Anal. Chem. 1992, 64, 2672-2677
2872
Solid-Phase Method for the Purification of DNA Sequencing Reactions Xinchun Tong and Lloyd M. Smith* Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706
A wiid-phase method for the puritkatlonof the singlestranded DNA molecules produced In enzymatic sequencing reactions has been developed. A prlmer oligonucleotideis synthesized contalnlng a biotin moiety at an Internal posttion. This primer k utilized In enzymatic extension reactions, and the resulting products are bound to dreptavidin-coated magnetic beads. Contaminating species such as protein, salts, template DNA, and unincorporated or degraded deoxy and didwxy nucleotide triphosphates may be removed by washing the beads after immobilizingthem in the sample tube with a fixed magnet. The resulting pure single-stranded DNA fragments are removed from the wild support by heatlng in 10 mM EDTA, 95% formamide, loading dye at 90 O C , and may then be dlrectly loaded onto a polyacrylamide gel for sequence analysis. This method was wed to investigate the effect of various contaminants upon DNA sequence data.
INTRODUCTION DNA sequence analysis continues to play a central role in modem molecular biology. Automated methods for sequencing promise eventually to remove the burden of routine DNA sequencing from the research laboratory, as well as permitting the successful performance of very large scale projects such as the analysis of the Human Genome.192 Although some apsects of sequencing have been effectively automated, others remain to be addressed. For example, although the separation and detection of DNA fragments in polyacrylamide gels has been automated,3*4most of the steps preceding this separation, such as the generation of subclones, growth and purification of template DNAs, and so on, are still largely performed manually. Much of the reason for this lies in the nature of the procedures involved. Traditional methods for sequencing are not readily amenable to automation, involving procedures such as the growth and plating of bacterial cultures, biochemical extractions, and centrifugations. In general, these processes need to be redesigned in ways that are more compatible with automation. One generic approach to many of these problems is offered by solid-phase chemistry. Solid-phase methods have a long history in molecular biology, in for example, the Southern and Northern hybridization procedures.5 More recently, solid-phase chemistries for DNA and peptide synthesis, and even total gene assembly, have been developed, with revolutionary consequences for the field.687 These procedures are (1) Hood, L. E.; Hunkapiller, M. W.; Smith, L. M. Genomic 1987,1, 201-212. (2)Smith, L. M.;Hood, L. E. BiolTechnology 1987,5,933-939. (3)Smith, L. M.;Sanders, J. Z.; Kaiser, R. J.; Hughes, P.; Dodd, C.; Connell, C. R.; Heiner, C.; Kent, S. B. H.; Hood, L. E. Nature 1986,321, 674-679. (4)Prober, J. M.; Trainor, G. L.; Dam, R. J.; Hobbs, F. W.; Robertson, C. W.; Zagursky, R. J.; Cocuzza, A. J.; Jensen, M> A.; Baumeister, K. Science 1987,238,336-341. (5)Gillespie, D.; Spiegelman, S. J.Mol. Biol. 1966,12,829-842. (6)Atkinson, T.;Smith, M. In Oligonucleotide synthesis-A practical approach; Gait, M. J., Ed.; IRL Press: Oxford, 1984;pp 35-81. 0003-2700/92/0364-2672$03.00/0
intrinsically amenable to automation and eliminate the problems with purification of intermediates that plague conventional approaches. Uhlen and co-workers have pioneered the use of such methods for problems in DNA sequence analysis, exploiting the high affinity and stability of biotinavidin chemistry to permit the immobilization and sequence analysis of double-stranded PCR products.Sl0 In this paper we describe a solid-phase DNA sequencing method for the purification of the single-stranded DNA molecules produced in enzymatic sequencing reactions. A primer oligonucleotide labeled a t the 5' terminus with 32P and containing a biotin group at an internalposition is utilized in the enzymatic extension reactions, and the resulting products are bound to streptavidin-coated magnetic beads. The template strand and other reaction contaminants are removed by denaturation and washing of the beads, and the remaining pure single-stranded fragments are eluted by heating in a EDTA/formamide mixture. In contrast to previously described methods, in which the biotinylated strand remains attached to the support particle, in this approach the biotinylated molecules are removed from the support particle and directly analyzed. This allows the purification method to be utilized for the sequence analysis of the single-stranded M13 template DNAs most widely used in enzymatic DNA sequencing. This solid-phase strategy was used to investigate the effects of template DNA, polymerase, glycerol, and salts upon DNA sequence data.
