**1. Introduction**

To date, nucleic acids have been used not only for biological studies but also as powerful biomarkers for clinical diagnosis, agriculture, forensic science, and so on. PCR is a well-known molecular biology tool for the amplification of target sequences. In PCR, DNA amplification relies on heating and cooling of nucleic acids followed by hybridization. It can efficiently amplify target sequences within a few hours in three temperature-dependent steps: initiation,

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annealing, and elongation. However, PCR requires thermocyclers for reaction temperature adjustment and trained personnel, which hinders its usage in resource-limited settings; and additionally, it has mispriming and sometimes inadequate template amplification. Therefore, the development of simple and inexpensive methods for nucleic acids amplification and detection is important for on-site inspections.-

Whilst several isothermal amplification platforms, such as nucleic acid sequence based amplification (NASBA) [1], strand displacement amplification (SDA) [2–5], loop-mediated isothermal amplification (LAMP) [6], rolling circle amplification (RCA) [7], recombinase polymerase amplification (RPA) [8], and Helicase dependent Isothermal DNA Amplification (HAD) [9, 10] have been developed. These amplification platforms can achieve linear or exponential dsDNA accumulation, and some of which can be purchased as kits and integrated in portable devices [5], so no expensive instruments are needed, but there are still challenges in multiplexing, primer design, and stringency in experimental design. In this chapter, we describe the development and current directions of SDA in detail. Prospects in analysis using fluorescence, colorimetry, and lateral flow biosensors are also discussed.-

Moreover, CRISPR-Cas system is also currently exploited for the detection of nucleic acids. For- instance, some CRISPR-Cas proteins have been found to exhibit collateral cleavage of target- nucleic acids and any nonspecific single-stranded nucleic acids in the solution. Therefore, if the- latter is labeled with a fluorescent or a specific antibody recognized molecule, detectable signal- can be generated [11–13]. This combination of isothermal amplification and various CRISPR-Cas-based signal readouts is simple, fast, specific, and sensitive and thus can elevate the specificity of nucleic acid diagnosis. In addition, CRISPR-Cas-based diagnosis can be multiplexed to- provide another convergent evolution and convenient point-of-care detection of nucleic acids- in low cost.-

### **2. Strand displacement amplification (SDA)-**

SDA is carried out under isothermal condition. It is inspired from normal physiological RNA transcription and DNA replication, which occurs at a constant temperature. Over the past 2 decades, SDA has been widely used as an alternative to PCR for the detection of pathogens [14, 15], hereditary diseases [16], and cancers [17–19]. Moreover, SDA amplified nucleic acids can be multiplexed and readily provide optical and visual readouts [14, 20, 21]. In a typical SDA, two pairs of primers are designed to specifically recognize two regions of a target sequence. One pair is bumper primers designed as standard PCR primers, and another pair is SDA primers, which bind immediately next to the bumper primers at the target sequences. In addition, a HincII restriction enzyme and an exonuclease deficient (exo<sup>−</sup> Klenow) polymerase are added. The reaction mixture is incubated in a single constant temperature of 37°C.-In this reaction, the *Hinc*II cleaves at the recognition site of the phosphorothioate (modifiedsubstrate, dATPαS) of the DNA probe, and the *exo*<sup>−</sup> Klenow initiates the replication of the sequence. Subsequently, the exponential reaction starts with the primer-triggered repeated cycle of nicking, extension, and strand displacement. This amplification is accelerated by additional primers franking the inner region of the target sequence, and this reaction exhibits a single and double nicking site cycle (**Figure 1**). The final new product chain yield can reach 10<sup>7</sup> fold amplification within 2h [2, 20]. However, unlike the original method of cutting doublestranded sequences using restriction enzymes, the modified SDA uses nicking endonucleases (engineered restriction enzymes) such as Nt.BsmAI, Nb.BsmI, Nb.BsrDI, Nt.BspQI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, and Nt.BstNBI (**Table 1**) to enhance the site-specific cleavage accuracy of target sequences and do not require modified dNTPs such as dATPαS at the cleavage site.-

These endonucleases effectively create a nick for Klenow fragment or *Bst* polymerase to- initiate a new strand replication and displace the downstream strand. The improved SDA- exhibits an enhanced exponential amplification at 37–55°C, with 10<sup>9</sup> -fold amplified DNA- in <30min [3]. The amplified nucleic acid products can be detected by photometry (turbidity), electrophoresis, SYBR Green's DNA-insertion dye, cross-flow text of a sequence-specific- hybridization capture probe, or visual inspection of white precipitate. As shown in **Figure 1**, DNA double-strand is denatured, and primers (S1, S2, B1, and B2) bind to a specific DNA- polymerase such as exo<sup>−</sup> Klenow or Bst and then extend through the strand displacement- activity. Upon the formation of strand containing the nicking site (linear amplification), the- nicking enzyme cleave it, and the DNA polymerase primes initiate a new round of replication (1st cycle). The exponential amplification continues with a cycle of single and doublenicking, extension, and displacement cycles, producing a targeting sequence (SDA product).-

**Figure 1.** Schematic illustration of strand displacement isothermal amplification principle. First, double strand DNA is heat denatured for primer binding. The SDA primers (S1 and S2) contain recognition sequence for nicking endonuclease (green and yellow), a linker (blue), and sequence complementary to a target sequence (red). Bumper primers (B1 and B2) are located upstream of the SDA primers (red) and are complementary to the target sequence (black and gray). These primers bind to target sequence and extend by specific DNA polymerase such as exo<sup>−</sup> Klenow or Bst with strand displacement activity. Upon formation of strands harboring a nicking site (linear amplification), the nicking enzyme nicks them, and the DNA polymerase primes a new round of replication (first cycle). Exponential amplification of the target sequence starts by cycle of single and double nicking, extension, and displacement.-


