4500-4504 Nucleic Acids Research, 1997, Vol. 25, No. 22
`
`© 1997 Oxford University Press
`
`New dye-labeled terminators for improved DNA
`sequencing patterns
`B. B. Rosenblum*, L. G. Lee, S. L. Spurgeon, S. H. Khan, S. M. Menchen, C. R. Heiner
`and S. M. Chen
`
`P E Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA
`
`Received August 14, 1997; Revised and Accepted October 3. 1997
`
`ABSTRACT
`We have used two new dye sets for automated
`dye-labeled terminator DNA sequencing. One set
`consists of four, 4,7-dichlororhodamine dyes (d-rhoda(cid:173)
`mines). The second set consists of energy-transfer
`dyes that use the 5-carboxy-d-rhodamine dyes as
`acceptor dyes and the 5- or 6-carboxy isomers of
`4'-aminomethylfluorescein as the donor dye. Both dye
`sets utilize a new linker between the dye and the
`nucleotide, and both provide more even peak heights
`in terminator sequencing than the dye-terminators
`consisting of unsubstituted rhodamine dyes. The
`unsubstituted rhodamine terminators produced elec(cid:173)
`tropherograms in which weak G peaks are observed
`after A peaks and occasionally C peaks. The number of
`weak G peaks has been reduced or eliminated with the
`new dye terminators. The general improvement in peak
`evenness improves accuracy for the automated base(cid:173)
`calling software. The improved signal-to-noise ratio of
`the energy-transfer dye-labeled terminators combined
`with more even peak heights results in successful
`sequencing of high molecular weight DNA templates
`such as bacterial artificial chromosome DNA.
`
`INTRODUCTION
`
`Sanger dideoxy DNA sequencing is the most commonly used
`method for DNA sequencing, particularly in large scale genomic
`sequencing ( 1 ). Automated DNA sequencing uses fluorescent
`dyes for the detection of the electrophoretically resolved DNA
`fragments. Two variations of automated DNA sequencing have
`evolved: dye-labeled primer sequencing (2-4), in which the
`fluorescent dyes are attached to the 5' end of the primer
`oligonucleotide, and dye-labeled terminator sequencing, in which
`the dyes are attached to the terminating dideoxynucleoside
`triphosphates (5,6). Each sequencing method has advantages and
`disadvantages.
`the
`Dye-labeled primer sequencing has benefited from
`development of DNA polymerases which do not discriminate
`between deoxy- and dideoxynucleotides (7,8). These polymer(cid:173)
`ases provide sequencing electropherograms with very even peak
`heights. Base-calling is easy and reliable, and the ability to call
`
`heterozygotes can be based on peak heights as well as the
`presence of two bases at a position (9, 10). The major disadvan(cid:173)
`tage oftbe dye primer method is the requirement for four separate
`extension reactions and four dye-labeled primers for each
`template.
`The major advantages of dye-labeled terminator sequencing
`are convenience, since only a single extension reaction is required
`for each template, and the synthesis of a labeled primer is
`unnecessary, allowing the use of preferred hybridization sites. In
`addition false terminations, in which the DNA fragments are
`terminated by a deoxynucleotide rather than a dideoxynucleotide,
`are not observed as these products are unlabeled. The major
`disadvantage of dye-labeled terminators is that with every
`polymerase the pattern of termination with dye-labeled termin(cid:173)
`ators has been found to be less even than for dye-labeled primers.
`The presence of very small or very large peaks can result in errors
`in automated base-calling.
`We have evaluated the use of two new dye sets, 4,7-dichloro(cid:173)
`substituted rhodamines (d-rhodamines), and a set of energy(cid:173)
`transfer dyes that were previously described for use on
`dye-labeled primers ( 11 ). Use of the new terminators, d-rhoda(cid:173)
`mine terminators and BigDyeTM terminators, using the energy
`transfer dyes, required optimization of the linker attaching the
`dyes to the nucleotides, the dye isomer used, and the choice of
`each dye on a particular nucleotide. A major objective with both
`dye-sets was to obtain more even peak patterns compared with
`current DNA sequencing terminators. The energy-transfer dyes
`also offer the advantage of increased signal. The improved peak
`evenness found in both new dye sets allows greater accuracy in
`base-calling, longer reads and the ability to use dye-labeled
`terminators for heterozygote analysis.
`
`MATERIALS AND METHODS
`
`Dye-labeled terminators
`
`The dye-labeled terminators were prepared by methods previous(cid:173)
`ly described ( 12). Briefly, the succinimidyl ester of each dye was
`mixed with the nucleoside triphosphates at pH 9. The products
`were purified on HPLC by anion-exchange chromatography to
`remove excess dye, followed by reverse-phase chromatography
`to separate unlabeled tripbosphates and to separate dye-isomers.