EXPERIMENTAL SECTION Enzyme, DNA Template, and Oligonucleotide Primer. Bst DNA polymerase was obtained from Bio-Rad (Richmond, CAI. Single-stranded M13mp19 template DNA was prepared by standard methods. A modified M13 sequencing primer (denoted B-BT) with the sequence 5'-CAT* GAC GTT GTA AAA CGA CGG CCA GT-3' was synthesized by the University of Wisconsin Biotechnology Center on an Applied Biosystems 380A DNA synthesizer (Foster City, CA). T* is the product obtained from use of the modified nucleoside phosphoramidite amino-modifier-dT (Glen Research, Sterling, VA), which has a primary amino group attached to thymine with a 10-atomspacer arm. This amino group was coupled to NHS-LC-Biotin(Pierce, Rockford, IL) as described." The biotinylated primer (denoted Bio-B-BT)was purified by reverse-phasehigh-performance liquid chromatographyand 5' end-labeled using polynucleotidekinase (USB, Cleveland,OH) and [y-32Plat 5000 Ci/mmol (Amersham, Arlington Heights, IL). Magnetic Beads. Streptavidin-coupled magnetic beads (Dynabeads M-280, Dynal, Inc., Great Neck, NY) were utilized as a solid support. A 1J2-in.X 1J2-in.neodymium-iron-boron magnet (BuntingMagnetics, Newton, KS)was used to immobilize the beads during the supernatant removal and washing steps. (7)Stewart,J.M.;Young, J. D. Solidphasesynthesis; W. H. Freeman: San Francisco, 1969. (8) Stahl, S.; Hultman, T.; Olsson, A.; Mob,T.; Uhlen, M. Nucleic Acids Res. 1988,16,3025-3038. (9)Hultman, T.; Stahl, S.; Homes, E.; Uhlen, M. Nucleic Acids Res. 1989,17,4937-4946. (IO)Hornes, E.; Korsnes, L. Genet. AMI. 1990 7 , 145-150. (11)Dynabeads M-280 Technical Handbook: Magnetic D N A Technology 6. 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
Table I. Protocol for Solid-Phase DNA Sequencing 1. label tubes A, C, G, T and aliquot 2 pL of the appropriate d/ddNTP mix to each tube; keep on ice 2.
3. 4.
5. 6. 7. 8.
9. 10. 11.
5
v ' 7 NH, Primer (B-BT)
mix the following cocktail (10 p L per set
of four reactions): 1.0 p L of ssM13mpl9 template DNA (2.0 pg/pL = 0.8 pmol/pL) 1.5 pL 10 X SB (100 mM MgC12,lOO mM Tris pH 8.5) 0.8 pL primer (0.8 pmol) 6.7 pL of H20 annealing: 65 "C (heatingblock) 2 min, 37 "C (heatingblock) 5 min, RT (air) 10 min add 0.5 pL of Bst DNA polymerase remove 2.5 pL of cocktail and add it to each nucleotide mix tube and immediately place at 65 "C for 5 min remove tubes from heating block and add 2 pL of 10 mM EDTA, 95% formamide heat tubes at 90 "C for 5 min; place on ice binding: to each tube add 2.0 pL of 5 X BB (5 X TES,1%Tween-20) 2.0 pL of beads (in 1 X TES) sit at room temperature for 15 min immobilize beads with a magnet, wash twice with 10 pL of 1 X TES, once with 10 pL of H20 elute DNA fragments from beads in 6.5-pL stop solution (10 mM EDTA, 9596 formamide, 0.05 ?6 bromophenol blue) at 90 "C for 5 min pipette off the supernatant and load on gel