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**Table 1.** Singlex and multiplex detection of nucleic acids using SDA-based assays.-

### **2.1 Experimental procedures**

## *2.1.1 Design of SDA primers*

Primers are prerequisite for the initiation of nucleic acid amplification. SDA requires two pairs of- primers, SDA primers (S1 and S2), and Bumper primers (B1 and B2). Bumper primers are similar- to standard PCR forward and reverse primers for the identification of specific sites and amplification of a target DNA sequence. The B1 and B2 primers are 18–23 nucleotides designed based- on the target strand from the 5′ end and a reverse complementary sequence of the 3' end strand,- respectively. However, the S1 and S2 primers are a pair of 5′ end-turned special primers. From- 5′ to 3′, both primers contain a protecting 10–15 nucleotides, a nicking recognition sequence (~5- nucleotides), linkage sequence (~4 nucleotides), and then a 10–18 nucleotides complementary to- the target sequence at the 3' end immediately adjacent to a bumper primer (**Figure 1**).-

### *2.1.2 Properties and choice of nicking endonuclease*

Restriction endonucleases are well-known endonucleases that recognize and cleave palindromic DNA sequences. Nicking endonucleases (NEases), on the other hand, cleave one- strand of a specific DNA sequence. Typically, homodimer restriction enzymes bind to two- half sites of a specific palindromic sequence, that is, each monomer cut one strand. However,- both the nicking endonucleases and one strand-cleaving restriction enzyme are heterodimers,- allowing only single nicking or cleaving at the asymmetric recognition sequences. Through- the genetic engineering of naturally occurring restriction enzymes, various nicking endonucleases, such as Nb.BbvCI and Nt.BbvCI, have been generated through modifications of the- catalytic activity of the asymmetric amino acid sequences of the enzymes. BbvCI, a heterodimeric Type IIS endonuclease recognizing 7bp of asymmetric DNA sequence, uses its R1 and- R2 subunits at the catalytic sites to cleave bottom strand and top strand (..CC↓TCAGC.. and- ..CCTCA↑GC..), respectively [3, 42]. The Nt.BbvCI is an engineered BbvCI with functional- R2 and a missing R1 domain which can only cleave top strand (..CC↓TCAGC..) [42, 43], while Nb.BbvCI was engineered from BbvCI, with functional R1 and deficient R2, which- can only cleave bottom strand (CCTCA↑GC..) [43]. However, other nicking endonucleases- were generated through mutations, truncation, and domain swapping. These nicking endonucleases include bottom strand nickase such as Nt.AlwI, Nb.BsmI [44], Nb.BsrDI, Nt.BspQI- [45], Nb.BtsI, and Nt.CviQII and top strand nickase such as Nt.CviPII [46–48]. For example,- Nt.AlwI was engineered from the dimeric AlwI (dsDNA cleaving REase) to cleave only- the top strand of the AlwI target sequence (..GGATCNNNNN↓N..). The Nt.AlwI monomer- structure derives from the dimerization swapping with nonfunctional domain of Nt.BstNBI- [49]. Thus, as shown in **Table 1**, the choice of nicking enzymes may depend on the preferred- DNA polymerase and the desired type of reaction to achieve an efficient amplification.-

### *2.1.3 Properties and choice of DNA polymerase*

The combinations of nicking enzymes and DNA polymerases have great effect on the amplification efficiency. For example, among several nicking enzymes and DNA polymerases studied, Nt.BspQI coupled with Sequenase 2.0 polymerase showed a higher linear SDA amplification [47], and Nt.BstNBI coupled with Bst DNA polymerase showed a 10 times higher exponential amplification compared to other combinatorial NEases. The authors attributed this property to the enzymatic conformation and concentration, as higher or lower concentration could be ineffective for the SDA reaction [50]. DNA polymerase such as exo<sup>−</sup> Klenow and Bst (Bst 2.0, Bst 2.0 WarmStart, and Bst 3.0) have good amplification performance and are good choices for SDA [3]. Although Exo<sup>−</sup> Klenow exhibits excellent performance after binding to specific nicking enzymes like Nb.BbvCI, the amplification efficiency decreases by 5–100 folds when the target nucleic acid sequence increases by 50 base pairs [47]. Bst exhibits a similar limitation. To address the limitation of Exo<sup>−</sup> Klenow and Bst, newly engineered DNA polymerases such as Bst 2.0 show higher efficiency, thermal stability, salt tolerance, and greater fidelity in SDA amplification. Xu etal. showed that Bst 2.0 polymerase highly prefers Nt.BbvCI rather than other conventional nicking enzymes, and this combination showed good results for the detection of viral, bacteria, and *BRCA1* gene sequences. However, various inhibitors can affect the activity of most polymerases. Fortunately, Bst 3.0 was designed to amplify both RNA and DNA with high activity even in the presence of amplification inhibitors.-

Furthermore, to encounter mispriming occurring at lower temperatures and isothermal amplification stringency, Bst 2.0 WarmStart was engineered. A specific aptamer (unique oligonucleotide sequence) targeting Bst DNA polymerase via noncovalent binding has been selected- using the systematic evolution of ligands by exponential enrichment (SELEX). The Bst DNA- polymerase with the bound aptamer cannot perform strand displacement unless the temperature is raised to 50°C, thereby minimizing the unwanted isothermal preamplification usually- occurring at room temperature, and mispriming is therefore prevented. Another attempt for- limiting undesired preamplification and spurious results is by performing all sample preparation steps in ice before starting the reaction. By this simple tuning combined with the best- DNA polymerase, the detections for *C. elegans*, *E. coli*, *λ-*phage specific genes, and Hela cell- genomic DNA showed rapid (~10min) and consistent isothermal amplification [51, 52].-
