.:) 1990 Oxford University Press
5914 Nucleic Acids Research, Vol. 18, No. 19
Use of primer pools for specific
gene
amplification
Uwe H.Dietz, Marita Gareis, Wolf M.Bertling Max-Planck Society, Clinical Research Units Rheumatology at the University of Erlangen, Schwabachanlage 10, 8520 Erlangen, FRG Submitted July 16, 1990 The selection of primers to use for sequence specific DNA amplification (1) depends on the availability of sequence information. Short oligonucleotides and highly degenerated oligonucleotides require a low stringency of the annealing process. A lower stringency generally results in a high background due to unspecific priming. Sequence information allowing only limited stringency is often available if backtranslating protein sequences or if generating a library specific for gene families, or if amplifying specific sequences out of libraries with only one primer being unique and the other one directed to a vector sequence. Here we report the observation that 3 or 4 primer systems increase the specificity under otherwise unspecific conditions.
upstream primer
primer complementary to upstream primer
primer complementary to downstream primer downstream primer 2
Figure 2. Schematic drawing of the four primer setup. Exon 2 of chicken collagen aI type H counts 211 bp (3), the upstream primer pair comprises nucleotide numbers 14 through 31, the downstream primer pair nucleotide numbers 188 through 205 of that sequence.
In addition to the normally used upstream and downstream primers this procedure uses complementary primer pairs 3' and 5' to the sequence which is to be amplified. Amplification in such a multi primer system yields mostly specific but only little unspecific amplification product. This procedure should also be particularly useful for specific DNA amplification if there are many secondary annealing sites of marginal homology or if one primer site is much more frequent than the other. The example described here is the amplification of exon 2 of chicken collagen al type H from genomic DNA. Low stringency can be due to a relatively low temperature annealing step or the presence of a number of secondary annealing sites in the genome. Instead of using single stranded upstream and downstream primers we used double stranded primers. The complementary strand of each downstream respectively upstream primer holds the primer in a pool and since it is of the same length and completely homologous, keeps it from annealing to secondary sites of lesser homology. This not only reduces the amount of primers needed, it also reduces the number of non-specifically primed amplification products.
REFERENCES 1. Saiki,R.K., Scharf,S., Faloona,F., Mullis,K.B., Horn,G.T., Erlich,H.A. and Anrheim,N. (1985) Science 230, 1350-1354. 2. Krawetz,S.A., Pon,R.T. and Dixon,G.H. (1989) Nucl. Acids Res. 17, 819. 3. Nah,H.D. and Upholt,W.B. (1990) J. Dental. Res. 69, 206.
Figure 1. DNA amplification products of a 192 bp fragment of chicken collagen II in a 2 primer (lane 2) and 4 primer (lane 3) setup. All conditions (2) were the same for the two setups (lane 2 and 3) except in setup no. 2 (lane 3) where 4 primers were used. All primers had the same length of 18 bp. In lane 2 (only 2 primers) appears a strong background and a larger number of unspecific bands with almost no primers left after 25 cycles. In lane 3, however, most of the unused primer is still visible and only one band is visible on the ethidium bromide stained gel. (The identity of this band has been confirmed by sequencing.) Lane 1 shows the marker DNA, OX174 DNA digested with HaeIIl.
5914 Nucleic Acids Research, Vol. 18, No. 19
Use of primer pools for specific
gene
amplification
Uwe H.Dietz, Marita Gareis, Wolf M.Bertling Max-Planck Society, Clinical Research Units Rheumatology at the University of Erlangen, Schwabachanlage 10, 8520 Erlangen, FRG Submitted July 16, 1990 The selection of primers to use for sequence specific DNA amplification (1) depends on the availability of sequence information. Short oligonucleotides and highly degenerated oligonucleotides require a low stringency of the annealing process. A lower stringency generally results in a high background due to unspecific priming. Sequence information allowing only limited stringency is often available if backtranslating protein sequences or if generating a library specific for gene families, or if amplifying specific sequences out of libraries with only one primer being unique and the other one directed to a vector sequence. Here we report the observation that 3 or 4 primer systems increase the specificity under otherwise unspecific conditions.
upstream primer
primer complementary to upstream primer
primer complementary to downstream primer downstream primer 2
Figure 2. Schematic drawing of the four primer setup. Exon 2 of chicken collagen aI type H counts 211 bp (3), the upstream primer pair comprises nucleotide numbers 14 through 31, the downstream primer pair nucleotide numbers 188 through 205 of that sequence.
In addition to the normally used upstream and downstream primers this procedure uses complementary primer pairs 3' and 5' to the sequence which is to be amplified. Amplification in such a multi primer system yields mostly specific but only little unspecific amplification product. This procedure should also be particularly useful for specific DNA amplification if there are many secondary annealing sites of marginal homology or if one primer site is much more frequent than the other. The example described here is the amplification of exon 2 of chicken collagen al type H from genomic DNA. Low stringency can be due to a relatively low temperature annealing step or the presence of a number of secondary annealing sites in the genome. Instead of using single stranded upstream and downstream primers we used double stranded primers. The complementary strand of each downstream respectively upstream primer holds the primer in a pool and since it is of the same length and completely homologous, keeps it from annealing to secondary sites of lesser homology. This not only reduces the amount of primers needed, it also reduces the number of non-specifically primed amplification products.
REFERENCES 1. Saiki,R.K., Scharf,S., Faloona,F., Mullis,K.B., Horn,G.T., Erlich,H.A. and Anrheim,N. (1985) Science 230, 1350-1354. 2. Krawetz,S.A., Pon,R.T. and Dixon,G.H. (1989) Nucl. Acids Res. 17, 819. 3. Nah,H.D. and Upholt,W.B. (1990) J. Dental. Res. 69, 206.
Figure 1. DNA amplification products of a 192 bp fragment of chicken collagen II in a 2 primer (lane 2) and 4 primer (lane 3) setup. All conditions (2) were the same for the two setups (lane 2 and 3) except in setup no. 2 (lane 3) where 4 primers were used. All primers had the same length of 18 bp. In lane 2 (only 2 primers) appears a strong background and a larger number of unspecific bands with almost no primers left after 25 cycles. In lane 3, however, most of the unused primer is still visible and only one band is visible on the ethidium bromide stained gel. (The identity of this band has been confirmed by sequencing.) Lane 1 shows the marker DNA, OX174 DNA digested with HaeIIl.