The beads were prewashed in 1X TES (10 mM Tris-HC1,l mM EDTA, 1 M NaCl, pH 8.2) three times and resuspended at 10 pg/pL in 1X TES. These beads are reported by the manufacturer to possess 0.3 pmol of biotin-bindingsites per microgram of beads. Single-Stranded Sequencing Reactions. DNA sequencing reactions were performed by mixing 0.8 pmol of template DNA, 0.8 pmol of 5'-32Pend-labeled primer, and 1.5 pL of 10 X SB (SB, or sequence buffer, is 10 mM MgC12 and 10 mM Tris-HC1 pH 8.5).12 Water was added to give a total volume of 10 pL. This mixture was placed at 65 "C (heating block) for 2 min, at 37 "C (heating block) for 5 min, and at room temperature (air) for 10 min to anneal the primer to the template. A 0.5-pL portion of Bst DNA polymerase (1unit/pL, Bio-Rad, Richmond, CA) was added, and 2.5 pL of the resultant enzyme/DNA mixture was placed in each of four tubes containing2.0 p L of d/ddNTP mixture (see below). The sequencing reactions were placed at 65 " C for 5 min, followed by the addition of 2.0 pL of a solution containing 10 mM EDTA (diluted from a 0.5 M solution of disodium EDTA which had been adjusted to pH 8.2 with NaOH) and 95% formamideto stop the reaction. They were denatured by heating at 90 "C for 5 min and immediately placed on ice before either loading on an acrylamide gel for analysis or binding to magnetic particlesfor purification. The following nucleotide mixtureswere used: 250 mM each of 3 dNTPS, one of 240 mM ddGTP, 480 mM ddATP, 500 mM ddTTP, or 200 mM ddCTP, and 25 mM of the corresponding dNTP. Solid-Phase Procedures. The newly synthesized DNA fragments were bound to streptavidin-coated magnetic beads by adding 2.0 pL of the bead suspension and 2.0 p L of 5 X BB (BB, or binding buffer, is 1X TES and 0.2 96 Tween-20)toeach reaction tube. The tube was placed at room temperature for 15 min to allow binding to take place. The supernatant was removed, and the beads were washed using the magnet to immobilizethe beads in the tubes. The beads were washed twice with 10 p L of 1 X TES buffer and once with 10 pL of H20. The DNA fragments were eluted from the beads in 6.5 p L of stop solution (10 mM EDTA, 953' 6 formamide,0.05 76 bromophenol blue) at 90 "C for 5 min. Approximately 1.5 pL of supernatant were loaded on a 6 % denaturing polyacrylamide gel for sequence analysis. Table I is a protocol for solid-phase DNA sequencing, in which steps 1-7 describethe DNA sequencingreactions. Kodak XAR-5film (Rochester,NY) was used for autoradiography, and scintillation (12) Mead,D.A.;McClary, J.A.;Luckey, J.A.;Kostichka,A. J.; Witney, F. R.;Smith, L.M.BioTechniques 1991,lZ.7 6 8 7 .
3
'
NHS-LC-Biotin
c
2673
'y3' NH-Riotin Internal Biotinylated Primer (Rio-R-RT)
Extension From
Primer
5:
" 7 -
Rind To Magnetic Reads With Streptavidin
(
Denaturing
Y
) c
Enzyme, dNTP & d d NTP
t n
Washing
/
\
Enzvme.
"e
* -
Purified D S A Fragments
ddNTP Elute D N A Frapments
Flgure 1. A schematic drawing of the basic concept for solid-phase DNA
sequencing.
counting was performed on a Beckman LS6000SE (Fullerton, CA).
