Table 1.

Comparison of one-step and two-step PCR approaches for NNK-based SSM

	Libraries	Number of sequences obtaineda	Number of amino acids presentb	Number of codons presentc	Percent wild typed	Qpool value from pooled plasmidse
One-step PCR method	Y51X	91	18	25	48	0.30
	S72X	92	15	24	34	0.40
	L75X	91	18	28	35	0.44
	L437X	93	14	22	43	0.59
	T438X	91	19	25	47	0.31
Two-step PCR method	Y51X	91	19	30	4	0.43
	S72X	92	19	28	5	0.47
	L75X	92	19	30	2	0.74
	L437X	93	20	30	5	0.82
	T438X	91	18	28	8	0.67

^aNumber of colonies with complete sequence, which was obtained after sequencing 96 colonies for each library

^bNumber of different amino acids found within the sequence data for a given library (NNK primers theoretically encompass all 20 amino acids with different distributions)

^cNumber of different codes found within the sequence data for a given library (NNK primers theoretically encompass all 32)

^dFraction of the sequenced clone that contains the starting codon at the targeted position (assumed to result from wild-type carryover)

^eCalculated for the entire codon from sequence data obtained from the pooled plasmids isolated after the initial transformation according to the method reported (Sullivan et al. 2013), the weighted average across the three bases of a codon yields a Qpool value between 0 and 1, with 1 indicating perfect randomization