RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 14, 950–959 (2000)

Analysis of single nucleotide polymorphisms by

primer extension and matrix-assisted laser
desorption/ionization time-of-flight mass
spectrometry
Zhengdong Fei† and Lloyd. M. Smith*
Department of Chemistry, University of Wisconsin, Madison, WI 53706-1396, USA

SPONSOR REFEREE: Dr. Patrick Limbach, Louisiana State University, Baton Rouge, LA, USA

A method for typing single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOFMS) is described, in which a mass-tagged dideoxynucleoside
triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside
triphosphate (ddNTP). The increased mass difference due to the presence of the mass-tag greatly facilitates
the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous
samples. Twenty commercially available mass-tagged dideoxynucleoside triphosphates were screened for
amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide
primer extension (SNuPE) reactions. Several sample preparation and purification methods were also
examined and compared. Float dialysis was found to be a simple, versatile, and effective method for
purification of the extension products. High specificity and sensitivity were obtained, and all six possible
biallelic SNP heterozygotes were determined accurately using a 44-mer synthetic oligonucleotide target
DNA as a model system. Further validation of the method was demonstrated in the analysis of five single-
base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR
amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five
variable positions, with results in 100% concordance with conventional sequencing. Genotypes were
determined accurately at five sequence-tagged sites (STSs). Copyright # 2000 John Wiley & Sons, Ltd.
Received 30 March 2000; Accepted 1 April 2000

Driven by recent competition and participation from the
private sector with a new sequencing strategy, and faster
DNA sequencers based on new DNA sequencing technol-
ogy, the Human Genome Project (HGP) funded by the US
government has accelerated its goals towards completing
the full human genome by 2003 and producing a working
draft by 2001.1,2 As the whole sequence is revealed in the
next several years, attention will be shifted towards
deciphering the nature of genetic variations and diversity
in the human population.
Single nucleotide polymorphisms (SNPs)3,4 are the most
common type of human genetic variation. A number of
known inherited diseases are caused by one SNP at a single
locus. Many complex disorders such as cancers may result
from cumulative effects of several deleterious SNPs. Given
the multi-factorial nature of most genetic disorders, there is
an increasing need for tools to enable analysis of hundreds
to thousands of SNPs across the entire genome. Determina-
tion of the clinical relevance of SNPs requires comparative
studies of the genetic differences between thousands of
*Correspondence to: L. M. Smith, Department of Chemistry,
University of Wisconsin, Madison, WI 53706-1396, USA.
Contract/grant sponsor: US Department of Energy; Contract/grant
number: DE-FG02-91ER.
†
Present address: Pharmacia, Structard, Analytical and Medicinal
Chemistry, MS 7255-209-017, 301 Henrietta Street, Kalamazoo, MI
49007, USA
Contract/grant sponsor: The National Science Foundation; Contract/
grant number: 952068.
affected and unaffected individuals.5 The analysis of such
variations is also of great interest to the pharmaceutical
industry, which is contemplating the design of drugs
customized for individual patient-specific responses.6 Con-
ventional gel-electrophoresis-based methods for analysis of
SNPs are too slow, labor intensive and inaccurate for these
applications.7 High throughput, accurate and more cost
effective methods are sought to bridge this gap.
A number of new methodologies are under development
to address this issue. One strategy is based on hybridization
of a labeled DNA target to allele-specific oligonucleotides
(ASO) arrays immobilized on solid surfaces (DNA chip).8,9
This technique requires substantial optimization of hybri-
dization conditions. Careful design and 8-fold duplication of
each array element and a statistical interpretation of the
complex hybridization patterns caused by cross-hybridiza-
tion are required. The advantages of this approach include
(1) parallel analysis of many sequence variations simulta-
neously; (2) easy solid-phase sample handling; and (3) high
sensitivity of fluorescence detection. Though generally
powerful, the DNA hybridization-based methods suffer
from the low discriminating power for hybridization
between sequences differing by only a single base.10 The
chip-based differential hybridization techniques rely on the
differences in thermal stability, as reflected in the melting
temperature (Tm) of the DNA duplexes, between a perfectly
matched and a single-base mismatched hybrid of the target
DNA with a pair of ASO probes. The Tm of DNA hybrids is

Copyright # 2000 John Wiley & Sons, Ltd.

