We therefore examined whether we could modify the specifics of the tailing and PCR reactions to prevent the amplification of endogenous homopolymer sites. While the dA:dT base pair involves only two hydrogen bonds, when the artificial base 2-amino deoxyadenosine (2-amino dA) pairs with dT, three hydrogen bonds can form (11-14). We reasoned that if we created a tail composed of 2-amino dA, the added stability from pairing of this tail with an oligo(dT) primer could enable priming at PCR annealing temperatures where priming of the endogenous poly(dA) stretches would not occur.
To test this hypothesis we added two different homopolymer tails to V. cholerae genomic DNA. In the first case an oligo(dA) tail with a 30 nt average length was added using TdT and a 29:1 ratio of dATP:ddATP. This tail was used as a surrogate for an endogenous dA stretch and was used to define the maximum annealing temperature at which oligo(dT) can prime from oligo(dA). The second case was identical to the first except that 2-amino dATP was substituted. Each tailed substrate was first ligated to an oligonucleotide that has seven dT nucleotides at its 3′ end and then subjected to PCR using this same oligonucleotide together with a second oligonucleotide that has 22 dT nucleotides at its 3′ end. For each tailed substrate, seven different PCR annealing temperatures were tested and the results are shown in Figure 3. The intensity of products generated with the 2-amino dA-tailed substrate at an annealing temperature of 62.4°C was very similar to that obtained with the dA-tailed substrate at 58.3°C (compare lanes 5 and 12 in Figure 3), whereas no product was formed for the dA-tailed substrate at an annealing temperature of 62.4°C (Figure 3, lane 13). Hence, the maximum allowed annealing temperature was increased by more than 4°C when 2-amino dATP was substituted for dATP in the tailing reaction. The exogenously added poly(dA) sequence is chemically equivalent to an endogenous poly(dA) sequence that might naturally occur within a genome. We therefore conclude that by using 2-amino dATP in the tailing reaction and an annealing temperature of 62.4°C during PCR, it is possible to prime from exogenous tails without priming from endogenous stretches.
In summary, we developed a new method, HTML-PCR and used it to accurately sequence bacterial genomes, even when only 1 nanogram or less of sample DNA was used. Furthermore, by using a homopolymer tail of synthetic nucleotides, we were able to find conditions in which endogenous genomic homopolymers were ignored and only exogenously added tails were used to prime synthesis. This modification should enable the use of HTML-PCR with any genome regardless of its endogenous homopolymer content. Compared with Nextera, HTML-PCR is more versatile as it can be applied to sequencing platforms other than Illumina and to applications in addition to sequencing. It is also more cost-effective and uses reagents that are readily available from numerous sources. Finally, unlike Nextera, HTML-PCR functions with templates over a very broad range in concentration, without minimum size constraints, and even when the GC content is low. Compared with adapter ligation methods, HTML-PCR requires fewer steps, only a few inexpensive reagents and minimal hands-on time. The method does not require adapter ligation and is not prone to generating adapter-dimers or primer-dimers even when template is extremely limiting. This feature obviates the need for gel purification and size selection for many applications, thus enabling the method to be compatible with high-throughput formats and robotic assistance. In addition to research applications, HTML-PCR is also ideally suited to medical and forensic applications where the supply or integrity of sample DNA may be limited.
This work was supported by Award Numbers AI45746 (A.C.) and AI055058 (A.C.) from the National Institutes of Health. A.C. is a Howard Hughes Medical Institute investigator. This paper is subject to the NIH Public Access Policy.
The authors have a patent pending on HTML-PCR.
Address correspondence to Andrew Camilli, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. e-mail: firstname.lastname@example.org
1.) Bentley, D.R., S. Balasubramanian, H.P. Swerdlow, G.P. Smith, J. Milton, C.G. Brown, K.P. Hall, D.J. Evers. 2008. Accurate Whole Human Genome Sequencing using Reversible Terminator Chemistry. Nature 456:53-59. 2.) Ranade, S.S., C.B. Chung, G. Zon, and V.L. Boyd. 2009. Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection. Anal. Biochem. 390:126-135. 3.) Rohland, N., and D. Reich. 2012. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22:939-946. 4.) Adey, A., H. G. Morrison, X. Asan, J. O. Xun, E. H. Kitzman, B. Turner, A. P. Stackhouse MacKenzie. 2010. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biol. 11:R119. 5.) Boule, J.-B., F. Rougeon, and C. Papanicolaou. 2001. Terminal Deoxynucleotidyl Transferase Indiscriminately Incorporates Ribonucleotides and Deoxyribonucleotides. J. Biol. Chem. 276:31388-31393. 6.) Klein, B.A., E.L. Tenorio, D.W. Lazinski, A. Camilli, M.J. Duncan, and L.T. Hu. 2012. Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis. BMC Genomics 13:578. 7.) Heidelberg, J.F., J.A. Eisen, W.C. Nelson, R.A. Clayton, M.L. Gwinn, R.J. Dodson, D.H. Haft, E.K. Hickey. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477-484. 8.) Tettelin, H., K.E. Nelson, I.T. Paulsen, J.A. Eisen, T.D. Read, S. Peterson, J. Heidelberg, R.T. DeBoy. 2001. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293:498-506. 9.). 2001. Initial sequencing and analysis of the human genome. Nature 409:860-921. 10.) Venter, J.C., M.D. Adams, E.W. Myers, P.W. Li, R.J. Mural, G.G. Sutton, H.O. Smith, M. Yandell. 2001. The Sequence of the Human Genome. Science 291:1304-1351. 11.) Howard, F.B., J. Frazier, and H.T. Miles. 1966. A new polynucleotide complex stabilized by three interbase hydrogen bonds, poly-2-aminoadenylic acid + polyuridylic acid. J. Biol. Chem. 241:4293-4295. 12.) Rackwitz, H.R., and K.H. Scheit. 1976. The Stereochemical Basis of Template Function. Eur. J. Biochem. 72:191-200. 13.) Scheit, K.H., and H.R. Rackwitz. 1982. Synthesis and physicochemocal properties of two analogs of poly(dA): poly(2-aminopurine-9-3-D-deoxyribonucleotide) and poly 2-amino-deoxyadenylicacid. Nucleic Acids Res. 10:4059-4069. 14.) Cheong, C., I. Tinoco, and A. Chollet. 1988. Thermodynamic studies of base pairing involving 2,6-diaminopurine. Nucleic Acids Res. 16:5115-5122.