`
`• To whom correspondence should be addressed. Tel: + I 650 638 5566; Fax: + I 650 638 6666; Email: rosenbbb@perkin-elmer.com
`
`Illumina Ex. 1046
`IPR Petition - USP 10,435,742
`
`

`

`Nucleic Acids Research, 1997, Vol. 25, No. 22
`
`4501
`
`Table 1. Linkers, fluorophores and final terminator concentration for each nucleotide of the three terminator sets: rhodamine, d-rhodamine and BigDye
`
`Terminators Rhodamine
`Linker'
`Nucleotide
`
`Dye
`
`Final cone. (µM)
`
`d-Rhodamine
`Dye
`Linker
`
`Final cone. (µM)
`
`ddATP
`ddCTP
`ddGTP
`ddTTP
`
`PA
`PA
`PA
`PA
`
`0.02
`S-R6G
`0.13
`6-ROX
`S-RllO
`0.01
`6-TAMRA 0.23
`
`PA
`EO
`EO
`EO
`
`dR6G-2
`dTAMRA-2
`dRll0-2
`dROX-1
`
`0.02
`0.12
`0.01
`0.18
`
`BigDye
`Linker
`
`PA
`EO
`EO
`EO
`
`Donor dye Acceptor dye
`
`Final cone. (µM)
`
`6-FAM
`6-FAM
`S-FAM
`6-FAM
`
`dR60-2
`dROX-2
`dRll0-2
`dTAMRA-2
`
`0.11
`0.16
`0.10
`l.12
`
`"The linkers are PA. propmgylamino and EO, propargyl ethoxyamino.
`
`DNA sequencing
`Dye-labeled terminator cycle sequencing with d-rhodamine and
`BigDye terminators, using the energy-transfer dyes, was per(cid:173)
`formed using AmpliTaq DNA polymerase, FS, according to the
`ABI PRISM™ sequencing manual (PE Applied Biosystems,
`Foster City, CA). With the BigDye terminators, dUTP was
`substituted for dTTP at the same concentration in the dNTP mix.
`The concentrations of the dNTPs in the reactions were 100 µM
`for dATP, dCTP and dTTP (or dUTP) and 500 µM for dITP. The
`concentrations of the terminators used in the reactions were
`determined by titrating each terminator in single-color terminator
`reactions and selecting the concentration which maximi7.ed the
`signal of the 700th nucleotide (12). The concentrations were
`adjusted according to the relative brightness of each dye in order
`to obtain approximately equivalent signal for each color in the
`four-color reaction (Table 1 ). The d-rhodamine and BigDye
`terminator sets used 10 nm virtual filters on the CCD camera of
`the ABI PRISM 310 and 377 centered at 540 or 545 (ABI PRISM
`310), 570, 595 and 625 nm.
`Bacterial artificial chromosomal (BAC) DNA was purified by an
`alkaline lysis protocol ( 13) and was sequenced with BigDye
`teoninators with a slight modification of the terminator protocol.
`Table 2 shows the reagents used per reaction for BAC sequencing.
`BAC samples were cycled in a Perkin-Elmer 9600 thennocycler
`according to the following protocol: initial denaturation at 95 °C for
`S min, followed by 30 cycles of 95°C for 30 s, 55°C for 20 s, and
`60°C for 4 min. Excess terminators were removed using Centri-Sep
`spin columns (Princeton). Samples were vacmun dried and then
`resuspended in 2 or4 µI of formamide, heated to 95 °C for 2 min and
`2 µI of the sample loaded on the ABI PRISM 377. The BAC clone,
`bWXD342, used in these studies contains an insert, 169 kb in length,
`from the human X chromosome, locus Xq21.3.
`
`Table 2. Reagent mix used for BAC sequencing
`
`Reagent
`Terminator mix
`SX sequencing buffer
`Amplitaq FS for dye terminators
`Primer
`BACDNA
`Distilled water
`Final volume
`
`Per reaction
`
`8.0µ1
`4.0µ1
`0.5µ1
`4.0pmol
`-400ng
`to volume
`40.0µl
`
`Single color analysis of the d-rhodamine and BigDye terminators
`was perfomied using the ABI PRISM 310 Genetic Analysis system.
`Single color sequencing reactions were prepared as descnbed earlier,
`except that in all cases excess temlinators were removed using
`Centri-Sep spin columns (Princeton). Samples were resuspended in
`
`ddA-PA-sdR6G
`
`.