 SNPS BY PRIMER EXTENSION AND MALDI-TOF
Scheme 1. The model system employed for the single nucleotide primer extension assay.
sequence dependent, and the difference due to a single base
change can be as little as 0.5 °C.11–13
New strategies using mass spectrometry (MS) have
emerged as a powerful alternative to gel electrophoresis
for the analysis of nucleic acids.14 Two ‘soft’ techniques
suitable for ionizing large biomolecules were developed in
the late 1980s,15 matrix-assisted laser desorption/ionization
(MALDI),16 and electrospray ionization (ESI).17 MALDI is
better suited for mixture analysis than ESI because it
produces predominantly singly charged ions.18 A typical
MALDI-TOF mass spectrometric analysis takes only a few
seconds, which is thousands of times faster than gel
electrophoresis. Mass spectrometry measures the m/z ratio,
an intrinsic parameter of the analyte, rather than electro-
phoretic mobility, an extrinsic parameter. The absolute
molecular mass and fragmentation patterns accessible in a
mass spectrum provide precise molecular and structural
information about the analytes in question, and no
fluorescent or radioactive labeling is needed.
Despite marked improvements over the last decade in
instrumental sensitivity and mass resolution of DNA, the
practical use of MALDI-TOFMS in nucleic acids analysis is
still limited in mass range and resolution. The longest DNA
sequenced by MALDI-TOFMS is about 100 nucleotides (nt)
in length.19 Recently Hillenkamp and co-workers reported
the successful detection of a 2180-nt restriction enzyme
fragment of plasmid DNA by IR MALDI-TOFMS.20
However, the mass resolution obtained for the 1280-nt
fragment was only 30, and the mass accuracy was about 1%,
which is far from adequate for DNA sequencing. The
negatively charged backbone and labile glycosidic linkage
of nucleic acids are believed to contribute to the poor mass
resolution and low sensitivity of detection for long DNA
fragments. The negatively charged phosphate backbone
tends to form adducts21,22 with alkali ions, and the
adduction causes the molecular ion peak to spread out or
disappear. The fragmentation23–25 of larger molecular ions
leads to decreased signal intensity and peak broadening.
MALDI-TOFMS has been applied to mutation detection
and polymorphism characterization in many different
formats.14 Direct analysis of polymerase chain reaction
(PCR) products by MS is one of the earliest methods
Copyright # 2000 John Wiley & Sons, Ltd.
951
developed.26,27 Since the mass difference between the
natural DNA bases is very small, the mass resolution of
typical time-of flight mass analyzers cannot detect single
base changes such as T to A (9 Da mass difference) in
typically sized PCR amplicons (>100 bp). Specially
designed PCR primers with restriction enzyme recognition
sites are needed to shorten DNA fragments to a length
suitable for mass spectrometric analysis.27–29 An approach
based on an invasive cleavage assay,30 which does not
require a PCR step, has been developed recently.31
Another strategy takes advantage of the high specificity
of DNA polymerase catalyzed primer extension. An
extension primer is annealed to template at the 5' position
immediately adjacent to the polymorphic site. A single
dideoxynucleotide complementary to the polymorphic
nucleotide is added to the 3' terminus of the primer by
polymerase extension. The prototype of this assay based on
gel electrophoresis is called single nucleotide primer
extension (SNuPE)32 or minisequencing.10 Due to the high
specificity and flexible assay conditions, it has been
employed in a number of different assay formats, such as
solid-phase minisequencing,33 genetic bit analysis (GBA),34
arrayed primer extension (APEX),35 and template-directed
dye-terminator incorporation (TDI).36 The PinPoint assay37
is a mass spectrometric version of SNuPE, in which the
extended primers carrying precise information about the
nature of the polymorphic nucleotide are characterized by
MALDI-TOFMS. Up to 12-fold multiplex SNP analysis
with PinPoint assay has been reported.38 However, further
increases in this multiplex capability is limited by the mass
resolution required for resolving A/T (9 Da) heterozygous
extension products beyond 30-mers (9000 Da).
