Kourosh Salehi February 10, 1998 This method provides a simple and rapid way of synthesizing medium size pools containing two random domains. In this method, two oligonucleotides are synthesized such that each contain a unique 18-20 base long region in the 5' end, a random region (e.g., N60-65) in the middle, and an 18 base constant region in the 3' end. The 3' end sequences of the two oligonucleotides are designed to be complementary to one another. The pool is constructed by annealing the two oligonucleotides at their 3' ends, then extending these by Taq polymerase at 72°C (Fig. 1). Following purification, the extended DNA is ready for amplification with a set of primers complementary to the 5' ends to generate multiple copies of the pool. One of the primers may also contained the T7 promoter which will allow transcription of the DNA by the phage T7 polymerase. Materials: Procedure: 1. Set up a 10 ml reaction mix as follows: 2. Dispense the mix into 0.7 ml PCR tubes, 500 µl each. 3. Program the PCR machine for 1 cycle of 10 min at 94 °C, 15 min at 55 °C, and 45 min at 72 °C. 4. During the 94 °C step, hot start the reaction by adding 5 units of Taq to each tube, then mix the contents of each tube by pipetting. (Hot starting is always recommended, but it was not determined if it is necessary) 5. After the extension reaction is completed, add 5 µl of 0.5 M EDTA to each tube to stop the reaction. Pool the reactions and ethanol precipitate. 6. Gel purify the extended DNAs in a denaturing polyacrylamide gel. Bear in mind that once the pool is denatured, its renaturation is essentially not possible. If this is not desirable, non-denaturing polyacrylamide gels or agarose gels are recommended. 7. The pool is now ready for PCR amplification to generate multiple copies. Discussion This method offers a rapid way of synthesizing a medium size pool in essentially a single step. The procedure avoids potentially problematic steps such as ligation, and is easy to do. When desired, the sequences of the middle region where the two oligos anneal, can be chosen to include an aptamer or other motifs. It should be noted that this method has not been extensively optimized or tested; however, it has been employed to generate a 184 base pair long pool with the complexity of approximately 1014 that has yielded active ribozymes. Pools of higher complexities, e.g. 1015, should also be possible to generate and are planned to be synthesized in the near future. Ways to further increase the efficiency of the extension reaction will also be explored. One of the most important parameters to be considered in this method is the extendibility of the oligonucleotides. Decrease in extendibility, which generally correlates with the increase in length of the oligonucleotides, not only causes poor yield in the extension step, but may also decrease the PCR amplifiability of the pool. For an oligonucleotide pair that consisted of two 101 base oligos, (30% overall extension was observed(Fig. 2). The use of oligonucleotides significantly longer than 100 bases are likely to be problematic and are not recommended. Good luck and have fun with your pool synthesis! Kourosh Salehi, February 10, 1998
Mutual Templating Method 10X PCR buffer 1.0 ml dNTP (2mM) 1.0 ml dH2O X ml Oligo 1 2500 - 5000 pmol Oligo 2 2500 - 5000 pmol Total volume 10.0 ml