Novel Synthetic Nucleotides of Notifiable Dengue (1–4), Japanese Encephalitis, Yellow Fever and Zika Flaviviruses

Figures & data

Table 1.  Profile of the seven notifiable flaviviral species.

Coding number	Flavivirus species	Accession number	Start	End
2	Dengue virus 2	NC_001474.2	9157	9388
3	Dengue virus 4	NC_002640.1	9150	9381
5	Dengue virus 1	NC_001477.1	9158	9389
8	Dengue virus 3	NC_001475.2	9149	9380
15	Zika virus	NC_012532.1	9245	9479
25	Yellow fever virus	NC_002031.1	9227	9461
27	Japanese encephalitis virus	NC_001437.1	9270	9504

Figure 1.  Overlap extension-polymerase chain reaction design algorithm.

This formulation was prepared to algorithmically plan and accurately calculate the desired product for OE-PCR. The process was initially done using manual algorithm whereby executable OE-PCR oligomers can be designed on the limitation of user-determined oligomer length. The platform made use of Linux (Ubuntu).

OE-PCR: Overlap extension-polymerase chain reaction.

Figure 2.  Diagram of the two-step cycle overlap extension-polymerase chain reaction (A: first step; B: second step).

1st PCR: For OE-PCR, 50–60mer oligomers were designed to have 19–23mer overlapping regions and make partial dimers (a). At the start of extension step of 1^st PCR, DNA was synthesized by polymerase which has a 3′→5′ exonuclease activity (b). From the exonuclease activity, some oligomers were digested and replaced by synthesized DNA strand (c). Whole length of DNA was synthesized (d).

OE-PCR: Overlap extension-polymerase chain reactions.

Figure 3.  First and second step overlap extension-polymerase chain reaction showing prominent DNA bands.

Note the almost similar product sizes of 253–256 bp. Each of the four Dengue virus has 253 bp; Zika, yellow fever and Japanese encephalitis has 256 bp DNAs.

OE-PCR: Overlap extension-polymerase chain reactions.

Table 2.  Genomic profile of newly synthesized dengue virus 1–4, Japanese encephalitis, yellow fever and Zika flaviviruses.

Number	DDBJ/NCBI gene bank accession number	Flavivirus species	Complete genome	Nucleotide sequence (synthetic)
1	LC227563	Dengue virus 1	9158–9410	ATGTATGCAGATGACACAGCCGGATGGGACACAAGAATAACAGAGGATGATCTTCAGAATGAGGCCAAAATCACTGACATCATGGAACCTGAACATGCCCTATTGGCCACGTCAATCTTTAAGCTAACCTACCAAAACAAGGTAGTAAGGGTGCAGAGACCAGCGAAAAATGGAACCGTGATGGATGTCATATCCAGACGTGACCAGAGAGGAAGTGGACAGGTTGGAACCTATGGCTTAAACACCTTCACCA
2	LC227561	Dengue virus 2	9157–9409	ATGTATGCCGATGACACCGCAGGATGGGATACAAGAATCACACTAGAAGACCTAAAAAATGAAGAAATGGTAACAAACCACATGGAAGGAGAACACAAGAAACTAGCCGAGGCCATTTTCAAACTAACGTACCAAAACAAGGTGGTGCGTGTGCAAAGACCAACACCAAGAGGCACAGTAATGGACATCATATCGAGAAGAGACCAAAGAGGTAGTGGACAAGTTGGCACCTATGGACTCAATACTTTCACCA
3	LC227562	Dengue virus 3	9149–9401	ATGTATGCTGATGACACAGCTGGTTGGGACACAAGAATAACAGAAGATGACCTGCACAATGAGGAAAAGATCATACAGCAAATGGACCCTGAACACAGGCAGTTAGCGAACGCTATATTCAAGCTCACATACCAAAACAAAGTGGTCAAAGTTCAACGACCGACTCCAACGGGCACGGTAATGGATATTATATCTAGGAAAGACCAAAGGGGCAGTGGACAACTGGGAACTTATGGCCTGAATACATTCACCA
4	LC227568	Dengue virus 4	9150–9402	ATGTATGCTGATGACACAGCAGGCTGGGACACAAGAATCACTGAGGATGACCTTCAAAATGAGGAACTGATCACGGAACAGATGGCTCCCCACCACAAGATCCTAGCCAAAGCCATTTTCAAACTAACCTATCAAAACAAAGTGGTGAAAGTCCTCAGACCCACACCGCGGGGAGCGGTGATGGATATCATATCCAGGAAAGACCAAAGAGGTAGTGGACAAGTTGGAACATATGGTTTGAACACATTCACCA
5	LC227560	Japanese encephalitis virus	9270–9525	ATGTACGCTGATGACACCGCCGGATGGGACACTAGAATTACCAGAACTGATTTAGAAAATGAAGCCAAGGTGCTGGAGCTCCTAGACGGTGAACACCGCATGCTCGCTCGAGCCATAATTGAACTGACTTACAGGCACAAAGTGGTCAAGGTCATGAGACCTGCAGCAGAAGGAAAGACCGTAATGGACGTGATATCAAGAGAAGATCAAAGGGGGAGTGGACAGGTGGTCACTTATGCCCTTAACACTTTCACGA
6	LC227567	Yellow fever virus	9227–9482	TTCTACGCGGATGACACCGCTGGATGGGACACGCGCATCACAGAGGCAGACCTTGATGATGAACAGGAGATCTTGAACTACATGAGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATGACATACAAGAACAAAGTGGTGAAAGTGTTGAGACCAGCCCCAGGAGGGAAAGCCTACATGGATGTCATAAGTCGACGAGACCAGAGAGGATCCGGGCAGGTAGTGACTTATGCTCTGAACACCATCACCA
7	LC227589	Zika virus	9245–9500	ATGTACGCAGATGACACTGCTGGCTGGGACACCCGCATTAGTAAGTTTGATCTGGAGAATGAAGCTCTGATTACCAACCAAATGGAGGAAGGGCACAGAACTCTGGCGTTGGCCGTGATTAAATACACATACCAAAACAAAGTGGTGAAGGTTCTCAGACCAGCTGAAGGAGGAAAAACAGTTATGGACATCATTTCAAGACAAGACCAGAGAGGGAGTGGACAAGTTGTCACTTATGCTCTCAACACATTCACCA

