Table 3. Transformation with two overlapping PCR products vs. a single full-length PCR product.
| Construct | Note | 5΄ complementaritya | DNA length | Amount (ng) used in transformation | Total no. of transformants |
|---|---|---|---|---|---|
| hisA(dup 59 bp A)b | Full-length | 29 | 3332 | 250 | ∼1700 |
| hisA(dup 59 bp A)b | Two fragments | 29 | 1647+1962c | 125+147 | ∼1900 |
| hisA(dup 59 bp B)b | Two fragments | 41 | 1653+1968c | 125+147 | ∼1500 |
| hisA(dup 75 bp) | Two fragments | 41 | 1663+1974c | 125+147 | ∼3000 |
aAs the primers direct recombination within the same short sequence (59 or 75 bp) there is considerable complementarity between the 5΄ ends within the primer pair. During PCR cycling, the new 3΄-ends will be complementary, potentially causing multimerization of the products.
bThe primer pairs for generating hisA (dup 59 bp A) and hisA (dup 59 bp B) differed in that the A primer pair (oligos 57 and 58 in Supplementary Data) had shorter 5΄ homologies than the B pair (oligos 59 and 60 in Supplementary Data). The resulting transformants were identical.
cThe two fragments overlapped with each other by 277 bp in the cat gene, requiring recombination between the two fragments to restore a full cat gene and chloramphenicol resistance.