Previously, we demonstrated that using a mass-tagged
ddNTP in place of an unmodified ddNTP in SNuPE
substantially improved the accuracy of analysis for mock
heterozygous samples in a synthetic oligonucleotide model
system (Scheme 1).39 Here we report more detailed
experimental results utilizing this mass-tagging strategy
for application in human genomic targets. Ten SNPs in PCR
products amplified from plasmid and human genomic DNA
in human tyrosinase gene exon IV and human STSs have
been typed successfully by this approach. It is shown that all
Rapid Commun. Mass Spectrom. 14, 950–959 (2000)
952 SNPS BY PRIMER EXTENSION AND MALDI-TOF
Table 1. Sequences of oligonucleotide primers used for single
) and their concentrations were
nucleotide primer extension reactions
Names Sequence
HTGE406 5' TAAACTTCTTGAAGA 3'
HTGE419 5' GCCAATGCACCCATT 3'
HTGE419-b 5'-biotin- GCCAATGCACCCATT3'
HTGE422 5' ACCATGTAGGATTCC 3'
HTGE446 5' TCATCCAAAGATCTG 3'
HTGE446-b 5'-biotin- TCATCCAAAGATCTG 3'
HTGE448 5' TGTAGATAGCTATAG 3'
STSWI-867b 5' AACGACGGCCAGTAA 3'
STSWI-681 5' GGTGCCATAGTATAA 3'
STSWI-921 5' TGTCAGATCTCTCCC 3'
STSWI-1325 5' GGAAGTTCTGAGGGT 3'
STSWI-1803 5' ATGCTCTACCCTACT 3'
STSWI-2032 5' AGCAATTACAGGAAG
STSWIAT-2057 5' ACTGGCTCCTGAATT 3'
a
The masses of the protonated, singly charged molecular ions were calculated
from the most stable isotopes.
b
The primer for 44-mer synthetic templates containing partial sequence of WI-
867 flanking a SNP.
six possible biallelic SNP heterozygotes may be determined
unambiguously with a large mass separation. The assay can
detect as little as 0.2 fmol of a rare allele target in the
presence of a 104-fold excess of wild-type allele. The
experimental results indicate that our approach is simple,
accurate, and robust, and has excellent potential for the
high-volume SNP analysis of PCR-amplified DNAs.
EXPERIMENTAL
Materials
Reagents. Fluorescein-12ddNTPs, FAM-ddNTPs, lissa-
mine-5-ddGTP, coumarin-5-ddUTP and tetramethylrhoda-
mine-6-ddUTP were purchased from NEN Life Science
Products (Boston, MA, USA). ddNTPs and dNTPs were
obtained from Pharmacia Biotechnology (Piscataway, NJ,
USA). AmpliTaq DNA polymerase-FS and dye terminators
were from a dye terminator DNA cycle sequencing kit (PE
Applied Biosystems, Foster City, CA, USA). Thermo-
Sequenase was from Amersham Life Science (Arlington
Heights, IL, USA). Exoÿ pfu DNA polymerase was from
Stratagene (La Jolla, CA, USA). Float dialysis membrane
filters VSWP 047 00 with 0.025 mm pore size were from
Millipore (Bedford, MA, USA). Streptavidin-coated mag-
netic beads Dynabeads M-280 were from Dynal (Oslo,
Norway) 3-Hydroxypicolinic acid (3HPA) was from
Aldrich Chemical Co. (Milwaukee, WI, USA). A mixture
of five individual human genomic DNAs (#G304A) was
obtained from Promega (Madison, WI, USA).
