Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins

Table 6 Comparison of different methods used to produce synthetic DNA fragments

Year	Method	Number of steps for gene synthesis	Oligo length	Oligo overlap	Oligo gap	Oligo purification	DNA polymerase	Accuracy or error rate per Kb	Reference
2016	This study	Single step: assembly PCR	60	20	20	No purification	Kod Hot Start	1.13	–
1995	Stemmer method	Single step: assembly PCR	40	20	NM	No purification	Taq	NM	[4]
2004	PTDS	Two steps: Assembly PCR & Overlap extension PCR	60	20	NM	PAGE	Pfu or pyrobest	1.26	[13]
2004	Two-step method	Two steps: Dual asymmetric PCR & Overlap extension PCR	50	10	NM	No purification	Pfu	3/4 clones contain 1–3 single base deletions	[7]
2006	PAS	Two steps: Assembly PCR & Overlap extension PCR	60	21	NM	PAGE	Pfu	~1	[18]
2006	SGS	Single step: assembly PCR	40	18–20	No	No purification	Kod	2.7	[9]
2010	IPS	Two steps: Assembly PCR & Overlap extension PCR	60 & 30	15	NM	PAGE	Pfu	1	[8]
2012	AOE	Two steps: Assembly PCR & Overlap extension PCR	30–50	NM	NM	PAGE	Pfu	<1	[19]
2015	RapGene	Single step: assembly PCR	57–60	20	NM	No purification	NM	1.67	[20]

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