DDBJ: DNA Data Bank of Japan; NCBI: National Center for Biotechnology Information.

Table 3.  Comparative features of oligomer design and assembly methods for overlap extension-polymerase chain reaction.

Program name	Study (year)	Platform	License	Execute	Problem	Ref.
Speedy genes	Currin A, Swainston N, Day P, & Kell D. SpeedyGenes: exploiting an improved gene synthesis method for the efficient production of synthetic protein libraries for directed evolution. In Synthetic DNA Humana Press, New York, NY, 63–78 (2017)	Web (http://g.gene-genie.appspot.com/?)	Free	OK	Design limitation from AA sequence	[13]
	Kalendar R, Lee D and Schulman A. Java web tools for PCR, in silico PCR and oligonucleotide assembly and analysis. Genomics 98(2), 137–144 (2011) Kalendar R, Tselykh T, Khassenov B, & Ramanculov E. Introduction on using the FastPCR software and the related Java web tools for PCR and ligonucleotide assembly and analysis. PCR: Methods and Protocols, 33–64 (2017)	Windows/MacOS/Linux/Web (http://primerdigital.com/fastpcr.html)	Free (web)/charge	OE-PCR oligomer can be designed	Some oligomer exceeds its size	[14]
SITICHER2.0	O’Halloran D, Uriagereka-Herburger I and Bode K. STITCHER 2.0: primer design for overlapping PCR applications. Sci. Rep. 7:45349 (2017)	Web (http://www.ohalloranlab.net/STITCHER_2_0/index.html)	Free	Mainly for designing PCR-primers	No function for OE-PCR	[16]
Primer mapper	O’Halloran D. PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection. Sci. Rep. 6:20631 (2016)	Linux (Perl)	Free	Mainly for designing PCR-primers	No function for OE-PCR	[17]
RGU-design OE-PCR^†	Camer and Endoh et al. 2018 Novel synthetic DNAs of notifiable dengue, Zika and other flaviviruses: algorithmic OE-PCR	Platform: Linux (Ubuntu) (https://bitbucket.org/dendoh_2/rgu-design-oepcr/src)	Free (distribute on BitBucket)	OE-PCR oligomers can be designed on the limitation of user-determined oligomer length.	Program language Ruby must be installed on the user’s computer. Skills for executing Ruby program is needed.	^†

† RGU-design OE-PCR refers to current report of Camer and Endoh et al., 2018 of Rakuno Gakuen University, Department of Radiation Biology, School of Veterinary Medicine, Japan.

OE-PCR: Overlap extension-polymerase chain reaction.

Currin A, Swainston N, Day P, Kell D. SpeedyGenes: exploiting an improved gene synthesis method for the efficient production of synthetic protein libraries for directed evolution. Methods Mol. Biol. 1472, 63–78 (2017).

 PubMedGoogle Scholar

Kalendar R, Lee D, Schulman A. Java web tools for PCR, in-silico PCR and oligonucleotide assembly and analysis. Genomics 98(2), 137–144 (2011).

 PubMed Web of Science ®Google Scholar

Kalendar R, Lee D, Schulman AH. FastPCR software for PCR, in-silico PCR and oligonucleotide assembly and analysis. In: DNA Cloning and Assembly Methods. Humana Press, NJ, USA, 271–302 (2014).

 Google Scholar

O’Halloran D, Uriagereka-Herburger I, Bode K. STITCHER 2.0: primer design for overlapping PCR applications. Sci. Rep. 7, 45349 (2017).

 PubMed Web of Science ®Google Scholar