3094 Nucleic Acids Research, Vol. 18, No. 10
.-_j 1990 Oxford University Press
Rapid generation of DNA fragments by PCR amplification of crude, synthetic oligonucleotides Richard W.Barnett and Heather Erfle DNA Chemistry Group, Allelix Biopharmaceuticals Inc., Mississauga, Ontario, L4V 1 P1 Canada Submitted March 20, 1990
The most time consuming, labor-intensive steps in total gene synthesis are the preparation and assembly of the oligonucleotide building blocks. Using a classical block ligation assembly strategy, even a medium-sized gene fragment of 650bp requires the synthesis, purification, 5'-phosphorylation and ligation of from 16 to 40 different oligonucleotides (1, 2). We wish to report an alternative method for synthesizing gene fragments which requires neither oligonucleotide purification nor ligation. This approach employs the Polymerase Chain Reaction (PCR) (3) to generate and amplify a double-stranded gene fragment directly from a crude oligonucleotide synthesis mixture. Briefly, an oligonucleotide synthesis mixture is comprised of fulllength product plus numerous 'failure sequences', all of which have common 3'-end sequences. In the presence of a primer complementary to this common 3'-end a series of double-stranded fragments, i.e. full-length + failure sequences, is generated in the first round of PCR. Using a second primer complementary only to the 3'-end of the nascent full-length complementary strands, subsequent PCR cycles result in the selective, exponential amplification of full-length product against a background of linearly replicated failure sequences. In the present work, two gene fragments of 180 and 254bp were prepared from crude, synthetic 179 and 234mer, respectively. The 179 and 234mer were synthesized on an Applied Biosystems 380B using CE-phosphoramidite chemistry and a small-scale synthesis cycle (0.1-0.15 micromole). The crude oligonucleotides were cleaved from their CPG supports and deprotected by standard ammonium hydroxide treatment. One quarter of the crude 179mer was desalted on a Waters C 18 SepPak cartridge (4). The crude 234mer was used directly. Aliquots of the crude 179mer (288ng) or 234mer (1 Ing) were subjected to PCR amplification (IOO1,L GeneAmp buffer, 125pmol primers (19 -35mers), 2U Taq polymerase; 25-31 cycles of 2min/94C, 2min/55C, 2min/72C) to generate the desired 180 and 254bp fragments, respectively (Figure 1 - Lanes A and B). The PCR fragments were subsequently isolated, digested with the appropriate restriction enzymes, cloned into plasmid pTZ and transformed into E. coli. Restriction digest and sequence analysis of several recombinant clones showed that each contained the expected gene fragment, and that most were of the correct sequence. A low mutational background (0.1-0.2% per nucleotide), possibly due to nucleotide misincorporation during amplification, was observed. Using this method small genes or
gene fragments can be synthesized, cloned and characterized in a matter of weeks instead of months. Extension of this approach to even longer synthetic strands ( > 250 -300mers) is currently in progress.
ACKNOWLEDGEMENTS We thank Gary Thompson and Raya Kuperman for their expert synthesis of the 179 and 234mer, respectively.
REFERENCES 1. Bell,L.D. et al. (1988) Gene 63, 155-163. 2. Wosnick,M.A., Barnett,R.W. and Carlson,J.E. (1989) Gene 76, 153-160.
3. Saiki,R.K. et al. (1988) Science 239, 487-491. 4. Applied Biosystems User Bulletin (1987) Issue No. 13- revised.
A
Mt
M
B
-254 190
180 0'
1s80 -160
Figure 1. Lane A l80bp product from PCR of crude, desalted 179mer. Lane M MspI-digested pBR322 marker DNA. Lane B 254bp product from PCR of crude 234mer. Lane M'-HaelII-digested phiX 174 RF marker DNA. -
.-_j 1990 Oxford University Press
Rapid generation of DNA fragments by PCR amplification of crude, synthetic oligonucleotides Richard W.Barnett and Heather Erfle DNA Chemistry Group, Allelix Biopharmaceuticals Inc., Mississauga, Ontario, L4V 1 P1 Canada Submitted March 20, 1990
The most time consuming, labor-intensive steps in total gene synthesis are the preparation and assembly of the oligonucleotide building blocks. Using a classical block ligation assembly strategy, even a medium-sized gene fragment of 650bp requires the synthesis, purification, 5'-phosphorylation and ligation of from 16 to 40 different oligonucleotides (1, 2). We wish to report an alternative method for synthesizing gene fragments which requires neither oligonucleotide purification nor ligation. This approach employs the Polymerase Chain Reaction (PCR) (3) to generate and amplify a double-stranded gene fragment directly from a crude oligonucleotide synthesis mixture. Briefly, an oligonucleotide synthesis mixture is comprised of fulllength product plus numerous 'failure sequences', all of which have common 3'-end sequences. In the presence of a primer complementary to this common 3'-end a series of double-stranded fragments, i.e. full-length + failure sequences, is generated in the first round of PCR. Using a second primer complementary only to the 3'-end of the nascent full-length complementary strands, subsequent PCR cycles result in the selective, exponential amplification of full-length product against a background of linearly replicated failure sequences. In the present work, two gene fragments of 180 and 254bp were prepared from crude, synthetic 179 and 234mer, respectively. The 179 and 234mer were synthesized on an Applied Biosystems 380B using CE-phosphoramidite chemistry and a small-scale synthesis cycle (0.1-0.15 micromole). The crude oligonucleotides were cleaved from their CPG supports and deprotected by standard ammonium hydroxide treatment. One quarter of the crude 179mer was desalted on a Waters C 18 SepPak cartridge (4). The crude 234mer was used directly. Aliquots of the crude 179mer (288ng) or 234mer (1 Ing) were subjected to PCR amplification (IOO1,L GeneAmp buffer, 125pmol primers (19 -35mers), 2U Taq polymerase; 25-31 cycles of 2min/94C, 2min/55C, 2min/72C) to generate the desired 180 and 254bp fragments, respectively (Figure 1 - Lanes A and B). The PCR fragments were subsequently isolated, digested with the appropriate restriction enzymes, cloned into plasmid pTZ and transformed into E. coli. Restriction digest and sequence analysis of several recombinant clones showed that each contained the expected gene fragment, and that most were of the correct sequence. A low mutational background (0.1-0.2% per nucleotide), possibly due to nucleotide misincorporation during amplification, was observed. Using this method small genes or
gene fragments can be synthesized, cloned and characterized in a matter of weeks instead of months. Extension of this approach to even longer synthetic strands ( > 250 -300mers) is currently in progress.
ACKNOWLEDGEMENTS We thank Gary Thompson and Raya Kuperman for their expert synthesis of the 179 and 234mer, respectively.
REFERENCES 1. Bell,L.D. et al. (1988) Gene 63, 155-163. 2. Wosnick,M.A., Barnett,R.W. and Carlson,J.E. (1989) Gene 76, 153-160.
3. Saiki,R.K. et al. (1988) Science 239, 487-491. 4. Applied Biosystems User Bulletin (1987) Issue No. 13- revised.
A
Mt
M
B
-254 190
180 0'
1s80 -160
Figure 1. Lane A l80bp product from PCR of crude, desalted 179mer. Lane M MspI-digested pBR322 marker DNA. Lane B 254bp product from PCR of crude 234mer. Lane M'-HaelII-digested phiX 174 RF marker DNA. -