RESULTS AND DISCUSSION Basic Concept. A diagram outlining this approach to solid-phase DNAsequencing is shown in Figure 1. Sequencing reactions are performed in solution by standard procedures, except that a biotinylated primer is employed. In this work the primer was a 26mer biotinylated at an amino-modified T nucleoside three bases from the 5'end. The primer sequence was chosen to be complementary to the M13 sequence a t all positions other than the modified T, which replaced a C normally at that position. The choice of an internal position for the biotin group left the 5' terminus of the primer free for labeling with 32Pusing polynucleotide kinase. The sequencing reactions are denatured to separate the newly synthesized fragments from the template strand, and the fragments are captured on streptavidin-conjugated magnetic beads in a 15-min incubation at room temperature. The inclusion of the detergent Tween-20 in the binding buffer was found to be important for minimizing nonspecific binding. Contaminating species are removed by washing the beads after immobilizing them in the sample tube with a fixed magnet. The resulting pure single-strandedDNA fragments are eluted from the solid support by heating at 90 "C in 10 mM EDTA and 95 % formamide, and may be loaded directly onto a denaturing polyacrylamide gel for sequence analysis. Binding and Elution Efficiency. A central issue in this solid-phase procedure is the efficiency of capture and elution of the extension reaction products. In order to quantify and optimize these efficiencies, biotinylated primers labeled at the 5'terminus with 32Pwere bound to and eluted from beads with radioactive counting of the beads and supernatants. The
2674
- -
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
90°C 2min 90°C Smin Room Temp. m C D E F A B Control Solid Phase Control Solid Phase Control Solid Phase ACGT ACGT ACGT ACGT ACGT ACGT
Table 11. Elution Efficiency kmp(OC), time (min)
90,lO
90,5 9092
65,5 65,2 37,lO 90,lO 90,lO 90,lO 90,lO 90,lO 90.10
Figure 2. The effect of a denaturatkm step prior to bead capture on binding efficiency. An autoradiograph of a 6% polyacrylamide, 8.3 M urea gel showsthe productsof sequencingsingle-stranded M 13mp 19 with Bst DNA potymerase. Sets B, D, and F were sequencing reactions obtained using the soiibphase method in which a denaturation step subsequent to the extension reactions but prior to bead capture is omitted (B), at 90 O C for 2 mln (D), and at 90 OC for 5 min (F), then to which 2.0 gL of 10 pg/gL beads suspended in 1 X TES and 2.0 pL of 5 X TES, 1 % Tween-20 were subsequentJy added. Following binding and washing procedures (described in Table I), the DNA fragments were eluted from the beads in 3.0 pL of stop solution. Sets A, C, and E show control reactions that were denatured in the same way but to which no beads were subsequently added. The comparable intensity of the sequence ladders in lanes E and F is indicative of a binding efficiency of about 50 % ,as the control reactions were twice as dilute; 1.5 pL of the sequencing reactions were loaded on the gel for each Of Wt A-F.
variables examined in the binding reaction were temperature, saltconcentration,the primer/bead ratio, and incubation time. Varying the temperature from ambient to 65 "C had little
elution solution 10 mM EDTA, pH 8.2 and 95% formamide 10 mM EDTA, pH 8.2 and 95% formamide 10 mM EDTA, pH 8.2 and 95% formamide 10 mM EDTA, pH 8.2 and 95% formamide 10 mM EDTA, pH 8.2 and 95% formamide 10 mM EDTA, pH 8.2 and 95% formamide H2O 10 mM EDTA, pH 8.2 95 7; formamide 30 mM NaOAc, pH 9 and 95% ' formamide 80 mM NaOAc, pH 9 and 95% formamide 140 mM NaOAc, pH 9 and 95% formamide
elution efficiencv (961 96.8 96.4 96.8 96.4 97.9 41.9 7.3 52.0 35.9 95.5 97.3 95.4