Oligonucleotides. Synthetic oligonucleotide templates and
primers (Table 1) were purchased from Integrated DNA
Technologies (Coralville, IA, USA) unless otherwise noted.
All oligonucleotides were purified by reversed-phase HPLC
with a 5 mm  300 mm C18 column (Rainin Instrument Co.,
Inc., Woburn, MA, USA) on a Shimadzu SCL-6A HPLC
system (Shimadzu Corp., Kyoto, Japan). The purified
oligonucleotides were dried down with a SpeedVac
Concentrator (Savant Instruments, Marietta, OH, USA).
The purity of oligonucleotides was checked with HPLC and
MALDI-TOFMS. Oligonucleotides were redissolved in
Rapid Commun. Mass Spectrom. 14, 950–959 (2000)
Milli-Q water (18 M
determined by UV absorption at 260 nm.
Massa (Da)
4573.83 PCR amplification of the human tyrosinase gene exon 4
4495.80
A short 182-nt fragment of human tyrosinase gene exon IV
4900.95
4550.81
was amplified from plasmid or genomic DNA as described
4534.81 previously.40 Biotinylated forward primers were used to
4939.96 allow solid-phase purification and single-stranded DNA
4629.83 preparation. The PCR reaction was performed in 50 mL
4593.80 reactions containing 1X reaction buffer (10X reaction buffer
4614.83 contained 200 mM Tris-HCl (pH 8.75), 100 mM KCl,
4477.78 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton X-100,
4687.83 and 1 mg/mL bovine serum albumin (BSA)), 0.2mM
4461.79 ddNTPs, 50 ng template DNA, 0.1–1 mM of primer and
4632.85
2.5 U exo pfu DNA. Thermal cycling conditions employed
4542.80
were: 3 min at 95 °C followed by 35 cycles of 30 s at 94 °C,
60 s at 55 °C and 120 s at 72 °C, followed by a 10 min
incubation at 72 °C.
PCR amplification of the human STSs
Human STSs were amplified by PCR in a 50 mL reaction
mixture with 0.1–0.5 mg human genomic DNA, 200 mM of
dNTPs, 0.4–1 mM each primer, 1 mM MgCl2, and 2.5 U
Amplitaq DNA polymerase, using the same cycling
conditions given above.
Purification of PCR amplicons
After PCR, the reaction mixture was diluted to 500 mL with
Milli-Q water, and the PCR product was purified and
concentrated by microcentrifugation with a Microcon-30
filter at 4 °C for 20 min at 14000 g-force (Amicon, Inc.,
Beverly, MA, USA). Alternatively, one strand of a PCR
product containing a 5'-biotin modification was purified by
solid-phase affinity capture with streptavidin-coated mag-
netic beads.41 Briefly, 2 mg (200 mL suspension) of beads
were washed with 200 mL 1X binding & washing (B&W)
buffer (5 mM Tris-HCl, 0.5 mM EDTA, 1.0 M NaCl, pH
7.5) and then resuspended in 100 mL 2X B&W buffer after
removing the supernatant. The washed beads were trans-
ferred to an Eppendorf microtube with 100 mL of PCR-
amplified product mixture and incubated for 15 min at 37 °C
with frequent shaking. The tube was placed in a Dynal
magnetic particle concentrator (MPC) to pull the beads with
the immobilized products to the side of tube and the
supernatant was removed with a micropipet. The beads were
washed twice with 50 mL 1  B&W buffer. 50 mL 0.1 M
NaOH freshly diluted from 10 M stock solution was added
to denature the DNA at room temperature for 10 min. The
beads were collected to the side of tube by applying an MPC
and the supernatant was transferred to a clean 1.5 mL
Eppendorf tube and neutralized with 0.1 M HCl. The beads
were washed with 1  B&W buffer three times to remove
the remaining NaOH.
