA fragments in this review. To distinguish the DNA assemblies according to their size, we defined the phase one assembly the protein- or RNA-coding cassettes (the genes) consisting of the coding sequences and the expression controlling elements like the promoter and the terminator; the phase two assembly a set of genes responsible for a specific function like a metabolic pathway; and the phase three assembly a cluster of the phase two assemblies either concertedly working in a cell or artificially recombined for a cell in this review. These assemblies at different phases may overlap each other (e.g., the enzymatic activity is also a specific function), and they may also include selection markers, reporters, the elements for their replication and stability and the expression of the genes they carried.

The improvements, modifications and combinations of the DNA assembling techniques have complicated the literature environment, and hardened our identification of the pioneering DNA assembling techniques and adoptions of the most appropriate assembling methods. The DNA assembling techniques evolves fast. Some of them were smash hits while others classic old songs representing the evolution trend of DNA assembling methodology. Identifying and outlining these pioneering techniques should be highly appreciated; such trial will provide researchers the convenience of using and flourishing these techniques, and assist them to set a clear direction for the future work. Recently, studies on the molecular biology and synthetic biology using the microalgae as the materials are burgeoning. In this review, we summarized the pioneering DNA assembling techniques available, extended them to their modifications, improvements and combinations (variants), and highlighted their applications in eukaryotic microalgae. We predicted the evolution trends of DNA assembling techniques in microalgae from genes to gene clusters and the gene stacking strategies on normal microalgal chromosomes, episomes and looming artificial chromosomes of microalgae (Table 1, Table 2; Fig. 1, Fig. 2, Fig. 3).