Reverse PCR is used to find a restriction endonuclease with more enzymatic sites and broader distribution in the mutant genome, but no enzymatic sites or only one enzymatic site in the transposon sequence or fragment the genome by enzymatic digestion. The DNA fragment is self-associated after enzymatic digestion by ligase to cyclize it. Specific primers can be designed from the transposons if the cyclized genome contains transposons. Specific primers can be designed from the transposon for amplification and sequencing to obtain the transposon insertion site if the transposon is included in the genome. The sequence obtained is on both sides of the insertion site if there is no enzyme cut site in the transposon (Figure 1A). Finally, the sequence obtained is on one side of the insertion site if there is a single enzyme-cut site (Figure 1B).

The reverse PCR method is simple in principle and operation, and its experimental cost is low. However, it is unsuitable for high-throughput sequencing. Additionally, it is not stable as a sequencing method because of its specific requirements for selecting restricted endonucleases and restrictions on the transposon insertion position. In addition, its sequencing length is not fixed. Feng et al. (2008) used reverse PCR to amplify the 5′ and 3′ flanking sequences of the TaCKX1 gene. These researchers obtained the full-length DNA sequence of TaCKX1 by cloning the TaCKX1 fragment from a conserved sequence of wheat cytokinin oxidase/dehydrogenase (CKX). Wang et al. (2021) used reverse PCR to amplify and detect the FSTA gene in transgenic zebrafish genomic DNA. This method may be used for gene doping detection in blood samples or to assess the safety of gene therapy and GMOs. Su et al. (2021) used this assay for transposase-accessible chromatin using the sequencing (ATAC-seq) method combined with the “Circle_finder” bioinformatic algorithm to predict extrachromosomal circular DNA (eccDNA) in human cancer cells. These researchers validated the detection of eccDNA using reverse PCR. Gutiérrez et al. (2021) used reverse PCR to identify chronic myeloid leukemia (CML) at the genomic level with the breakpoint sequence of the signature fusion gene BCR-ABL1. They applied this method to seven real cases.

The principle of plasmid rescue is similar to reverse PCR. The standard operation is (1) to insert the transposon into the genome, (2) digest it with a restriction endonuclease, (3) ligate it into a cloning vector, (4) transform it into an Escherichia coli culture, (5) select positive bacteria according to the label carried by the plasmid, (6) culture it, (7) extract its plasmid, and (8) sequence it according to the specific primers of the transposon and plasmid (Tsurumaru et al., 2008; Figure 2).

The advantage of the plasmid rescue method is the high specificity of the fragments obtained. The disadvantages of this method are the requirement for the selection of endonucleases, the lack of experimental stability, and the unsuitability for large-scale high-throughput sequencing. Huang et al. (2009) used a combination of plasmid rescue and reverse PCR to simultaneously determine the sequences on both sides of the Drosophila P-transposon insertion site. Kemppainen et al. (2008) randomly determined the genomic DNA sequences of the T-DNA right border (Rb) of 51 strains from a considerable number (~500) of T-DNA insertion mutants of Laccaria bicolor using plasmid rescue. Sixty-nine percent of the flanking sequences of this species were successfully determined. At the same time, 87% of these sequences were successfully localized in the genome.

Some specific restriction endonucleases are used in this method. These endonucleases have a common point: the enzymatic cut site is located after several bases of the recognition site. Therefore, a segment of the base sequence of the recognition site can be left after the enzymatic cut. For example, MmeI (Figure 3A) and EcoP15I (Figure 3B) can retain about 18 ~ 27 bp after the recognition site after enzymatic cleavage. Therefore, these enzymatic cleavage sites can be inserted at both ends of the transposon. The insertion site was sequenced by amplifying the specific sequences on the specific motifs by enzymatic ligation or by directly ligating special connectors. Finally, the insertion site was found against the target genome (Figure 3C).