Single nucleotide primer extension
For extension reactions from synthetic oligonucleotide
templates with Taq DNA polymerase, a typical primer
extension reaction mixture (20 mL) contained 2.0 mM
primer, 1.0 mM each template, 0.5–1 mM ddNTP, 10–
20.0 mM mass-tagged ddNTP, 4U of AmpliTaq DNA
polymerase FS, and 2 mL 10X sequencing buffer from a
dye terminator cycle sequencing core kit. The primer
Copyright # 2000 John Wiley & Sons, Ltd.
 SNPS BY PRIMER EXTENSION AND MALDI-TOF
extension reaction was carried out in a thermal cycler. The
thermal cycling conditions employed were 15 s at 96 °C,
60 s at 37 °C and 120 s at 60 °C for 1–25 cycles.
The following reaction conditions were used for the
Thermosequenase catalyzed primer extensions of PCR
amplicons, except as noted. The reaction mixture (20 mL)
contained 2 mM primer (Table 1), 5 mL purified PCR
amplicon, 10 mM mass-tagged ddNTPs, 0.5–1 mM ddNTPs,
25 mM NH4OAC (pH = 9.3), 2 mM Mg(OAC)2, and 4U
ThermoSequenase. The primer extension reactions were
performed using 25–50 cycles of 15 s at 94 °C, 30 s at 37 °C
and 60 s at 72 °C.
Sample preparation for MALDI-TOFMS
The purification of extension products with streptavidin-
coated magnetic beads is similar to the purification of PCR
products described above except for replacing the 1 M NaCl
with 1 M NH4OAc in B&W buffer. The extension products
were washed with 40 mL 1 M NH4OAC (pH = 7) once and
with 40 mL 0.1 M NH4OAc twice, and then the extended
primers were denatured from the beads at 90 °C in 2–5 mL
Milli-Q water. Alternatively, the extension mixture was
desalted by float dialysis. The extension reaction mixture
was placed on top of an MF (mixed cellulose esters)
membrane filter with 0.025 mm pore size (VSWP 047,
Millipore, Bedford, MA, USA) floated on 500 mL Milli-Q
water in a beaker. Dialysis was executed for several hours or
overnight to reach equilibrium. The purified samples were
dried down with a Savant SpeedVac concentrator and
redissolved in 2 mL of Milli-Q water. 0.5 mL of MALDI
matrix (saturated 3-HPA in a 1:1:2 mixture of water,
acetonitrile and 0.1 M diammonium citrate treated with
cation exchanger in ammonium form) was mixed with
0.5 mL purified extension products on a piece of Parafilm.
The mixture was spotted on a polished stainless steel
MALDI sample probe and air-dried.
MALDI-TOFMS
Mass spectra were taken by a Bruker Reflex II time-of-flight
mass spectrometer (Billerica, MA), equipped with a N2 laser
(337 nm) and operated in reflectron, positive ion mode at a
25 kV acceleration voltage and 2 ms delay time. The
instrument was initially calibrated with external standards.
The excess unextended oligonucleotide primers left or
added later can also be used as internal standards for
calibration if necessary.
RESULTS AND DISCUSSION
There are six possible heterozygous biallelic SNPs, that is
A/C, A/G, A/T, C/G, C/T, G/T. The A/G and C/T
transitional mutations are the most common, differing in
mass by 16 and 15 Da, respectively. The A/T heterozygote
is the most challenging for mass spectrometric analysis, as
the masses of these two nucleotides differ by only 9 Da. In
order to determine all six possible heterozygous SNPs, a
model system using mock heterozygotes made from
mixtures of equal concentration of 44-mer synthetic
oligonucleotide DNA templates was designed (Scheme 1).39
Design of synthetic oligonucleotide target DNA as a
model system
The 44-mer template, as shown in Scheme 1, contains a
Copyright # 2000 John Wiley & Sons, Ltd.