effect upon the binding efficiency. The binding efficiency was 89% in 1 X TES, 0.2% Tween-20 compared to 74% in 10mM Tris-HCI,pH 8.2,0.2 % Tween-20. An &fold variation in the primer/bead ratio (0.0025-0.02 pmol of primer/pg of beads) had no significant effect upon binding efficiencies, nor did incubation times of longer than 15 min. In early experiments the binding efficiencies obtained for sequencing reactions were substantially lower than those found for the free biotinylated oligonucleotides. Experiments were performed to evaluate whether this lower efficiency was due to interference in binding from the template strand. Figure 2 shows the effectof a denaturation step prior to bead capture. Reactions B, D, and F show the sequencing ladders obtained using the solid-phase protocol in which a denaturation step subsequent to the extension reactions but prior to bead capture is either omitted (B), 2 min a t 90 "C (D), or 5 min a t 90 "C (F). Reactions A, C, and E show control reactions treated the same way but to which no beads were subsequently added. In this experiment the control reactions were dissolved in twice the volume as the solid-phase reactions, but the same volumes were applied to the gel. The data show clearly that the denaturation step is critical to obtaining a good efficiency of binding to the beads. The comparable intensity of the sequence ladders in lanes E and F is indicative of a binding efficiency of about 50%, as the control reactions were twice as dilute. This was confirmed by cutting out the gel region and measuring the radioactivity by scintillation counting. The modest effect of the 2-min denaturation relative to the 5-min denaturation is probably due to the time required to bring the sample tube to temperature after placing it in a hot block.13 The need for a denaturation step prior to binding presumably reflects some form of steric hindrance between the template DNA and the streptavidin-coated magnetic particle, although this has not been investigated in detail. In order to further increase the binding efficiency, the denaturation conditionswere investigated. As expected,more stringent denaturation conditions produced by lower salt concentrationsand/or added formamideincreasedthe binding efficiency. It was also found to be important to place the reactions on ice immediately after denaturation, presumably to inhibit renaturation prior to bead capture. The best conditions found employed a 5-min denaturation a t 90 "C in the presence of 29 % formamide and 3.0 mM EDTA followed (13) DCunha, J.; Berson, B. J.; Brumley, B. L.; Wagner, P. R.; Smith,
L. M. BioTechniques
1990,9,80-90.
-
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
Conventional
A Not Denatured ACGT
B Denatured
ACGT
2675
Solid Phase I
C Not Denatured ACGT
I
D Denatured ACGT
30000-
15 L
10
O
Sample well region Main body of gel
20
25
3I5
30
Rehybridization Time ( m i d
2 f 2m
B)
30000
.r(
2
2
lsoob
-
0 4k -5000
.
I
.
,
-
Sample well region Main body of gel 0
I
.
,
.
I
.
,
,
.
by immediate incubation on ice (see Table I for protocol) and resulted in a binding efficiency of -80 % The degree of nonspecific binding of DNA to the streptavidin-coated beads was determined in mock binding experiments using unbiotinylated B-BT primers. Somenonspecific binding (12.4 % ) was observedin early experiments using TES as a binding buffer, but was rendered negligible (0.9%) by inclusion of the detergent Tween-20 in the binding buffer. In order to have a high overall efficiency, both the binding and the elution efficiencies must be high. A variety of conditions were investigated for elution of the immobilized DNA fragments. As before, initial studies were performed with radiolabeled biotinylated primer oligonucleotides. The results are shown in Table 11. A 10 mM EDTA, 95% formamide solution gave efficient elution for temperatures of 65 "C or greater. Even a t a temperature of 90 "C for 10 min, very poor elution efficiency was observed in water alone, only a 52 % efficiency in 10 mM EDTA alone, and only a 36 % efficiency in 95% formamide alone. It was suspected that the effect of the EDTA might simply be to provide ionic strength to the elution buffer. To test this hypothesis other salts were tested in the elution buffer. Sodium acetate a t 30 mM or higher concentration also gave good elution efficiency (Table 11). Other salts such as triethylammonium acetate and Tris buffer of varying concentrations improved elution efficiency, but for reasons that are unclear led to some
.