`
`cldQ.EO-SdR.110 ·
`
`Figure I. Structure of the d-rhodamine dye-labeled terminators.
`
`Template Suppression Reagent (TSR; PE Applied Biosystems,
`Foster City, CA) and heated to 95°C for 2 min. A computer program
`has been developed to determine peak heights and to calculate the
`mean, standard deviation and relative error, where the relative error
`is the ratio of the standard deviation to the mean.
`
`RESULTS AND DISCUSSION
`
`Dichlororhodamine terminators
`In order to optimize the performance of the new d-rhodamine
`terminators, we synthesized and tested 39 out of a possible 64
`combinations of the four dichlororhodamine dyes (both 5- and
`6-carboxy isomers), propargylamino (PA) or propargyl ethoxy(cid:173)
`amino (EO) linker, and nucleotide terminators (8 x 2 x 4 = 64).
`The structure of both the dye and the linker between the
`nucleotide and the dye affected the pattern of termination
`(manuscript in preparation). We chose the dye set which
`maximized the evenness of the peaks in the sequencing pattern.
`The final d-rhodamine terminator set had mobility shifts within
`the half base requirement for minimal artifacts. (The set of all 32
`possible rhodamine dyes with the EO linker was tested but a
`4-dye set was not found that had acceptable mobility characteris(cid:173)
`tics.) The structures of the d-rhodamine dye-labeled terminators
`are shown in Figure 1. Only the A-terminator retains the
`propargylamino linker of the original terminators (Table I).
`
`

`

`4502 Nucleic Acids Research, 1997, Vol. 25, No. 22
`
`BigDye terminators
`
`We have previously reported on a set of energy-transfer dyes for
`dye-labeled primer sequencing that uses the 5-carboxy-dichloro(cid:173)
`rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers
`of 4'-aminomethylfluorescein as the donor dye. These dyes show
`both improved spectral resolution and improved brightness
`compared with the standard dyes used for dye-primer sequencing
`( I 1 ). Here, we have investigated the use of the energy-transfer
`dyes on dye-labeled terminators.
`We synthesized and tested 18 out of a possible 64 combinations
`of energy-transfer dyes (four 5-carboxy-d-rhodamines with both
`5- and 6-carboxy isomers of 4'-aminomethylfluorescein), propargyl(cid:173)
`amino (PA) or propargyl ethoxyamino (EO) linker, and four
`nucleotide terminators (8 x 2 x 4 = 64). Again, we chose the dye
`set which maximized the evenness of the peaks in the sequencing
`pattern and minimized the dye-related mobility effects. The final
`BigDye terminator set had mobility shifts within the half base
`requirement for minimal artifacts. The structure of one of the four
`BigDye terminators, the ddT-E0-6CFB-dTMR, is shown in
`Figure 2. In addition to varying the terminator structure, we found
`that varying the structure of the dNTPs also affected the pattern
`of termination. By substituting dUTP for dTTP the termination
`pattern for energy-transfer dye-labeled ddT terminators was
`improved for each of the seven different energy-transfer dye/
`linker/ddT compounds tested.
`A feature of the BigDye terminator set is that the total signal is
`increased compared with either the rhodamine or d-rhodamine
`dye sets. BigDye terminators can be used with advantage in cases
`when template molar equivalents are limited. BAC DNA
`fragments typically have very high molecular weights and cannot
`
`+
`NMe,
`
`~o NH
`
`·o
`
`~'-"'o
`0 ~t
`>ao,P.,oD .o
`
`ddT-E0-6CFB-dTMR
`
`Figure 2. Strucrure of the ddT-BigDye tenninator.
`
`be sequenced with single dyes. Energy-transfer dye-labeled
`primers (9, 14-17) have proven useful for these templates (13).
`Here we demonstrate the signal advantage of energy-transfer
`dyes combined with the convenience of dye-labeled terminators.
`Figure 3 shows a sequencing pattern for a bacterial artificial
`chromosome template (clone:bWXD342; sequencing reaction
`Table 2) using the BigDye terminators.
