Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10162-10166, October 1995 Biochemistry A DNA sequencing strategy that requires only five bases of known terminal sequence for primiing (primer extention/stacking interaction/fluorescein/solid state/duplex probe) DONG-JING FU*, NATALIA E. BROUDEt, HUBERT K6STERt, CASSANDRA L. SMITHt, AND CHARLES R. CANTORt *Sequenom, Inc., 101 Arch Street, Boston, MA 02210; tCenter for Advanced Biotechnology and Department of Biomedical Engineering, Pharmacology, and Biology, Boston University, 36 Cummington Street, Boston, MA 02215; and tFaculty of Chemistry, Department of Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany Contributed by Charles R. Cantor, July 11, 1995 ABSTRACT We have previously reported an enhanced single-stranded probes. Stacking interactions between the version of sequencing by hybridization (SBH), termed posi- duplex probe and a single-stranded target provide enhanced tional SBH (PSBH). PSBH uses partially duplex probes stringency in discriminating matched and mismatched targets. containing single-stranded 3' overhangs, instead of simple Two enzyme-catalyzed steps (DNA polymerase and T4 DNA single-stranded probes. Stacking interactions between the ligase), in addition to pure physical hybridization, are used to duplex probe and a single-stranded target allow us to reduce enhance the accuracy of sequence information obtained. Here, the probe sizes required to 5-base single-stranded overhangs. we report a completely different application of the PSBH Here we demonstrate the use of PSBH to capture relatively approach. Partially double-stranded probes are used to capture long single-stranded DNA targets and perform standard DNA targets and form primer-template complexes. These solid-state Sanger sequencing on these primer-template com- complexes are then used as substrates in conventional solid- plexes without ligation. Our results indicate that only 5 bases state Sanger sequencing. The potential advantages of this of known terminal sequence are required for priming. In approach are much more efficient methods for DNA sample addition, the partially duplex probes have the ability to preparation and a smaller set of oligonucleotide primers than capture their specific target from a mixture of five single- needed for segmented primer walking. The PSBH approach stranded targets with different 3'-terminal sequences. This appears capable of sequencing mixtures of DNA fragments indicates the potential utility of the PSBH approach to se- without prior purification. One example would be DNA frag- quence mixtures of DNA targets without prior purification. ments generated by restriction enzymes such as BstXI, which leave DNA overhangs that are not part of the recognition site. The success of the human genome project is largely dependent on improving the efficiency and lowering the cost of DNA MATERIALS AND METHODS sequencing. Current strategies of DNA sequencing include the directed approach such as primer walking (1-6) and the Oligonucleotides were purchased from Operon Technologies random, shotgun, approach (7, 8). Two very different strate- (Alameda, CA) in an unpurified form. Sequencing was carried gies for using oligonucleotides to increase the efficiency of out either by using a Sequenase version 2.0 kit (United States DNA sequencing have recently been described and tested. The Biochemical) or by using an AutoRead sequencing kit (Phar- first strategy is a form of primer walking where three short macia) employing T7 DNA polymerase. oligonucleotides are stacked together and used as a segmented Immobilized single-stranded DNA targets for solid-phase primer (2, 5, 6). The advantage of this approach is that a DNA sequencing were prepared by PCR amplification using complete set of the short oligonucleotides can be made in procedures similar to those reported before (17, 18). PCR was advance, and no additional DNA synthesis is required as the performed on a Perkin-Elmer/Cetus DNA Thermal Cycler. sequencing project proceeds. This allows a much more parallel Vent (exo-) DNA polymerase was purchased from New sequencing strategy to be employed than in conventional England Biolabs, and the dNTP solutions were from Promega. primer walking. The second strategy is a completely different Plasmid NB34, a pCR II plasmid (Invitrogen) with a 1-kb approach, called sequencing by hybridization (SBH) (9-15). In anonymous human DNA insert, was used as the DNA template SBH a set of short oligonucleotide probes with known se- for amplification. PCR was performed using an 18-nt upstream quences is used to identify the complementary sequences in an primer and a downstream 5'-end-biotinylated 18-nt primer in unknown long DNA target. The hybridization results are then a 100-,ul or 400-,ul volume containing 10 mM