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We then compared cloning efficiency, in terms of number of colonies produced after transformation, for optimal ratio of vector and insert during Dpn I enzyme digestion and optimal amount of the vector-insert mixture used in transformation. The number of colonies that grew on each plate was counted next day. The result shows that the cycle PCR amplification of vector and insert produced many more colonies than the cycle and cycle amplifications 31, , 43 colonies for 12, 18, and 24 cycles respectively.

For incubation duration in Dpn I digestion, 1, 2, and 4 hours of incubation had similar colony production efficiency with colony numbers of , , and for 1, 2, and 4 hour-incubations, respectively. However, shorter incubation time is not recommended. In our QuickChange mutagenesis experiment, 30 min incubation in Dpn I digestion could not adequately remove the wild-type template background and resulted in a significantly higher fraction of wild type constructs.

Optimization of cloning conditions. B Comparison of number of colonies grown on the plates after transformation. See text for details. D Clone validation by restriction digestion to exclude unusual constructs. In addition, the PCR-based method could produce some unusual constructs. With our FastCloning method in the past few months, we have not seen any unusual construct yet.

Thus, if our cloning method produces unusual constructs, their occurrence must be very low. Figure 4D is an example of using restriction digestion to screen clones to exclude unusual constructs. It would be a good practice to perform such a screening for all resulting clones before DNA sequencing. The results further validate the versatility and reliability of this new technique. Because the PCR amplification of vector can be controlled by primers to exact positions, our FastCloning method is truly sequence-independent.

Thus, one can put an insert to any position and in any frame. This feature, although with only a small modification of standard cloning protocol, makes it easy to construct cDNAs for fusion proteins or chimeras.

Furthermore, a minor variation of this technique can be applied for insertion of a short DNA fragment directly from two relative long primers for PCR amplification of a cDNA along with its vector. Note that each primer contains only slightly more than half of the insert. The primer pairs have a base overlap region. An example of chimera construction with a short bp DNA fragment replaced by a synthesized insert of 99 bp included in two primers.

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Two colored arrows indicate two primers with a 15 bp overlapping region highlighted. Note that the entire insert of the 99 bp fragment is included in the two primers. Thus, there is no need to amplify the insert. The length of each primer is 81 bases for the forward primer and 76 bases for the reverse primer. Making multiple mutations in a stretch of DNA is equivalent to making short insertions.

With this relatively short primer pair base forward primer and base reverse primer , we have successfully obtained this multiple mutations spanning a codon stretch in the cDNA. In addition, we have successfully made a quintuple mutant 5 consecutive prolines mutated to 5 alanines of human 5-HT 3 A receptor subunit with a base forward primer and base reverse primer.

An example of making multiple mutations across a wide region. Mutated nucleotides are indicated in red. B Actual forward base and reverse base primers with 16 bp overlapping in their 5' ends. It is important to be aware that with PCR-based cloning, the synthesized primers may not be completely uniform with correct sequences.

This is especially true for short insertion with long primers. Thus, sequencing across the primer region is required for ultimate confirmation of each clone. If random mutation happens in one clone, picking up another clone often solve the problem. The random mutation out of primer region is rare with high fidelity DNA polymerases. However, DNA sequencing of the entire coding region is still necessary.

In the past 8 months, we have successfully used our new cloning method to obtain 21 constructs. Among the 63 sequenced clones, we found 2 deletions in 2 chimeras constructed with long primer pairs, but found no mutations in the entire regions of all constructs beyond primers. Finally, the cloning efficiency of our FastCloning method is high. Of the 21 cloning constructs, we obtained 19 desired constructs in single runs. In 2 experiments, we needed to repeat the procedure to get the final clones. With only one additional construct, we have not obtained final clones after two attempts.

It is ligation-independent and does not require specific sequence in the vector. Thus, one can insert a DNA fragment into a vector at any desired position without considering restriction sites. This feature also makes it extremely easy to make constructs for fusion proteins and chimeras. In addition, it can be used to make short insertions and multiple mutations spanning a wide region up to bp in a cDNA. Finally, it is a highly efficient and reproducible method.

Nucleic Acids Research. Methods in Enzymology. Current Issues in Molecular Biology. Download references. If a Southern blot analysis or reamplification using primers located within the original target nested PCR reveals that the desired product is present at a very low level, the explanation may be that too few cycles of amplification were used for the starting target copy number see Notes 20 and Adjustments to enzyme concentration can be made in increments of 0.

Optimization of the Mg OAc 2 concentration should be car- ried out in increments of 0. Carryover contamination see Chapter 1 and dNTP stock solutions of poor quality can both significantly reduce the apparent optimal range for the Mg OAc 2 concentration. These problems may not be observed initially, but may become apparent during late amplifications with targets and primers previously observed to work well.

The 3' ends of the PCR may have an additional one or two nontemplated nucleotides 28, References 1. Cheng, S. PCR Meth. Proceedings of the National Academy of Sciences.

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    PCR Cloning Protocols

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