Figure 3. Sequencedata examiningthe effect of a sample denaturation step prior to loading on the sequencing gel in both solid-phase purified DNA fragments and conventional sequencing reactions. Sets A and B: conventional sequencing reactions were denatured by heating at 90 OC for 5 min and immediatety placed at room temperature for longer than 1 h (A), or on ice (B). Sets C and D: the sequencing reactions employing a biotinyiated primer (see text) were denatured by heating at 90 OC for 5 min and immedlatety placed on ice before bindingto magnetk particles for purification(step 7 of Table Iprotocol). They were then eluted from the beads in 10 mM EDTA, 95 % fommide, 0.05% bromophenol blue and (C) were placed at room temperature for longer than 1 h or (D) were heated at 90 OC for 5 min and placed on ice. The radioacthe signal evident in the well of Figure 2 lane F but absent in the wells of Figure 3 lanes C and D is presumabty due to rehybridization of the sequencing products to the template DNA. This template DNA is not removed by the step employed for the experiments of Figure 2, but was removed by the procedureemployed for Figure 3.
.
2676
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992 Solid Phase
Conventional 1
Solid Phase Purified Sequencing Reactions (in 30mM NaOAc) 1 A
1
A
C Rsr
D Glycerol
ACGT
Polymcrxc AGCT
A C G 'I'
1
C
R
l0mM Mg2' 20mM Mg"
ACGT w
.
ACGT
ACGT
-*I I- ,
D
E
I:
! h M Mg2' S0mM Mg" IWmM Mg?
ACGT
ACGT
ACGT
- w -
'P n
- -
I
Figure 5. The influence of glycerol on sequence data. Set A was a control conventional sequencingreaction which includesapproximately 0.019 untts/pL or 5 ng/pL of Bst DNA polymerase and 1% glycerol. 8-D were solklphase purifiedDNA sequencing reactionsprepared as described In Table I, to which were added nothing (8). Bst DNA polymerase (0.057 unlts/pL or 15 ng/pL, C), or glycerol (3%. D).
difficultieswith reproducibility. The pH of the elution buffer may also have an effect upon elution efficiency, but this issue was not examined in detail. The mechanism of elution is presumed to involvethe denaturation of streptavidin, causing a loss of biotin-binding activity. Denaturation Experiments. Figure 3 presents data showingthe effect of a sample denaturation step immediately prior to gel loading upon both solid-phase purified DNA fragments and conventional sequencing reactions. Two observations may be made. First, data obtained from the
-
Fbwe 6. Salt effects in DNA sequencing. Single-stranded solidphase DNA sequencing reactions produced according to the protocol in Table I, except that DNA fragments were eluted in 3.0 pL of 30 mM NaOAc, 95% formamide by heating at 90 OC for 10 min. Addltional satts as indicated were added to the supernatants removed from the beads. These samples were dried on a SpewWac Concentrator (Savant) and resuspended in 3.0 pL 95% formamlde, 0.05% bromophenol blue. Aliquots (1.5 pL) were loaded on a 6% polyacrylamlde, 8.3 M urea gel and separated by electrophoresis.
solid-phase purified material did not suffer from the absence of a denaturation step, in marked contrast to data obtained from conventional reactions treated similarly. Second, the large mass of radioactive material present in the well of the conventional sequencing reactions is absent in the wells on which the solid-phase purified material was applied. Both these observations presumably reflect the absence of a DNA template strand to which the newly synthesized fragments can bind. Rehybridizationof the newly synthesizedfragments to the template strand increases their effective molecular weight greatly, preventing them from entering the gel and
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
thus producing the large amount of radioactive signal in the sample well. Independent evidence confirming the efficient removal of the template DNA strand in the solid-phase procedure was obtained in agarose gel electrophoresis experiments (data not shown). In further experiments, unpurified and purified sequencing reactions were left at room temperature for varying amounts of time after a denaturation step. They were then loaded on a sequencing gel, the products separated by electrophoresis, and the regions of the gel corresponding to the sample well and the separatedDNA fragments were excised and quantified by scintillation counting. The results are shown in Figure 4. Whereas for unpurified reactions the counts present in the sample well increased with time while those present in the gel decreased,for the solid-phasepurified reactions the counts present in both regions remained roughly constant.