`
`Table 3. Comparison of number of sequencing accuracy, read length and total signal for different templates with the three dye terminator chemistries
`
`Template
`
`%GC
`
`Errors to 720 bases
`
`Read length at 98.0% accuracy
`
`Signal strength
`
`Rhod
`
`dRhod
`
`BigDye
`
`Rhod
`
`dRhod
`
`BigDye
`
`Rhod
`
`dRhod
`
`BigDye
`
`349, -21Ml3
`349, -21Ml 3
`4009, -21Ml3
`4009, Reverse Ml3
`ABO 114, Reverse M 13
`pGEM, -21M13
`pGEM, -21Ml3
`
`pGEM, Reverse Ml3
`pcONA, Reverse M 13
`OJ2, Reverse M 13
`AB0116,-2 1Ml3
`ABO 116, -21M l 3
`ABO 116, Reverse M 13
`ABO 116, Reverse M l 3
`ABO 100, -21Ml3
`ABO 100, Reverse Ml3
`ABO 90, -21Ml3
`ABO 90, Reverse M 13
`MEAN
`
`65.3
`
`71.4
`
`59
`50.8
`
`33
`30
`37.3
`
`48
`
`36.3
`
`50
`45
`44
`28
`16
`I I
`21
`12
`4
`7
`12
`25
`6
`3
`26
`2 1
`33
`
`3
`20
`
`14
`15
`18
`14
`7
`4
`6
`
`9
`4
`3
`6
`11
`2
`
`6
`8
`12
`18
`
`8
`9
`
`16
`34
`26
`10
`7
`8
`6
`0
`2
`2
`
`12
`2
`
`5
`2
`6
`4
`12
`
`9
`
`394
`543
`586
`616
`691
`718
`
`654
`785
`790
`743
`744
`612
`788
`807
`605
`
`659
`364
`834
`663
`
`704
`
`688
`692
`701
`
`781
`796
`
`784
`727
`790
`798
`791
`736
`788
`740
`785
`
`727
`671
`773
`748
`
`683
`613
`646
`741
`
`733
`723
`
`776
`831
`843
`790
`
`810
`769
`776
`807
`813
`
`758
`835
`720
`759
`
`1453
`2812
`
`1098
`693
`
`2830
`3478
`
`3548
`3202
`3929
`1874
`2268
`1974
`2397
`21 02
`877
`1336
`1172
`1064
`2117
`
`732
`1182
`
`333
`397
`1413
`
`1447
`1374
`
`1374
`1366
`724
`948
`502
`1090
`650
`375
`498
`442
`602
`858
`
`1422"
`2993
`2056•
`2692'
`3698°
`
`4865
`3834°
`42688
`5184°
`4434•
`2750
`
`1346°
`4385
`2384'
`1118'
`1606°
`1884•
`15523
`2915
`
`'BigOye tenninator separations were perfonned with half the sample load compared with rhodarnine separations. Signal value has been nonnalized by doubling
`the signal value for the BigOye tenninators for these separations.
`
`

`

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`
`Nucleic Acids Research, 1997, Vol. 25, No. 22
`
`4503
`
`, ,. , , ,
`1:$
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`Figure 3. Sequencing ofBAC DNA (clone:bWXD342) with BigDye terminators.
`
`Comparison of the three terminator sets
`
`Table 1 shows the matching of linker and fluorophore for the
`original rhodamine, the d-rhodamine, and the BigDye tennin(cid:173)
`ators. Table 3 compares the sequencing accuracy, read-lengths
`and signal for the three tenninator sets for different templates. As
`a result of the better peak evenness of the d-rhodamine and
`BigDye tenninator sets both base-calling accuracy and read(cid:173)
`lengths are improved. Although the total signal for the d-rhoda(cid:173)
`mine tenninators is reduced compared with the rhodamines
`(equal amounts of template were loaded on the sequencing gels),
`the multicomponent noise is also reduced due to the better
`spectral resolution of the d-rhodamine dye set. The signal strength
`for the BigDye tenninators with most of the templates is higher
`than the signal strength for the rhodamine tenninators as expected
`based on the brightness of the energy-transfer dyes ( I I). The
`2-fold reduction in multicomponent noise for the d-rhodamine
`and BigDye tenninators compared with the rhodamine tennin(cid:173)
`ators is not reflected in the signal number ( 11).
`We have analyzed a series of different templates with three sets
`of dye-labeled tenninators, rhodamine, d-rhodamine and Big(cid:173)
`Dye, to compare peak evenness and to identify sequence context
`effects. The sequencing patterns of a portion of template DJ2
`using the three sets of dye-tenninators are shown in Figure 4. In
`the rhodamine set, very weak G peaks after A peaks are observed,
`with some weak G peaks after C peaks. There are also some very
`strong peaks that result in the smallest peaks being > 10-fold
`
`smaller than the largest peaks, with a small peak frequently
`appearing just before or after these large peaks. In both the
`d-rhodamine and the BigDye tenninator patterns, the peaks are of
`more even heights, so that in general the adjacent peaks are
`<5-fold different in size. The small G peaks after A peaks or C
`peaks has also improved, with the BigDye tenninators showing
`no weak G peaks and the d-rhodamines showing a few weak G
`peaks after A peaks or C peaks. These small G peaks are relia?ly
`called by the automated software due to the better balance in the
`peak heights. The BigDye tenninators show weaker T peaks after
`G peaks, but again, because the overall pattern is more balanced,
`these T peaks are called by the automated software.