KCl, 20 mM assembled to generate the complete sequence of the target Tris HCl (pH 8.8), 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% DNA. The advantages of the SBH method are its potential Triton X-100, 250 ,tM dNTPs, 2.5 ,tM biotinylated primer, 5 application for automated large-scale sequencing and its in- ,tM nonbiotinylated primer, <100 ng of plasmid DNA, and 6 trinsic power of determining many sequence regions in par- units of Vent (exo-) DNA polymerase per 100 gl of reaction allel. In one SBH format, a complete set of all possible short volume. Thirty PCR temperature cycles were performed; each oligonucleotide sequences is immobilized as an ordered array cycle included a heat denaturation step at 94°C for 1 min, an on a surface, and the unknown DNA fragment is hybridized to annealing step for 1 min at 60°C, and DNA chain extension this array. Alternatively, a set of unknown DNA fragments can with Vent (exo-) polymerase for 1 min at 72°C. PCR ampli- be immobilized on a surface and hybridized with one probe at fication with the tagged primer used a 1-min 45°C incubation a time. for primer annealing. The PCR product was purified by passing We have developed an enhanced version of SBH, termed through an Ultrafree-MC 30,000 NMWL filter unit (Millipore) positional SBH (PSBH) (16). PSBH uses duplex probes con- or by electrophoresis and extraction from a low-melting-point taining single-stranded 5-base 3' overhangs, instead of simple agarose gel. About 10 pmol of purified PCR fragment was then mixed with 1 mg of prewashed Dynabeads M280 with strepta- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: SBH, sequencing by hybridization; PSBH, positional accordance with 18 U.S.C. §1734 solely to indicate this fact. SBH. 10162 Downloaded by guest on September 26, 2021 Biochemistry: Fu et aL Proc. Natl. Acad. Sci. USA 92 (1995) 10163 A S' 23 mer NNNNN 18 mer s' -%?NNNNN annealing s 23 m F NNNN k 3 18 mer s 3 Sanger Sequencing D | Sanger Sequencing B s5NNN biodns' PCR Dynal Streptavidin Beads 1k 5NNNN iNNNNN- *s105' 0.1 M NaOH treatment and then remove the supernatant 3TQNN \E biotins' FIG. 1. Schematic of procedures used. (A) Solid-state Sanger sequencing with partially duplex probe. Outlined letters indicate the overhang sequence from the partially duplex probe. (B) Generating sequencing targets by PCR amplification. (C) Adding known sequence at the 3' end of a target to facilitate subsequent capture and sequencing by procedures in A and B. F, fluorescein; b, biotin. vidin (Dynal, Oslo) in 100 ,ul of 1 M NaCl and TE (10 mM slowly to 37°C over a 20- to 30-min time period. The duplex Tris-HCl, pH 8.0/1 mM Na2EDTA, pH 8.0) and incubated at primer was then annealed with 1-5 pmol of the immobilized 37°C or 45°C for 30 min. The immobilized biotinylated double- single-stranded DNA target by adding the annealing mixture stranded DNA fragment was then converted to a single- to the DNA-containing magnetic beads, and the resulting stranded form by treating with freshly prepared 0.1 M NaOH mixture was further incubated at 37°C for 5 min, room at room temperature for 5 min. The magnetic beads, with temperature for 10 min, and finally 0°C for at least 5 min. For immobilized single-stranded DNA, were washed with 0.1 M Sequenase reactions, 1 ,ul of 0.1 M dithiothreitol solution, 1 ,ul NaOH and TE. Sequencing of the immobilized single-stranded DNA target was performed with reagents from the sequencing kits for primer 5'-F-GGCCCTTTGACGGGAGCC Sequenase version 2.0 or T7 DNA polymerase. A DNA duplex primer 2 5'-F-GATGATCCGACGCATCAAGGCCC probe containing a 5-base 3' overhang was used as a sequenc- 3'-CTACTAGGCTGCGTAGTT ing primer. The duplex has a 5'-fluorescein-labeled 23-mer, containing an 18-base 5'-constant region and a 5-base 3'- primer 3 5'-F-GATGATCCGACGCATCACAGCTC variable region (which has the same sequence as the 5' end of - 3'-CTACTAGGCTGCGTAGTG the corresponding nonbiotinylated primer for PCR amplifica- primer 4 5'-F-GATGATCCGACGCATCAGAATGT tion of target DNA as described above; see Fig. IA) and an 3'-CTACTAGGCTGCGTAGTC 18-mer complementary to the constant region of the 23-mer. The duplex was formed by annealing 20 pmol ofeach of the two primer 5 5'-F-GATGATCCGACGCATCACTCAAC oligonucleotides in a 1O-plJ volume containing 2 pl of Seque- 3'-CTACTAGGCTGCGTAGTG nase 5 x buffer (200 mM Tris'HCl, pH 7.5/100 mM MgCl2/250 primer 6 5'-F-GATGATCCGACGCATCAGCCTAG mM NaCl) from the Sequenase kit or in a 13-,ul volume 3'-CTACTAGGCTGCGTAGTC containing 2 ,ul of the annealing buffer (1 M Tris'HCl, pH 7.6/100 mM MgCl2) from the AutoRead sequencing kit. The FIG. 2. Sequencing primers. The "F" at the 5' end indicates annealing mixture was heated to 65°C and allowed to cool fluorescein. Downloaded by guest on September 26, 2021 10164 Biochemistry: Fu et al.