Effects of Additional Components upon Sequencing Reactions. Since the solid-phaseprocedure described above yields pure single-stranded DNA fragments, it can be used to determine the effects of other components upon sequence data. Experiments were performed in which various components were added to solid-phase purified DNA sequencing reactions prior to their electrophoretic separation. The componentsexaminedwere DNA template, DNA polymerase, glycerol, and both sodium chloride and magnesium chloride. The effect of DNA template was as described above. Figure 5A,B shows control conventional and solid-phase purified sequencingreactions, and Figure 5C shows the effect of added DNA polymerase, whose only apparent effect was to produce a ‘bulge” in the sequence ladders near the top of the gel. It was suspected that glycerol present in the Bst storage buffer might be responsible for this effect.14 This hypothesis was tested by addition of glycerol to solid-phase purified sequencing reactions and found to be correct as shown in Figure 5D. Apart from the effect of the glycerol, the Bst polymerase did not appear to have an adverse effect upon the sequence data. Another major contaminant often present in sequencing reactions is salts. In protocols generally employed in fluorescence-based automated DNA sequencing,for example,l5Js an ethanol precipitation step to concentrate samples and remove salt is required prior to gel loading. Thus, the effects (14) Fuller, C. W. United States Biochemical Corp. Editorial Comments 1989, 16 (2), 19. (15) Applied Biosystema Taq DyeDeory Terminator Cycle Sequencing Kit User’s Manual; Part Number 901497,11& Al-A3. (16) Applied Biosystem 373A DNA Sequencing System User’s Manual,-Section 3, 10. (17) Drossman, H.;Luckey, J. A.;Kostichka, A. J.; D’Cunha, J.;Smith, L. M. Anal. Chem. 1990.62.900-903. (18) Luckev. J. A.: Drossman. H.: Kostichka. A. J.: Mead. D. A.: Ddunha, J.; Norris, T. B.; Smith, L.‘ M. Nucleic Acids Res. 1990, 18; 4417-4421.
2677
of added NaC1, MgC12, EDTA, and Tris-HC1upon sequence data were examined. In the addition experiments involving MgC12, the purified DNA molecules were eluted from the support in 30 mM NaOAc, 95% formamide to avoid interactions with the EDTA normally employed in elution. Interestingly, neither added sodium chloride up to 200 mM in concentration nor Tris-HC1 up to 400 mM had any discemable effect upon the sequence data (not shown). In contrast,both MgC12(Figure6)and EDTA (not shown)caused a ‘focusing” of the sequencing ladders, that is a narrowing together of the four lanes evident near the bottom of the autoradiogram. This ‘focusing” phenomenon was accompanied by a slow movement of the blue loading dye into the gel matrix from the wells and diffusion of the dye at the beginning of electrophoresis, followed by a subsequent sharpening later in the separation. It was initially surprising that these effects were specific to the salts employed and were thus not a function solely of either ionic strength or ionic conductivity. One explanation consistent with these observations,however, is that since EDTA molecules, either free or bound to Mg2+,have a relatively low electrophoretic mobility, they remain near the top of the gel much longer than do the fast moving chloride ions introduced with either sodium chloride or Tris-HC1. Their presence in the gel decreases the field strength locally, causing the marker dye to migrate slowly as well as bending the electric field lines and causing the “focusing”effect observed. In summary, we have described a simple system for the purification of DNA sequencing reactions on a solid phase. The ability to generate pure single-stranded DNA fragments for analysis permits the effect of contaminants upon sequence data to be determined, increases signal by eliminating rehybridization of the synthesized fragments to template which does not enter the gel, and offers a simple alternative approach to the automated production of clean samples for large-scale DNA sequencing. It is expected to prove useful in capillary electrophoreticmethods for DNA sequencing”J8 in which contaminants are believed to substantially decrease capillary gel stabilities and lifetimes. The utility of this method for sample preparation in automated fluorescencebased DNA sequencing methods is also under investigation.
ACKNOWLEDGMENT This work was supported by NIH Grant GM 42366. We wish to thank Mr. Robert L. Brumley, Jr. for many helpful discussions in the course of this work, and Ms. Amy Doubles for technical assistance.
RECEIVED for review May 11, 1992. Accepted August 17, 1992. Registry No. Streptavidin, 9013-20-1.