The advantage of this method is that it is simple. Only particular enzyme cleavage sites must be added on both sides of the transposon. It can support high-throughput large-scale sequencing by connecting Illumina adapter sequences. The disadvantage of this method is related to the short localization of the sequence, which is only about 38~52 bp. Therefore, sometimes even the genome length that can be measured is less than 38 bp to consider joint connection and sequencing problems. The specificity of the method is weak, and the position of the transposon insertion mutation cannot be accurately determined in some genomes with more repetitive sequences or palindromic sequences. Additionally, the particular enzyme cleavage method cannot be used for transposons, in which transposase recognition sites are at both ends because the enzyme cleavage sites cannot be added.

Regarding the application of the method, Ng et al. (2005) investigated a more accurate and efficient way of determining cDNA using the MmeI endonuclease in conjunction with other common endonucleases. These researchers mapped the cDNA to genomic sequences to delineate the transcriptional boundaries of each gene. Matsumura et al. (2003) used the EcoP15I endonuclease to analyze the sequence of cDNA applied to monitor the genome sequences of rice and P. aeruginosa. These researchers found that hydrophobic protein genes were the most actively transcribed in P. aeruginosa leaves. They also studied gene expression changes in Benthamiana, a model organism, before the hypersensitive response induced by INF1, allowing the rapid identification of genes that were up- or down-regulated by the induction. Zhang et al. (2014) investigated a method to determine the transcriptional boundaries of a high-throughput sequencing method for determining transposon insertion sites in Chlamydomonas reinhardtii. The species was investigated and applied to a mutant library, and 11,478 insertion sites were identified.

The basic principle of TAIL PCR is the same as nested PCR. TAIL PCR is based on designing multiple sets of nested specific primers with a higher annealing temperature (Tm) on the transposon and a shorter and lower Tm value of random simpler primers. The target sequence was amplified by amplifying different specific primers and simpler primers using the difference in Tm values (Liu et al., 1995; Liu and Wittier, 1995).

The commonly used TAIL PCR amplification method consists of three cycles of PCR reactions (Table 1; Liu and Huang, 1998). In the first cycle, products amplified by a specific primer one and simplex primer are yielded by the PCR reaction (type I). The products were amplified by a particular primer (type II). The products are amplified by a simplex primer (type III). During the second PCR reaction cycle, the product of the first cycle was diluted as a template. The product of the type I primer is selectively amplified using specific primer two. This primer was made using a simplex primer in a thermally asymmetric supercycle. The product of the second PCR reaction cycle was diluted in the third PCR reaction cycle. A template and a specific primer from the third cycle with a simplex primer are used in a normal PCR reaction cycle or a thermally asymmetric supercycle. The target fragment was further amplified to obtain the sequence on one side of the transposon insertion site (Figure 4).

The main advantage of the TAIL PCR method is that it does not require DNA manipulation before PCR. Cyclization and ligation are avoided and have a faster reaction speed, higher specificity, and higher efficiency. However, nonspecific binding due to low temperature can still exist and may sometimes lead to amplification and sequencing failures or situations where the amplified sequence length is insufficient. Various improved versions of amplification protocols are constantly updated as TAIL PCR continues to develop. For instance, the success rate and amplification sequence length are improved by the method by setting multiple sets of more extended simplex primers, increasing the success rate to 90% and the amplification sequence length to 1–3 kb (Liu and Chen, 2007).

Yuan et al. (2009) used a combination of TAIL PCR and plasmid rescue to efficiently identify eight insertional mutation sites in a library of rice streak transposon Tn5 insertional mutants with attenuated virulence on rice. These researchers used this method to determine the corresponding functional genes efficiently. Oranab et al. (2021) used the TAIL PCR technique to examine the T-DNA insertion sites of the activation marker mutants of the CNGC19 and CNGC20 genes in Arabidopsis cyclic nucleotide-gated ion channels (CNGCs) under salt stress conditions. Thus, it lays the groundwork for studying the role of CNGC19 and CNGC20 in Arabidopsis under salt stress regulation. Wang et al. (2013) used a modified TAIL PCR technique to examine the genome of Wolbachia. The WO genome of the mild phage on Wolbachia was determined using a modified TAIL PCR technique. The evolution of the WO genome was also assessed by comparing the WO genomes of infested fig wasps with those of infected insects. The following species were considered: the pink spotted borer moth, Culex mosquito, Drosophila melanogaster, Drosophila anthropomorphis, and the lyre fly nymphal set of golden wasps.