`
`Table 4. Relative errors" of peak heights for bp 10-315 in pGEM with the
`three tenninator sets and dye primers
`
`Dye· primer Rhodomine
`
`d·Rhodnminc BigDyc
`dT
`
`A
`c
`G
`T
`Average
`
`0.27
`0.26
`0.31
`0.22
`0.26
`
`0.83
`0.60
`0.83
`0.52
`0.69
`
`0.49
`0.33
`0.42
`0.36
`0.40
`
`0.23
`0.28
`0.37
`o.ss
`0.36
`
`dU
`
`0.23
`0.3 t
`0.3t
`0.40
`0.32
`
`The dye-primer reactions use c7dGTP in the dNTP mix, while the terminator
`reactions use c!ITP in the dNTP mix instead of dGTP.
`"The relative error is the ratio of the standard deviation of the peak heights to the
`mean of the peak heights for a selected group of peaks.
`
`

`

`4504 Nucleic Acids Research, 1997, Vol. 25, No. 22
`
`were generated using dye-primers, supplemented by dye-termin(cid:173)
`ators only when necessary. These requirements may now possibly
`be met with the two new dye-terminator chemistries, eliminating the
`need for dye primers. Sequencing for heterozygote analysis may be
`able to be performed with these new dye-terminators rather than
`dye-primers. Future work in enzyme engineering and dye synthesis
`to further enhance the performance of dye-labeled terminators will
`likely render obsolete traditional dye-labeled primer sequencing.
`
`Rhedamino Temunaiors
`
`ACKNOWLEDGEMENTS
`
`Oicll'ororhodDmine Tcnnlnotors
`
`BigOyo Termlna!OI&
`
`Figure 4. Comparison of sequencing patterns of rhodarnine, d-rhodamine and
`BigDye terminators on an AT rich template, DJ2. Arrows in (A) are G peaks
`with weak signal. Many of these G peaks are similar in size to noise peaks under
`adjacent peaks. In (B) and (C), all of the G peaks are much larger than any noise
`peaks. In (C), the arrows indicate weaker T peaks following G peaks. These T
`peaks are larger than any noise peaks and are called by the automated software.
`
`The peak patterns can be quantitatively evaluated by measuring
`the peak heights of a given sequence and calculating an average
`and standard deviation. The data are normalized by defining the
`relative error as the ratio of the standard deviation to the average
`peak height. A completely uniform series of peaks would yield a
`relative error value ofO. This is unlikely to occur for any type of
`Sanger sequencing except over a very short group of peaks,
`because of the exponential decay of the terminal events with
`increasing fragment size ( 12), results in decreasing peak height
`with increasing fragment length over a large group ofbases. Thus,
`for a group of> 100 bases, a value of0.15--0.3 would be expected
`for dye primer sequencing (Table 4). Table 4 shows the results of
`peak height evaluation for the rhodamine, d-rhodamine and
`BigDye terminator sets, along with the relative error values for
`dye-labeled primer sequencing for the same region.
`
`CONCLUSIONS
`
`We have developed two new dye-terminator sets that are both
`improvements over previous dye-terminators. The peak patterns of
`these chemistries are nearly as even as dye-labeled primer
`sequencing patterns. The genome sequencing community requires
`data with a high confidence of base-calling, and data generated by
`two different sequencing approaches in areas with single-orientation
`coverage ( 18- 22). In the past this has meant that the bulk of the data
`
`We thank Jim Bowlby for developing software for peak height
`evaluations and Bill Efcavitch for helpful discussions. We are
`grateful to Mike Hunkapiller for expediting the dye-terminator
`effort, to Krishna Upadhya, Tony Constantinescu, Ron Graham,
`Paul Kenney, Brian Evans, Scott Benson, Pete Thiesen and Mary
`Fong for performing the dye syntheses and purifications, to Pavel
`Cotofana and James Liang for synthesis of dye terminators and
`to Gilbert Amparo for analysis of base-calling accuracy. The
`clone DJ2 was a gift from Drs Simon Plyte and Jim Woodget. The
`purified BAC clone was prepared by Dr B.H. Brownstein at
`Washington University Medical School.
`
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