RACE is a technique based on reverse transcription PCR to rapidly amplify the 5′ and 3′ ends of cDNA from samples (Chutia et al., 2020). Since cDNA differs in prokaryotic and eukaryotic algae, and the situation is different at the 5′ and 3′ ends, various amplification methods are described below.

Reverse transcription primers were designed for eukaryotic microalgae to reverse the transcription of the first cDNA strand based on the naturally occurring poly(A) tail at the 3′ end of mRNA (Passmore and Coller, 2021). Specific primers were designed to synthesize the second cDNA strand based on transposon sequences. Subsequently, PCR amplification of the obtained cDNA strand was performed with the specific primer and the 3′ end primer of the righteous strand as a pair of primers to obtain the 3′ end sequence of cDNA (Figure 5A). In contrast, it is necessary to design specific primers based on transposon sequences, since there is no naturally recognizable sequence at the 5′ end of eukaryotic microalgae mRNA. Therefore, it will be possible to reverse transcribe it to obtain the first cDNA strand. At the same time, primer sequences at the 3′ end of cDNA by enzymatic linkage will be added, often with a poly(C) tail, and specific primers will be designed to synthesize the second cDNA strand based on the added sequence. The second cDNA strand was used as a template to synthesize double-stranded cDNA using transposon-specific primers. Finally, the cDNA 5′ end sequence was obtained by PCR amplification using transposon-specific primers and antisense strand 3′ end primers (Figure 5B).

The 3′ end of mRNA does not have a special structure similar to the poly(A) tail for prokaryotic microalgae. Therefore, a splice sequence must be directly attached to the 3′ end of the mRNA to replace the poly(A) tail. The other operations are consistent with the eukaryotic microalgae 3′ end in RACE (Figure 6A). The 5′ end of prokaryotic microalgae in RACE is the same as that of eukaryotic microalgae. This requires the addition of a splice sequence at the 5′ end of the cDNA after reverse transcription and amplification (Figure 6B).

Meslet-Cladière and Vallon (2012) determined the flanking sequences of 38 randomly selected insertion mutants in a transposon insertion mutation library of the model organism C. reinhardtii. These authors used the 3′ end of the RACE technique. Twenty-seven (71%) were valid flanking sequences, and 23 could be accurately localized in the genome. Hu et al. (2017) identified small regulatory RNAs (SRNAs) in Synechocystis sp. PCC 6803 uses 5′ and 3′ ends in the RACE method, naming it RblR. RblR positively regulates the gene rbcL. rbcL encodes a large chain of Rubisco, an enzyme that catalyzes carbon fixation under different stress conditions. Thus, it affects photosynthesis regulation in PCC 6803. Li et al. (2022) determined the sequence of small antisense RNA (ThfR) on the reverse complementary strand of the sll1414 (thf1) gene in PCC 6803 was used in the 5′ and 3′ ends in the RACE technique. These researchers investigated the relationship between ThfR and gene thf1 by examining its high- and low-expression mutants.

Target products are obtained in the linear amplification mediated-PCR (LAM-PCR) method by designing multiple primer sets amplified step by step. The first step is to amplify single-stranded DNA using transposon-specific primers with biotin. The amplified single-stranded DNA is captured by the adsorption of biotin by streptavidin magnetic beads. The insertion site flanking sequence was obtained by amplification and sequencing (Figure 7). This method is precise, has a high success rate, and may be designed to link Illumina junctions in the second round of amplification primers if needed (Carette et al., 2011). However, this method is more expensive for sequencing individual mutants, if not high-throughput sequencing.

Schmidt et al. (2007) used LAM-PCR to detect integration sites representing unique molecular markers for each transduced cell and its clonal progeny in the cells of an integration vector system for clinical gene therapy. Gabriel et al. (2014) used LAM-PCR to demonstrate that leukemia originated from the provirus-induced overexpression of adjacent proto-oncogenes in gene therapy patients. It was possible to bypass restriction digestion with LAM-PCR, eliminating retrieval bias at the integration site. This enabled a comprehensive analysis of the provirus location in the host genome, detailing a stepwise amplification method that integrates adjacent 3′ and 5′ sequences of the lentiviral vector.