953
polymorphic site at the sixth nucleotide from the 5' end. An
8-nt sequence taken from human STS WI 86742 was
appended to the 5' end of a 36-nt sequence from M-13
bacteriophage containing a universal M-13 primer binding
site.43 The 5' end of the template was modified with either a
biotin (shown) or fluorescent label. The biotin group was
used for the solid-phase purification strategy discussed
above. The fluorescent label (HEX or TET) at the 5'
terminus was used to follow the primer extension reaction
by electrophoresis of the reaction products on an ABI 370A
DNA sequencer.44 A 15-mer extension primer complemen-
tary to the target strand was annealed immediately 3' to the
variable base N. The DNA polymerase incorporated a
dideoxynucleotide X complementary to N at the 3' end of
the primer. A short terminated fragment (16-mer) was
produced because the incorporated ddNTP lacks the 3'-OH
required for further extension. The type of incorporated base
can be determined by the mass increase of the extended
primer and, thus, the nature of base(s) at the polymorphic
site in the template is disclosed. The short extension
products are analyzed by MALDI-TOFMS in an m/z range
yielding high instrumental mass resolution and sensitivity.
The fluorescent dye terminators were chosen as mass-tags
not only because of the large masses of the dyes (Table 2),
but also because they are commercially available and
known to be compatible with polymerase extension. Both
AmpliTaq FS DNA polymerase and ThermoSequenase
incorporate such modified ddNTPs, and are therefore the
enzymes utilized in these experiments. The primer exten-
sion reactions may be performed for multiple cycles to
accumulate target specific signal molecules (extension
products) in a linear fashion with time (number of cycles).
For synthetic oligonucleotide templates, one cycle was
sufficient to produce enough signal molecules for analysis
by MALDI-TOFMS, due to the large amount of template
that may be employed. The extension products were
purified by solid-phase methods as described above.39
Typing the six types of heterozygotes
Each of the six possible heterozygous biallelic SNP
genotypes have been determined unambiguously by using
six mass-tagged ddNTP/ddNTP pairs (Fig. 1). A large
excess of mass-tagged ddNTP (20-fold excess relative to
unmodified ddNTP) was used to increase the peak intensity
of the corresponding extension product.
Ionization efficiency
We found that modified nucleoside triphosphates were
incorporated efficiently by both AmpliTaq FS and Thermo-
Sequenase DNA polymerases (data not shown). However,
the peak intensity obtained from an oligonucleotide which
incorporated a ddNTP was greater than that of one which
incorporated a mass-tagged ddNTP. Initially, we thought
this was caused by inefficient incorporation of mass-tagged
ddNTPs; however, careful examination of the extension
products by gel electrophoresis showed that the extension
primers were converted quantitatively to single-base
extended product in the model system. This suggested that
the mass-tag itself was directly reducing detection effi-
ciency. To confirm this, a pair of synthetic oligonucleotides
identical except for an extra 5' FAM modification on one
were synthesized and analyzed by MALDI-TOFMS. A 1:1
mixture of the unmodified and modified oligonucleotides
Rapid Commun. Mass Spectrom. 14, 950–959 (2000)
954 SNPS BY PRIMER EXTENSION AND MALDI-TOF

Table 2. Selected mass-tags and dideoxynucleotide triphosphates

Modified ddNTPs Formula Exact Massa (Da) Formula Mass shiftb (Da)
ddATP C10H16N5O11P3 475.01 C10H12N5O4P 297.06
ddCTP C9H16N3O12P3 450.99 C9H12N3O5P 273.05
ddGTP C10H16N5O12P3 491.00 C10H12N5O5P 313.06
ddTTP C10H17N2O13P3 466.17 C10H13N2O6P 288.05
ABI A dye C42H43N7O15P3 978.20 C42H39N7O8P 800.26
ABI C dye C45H43N6O16P3 1016.19 C45H39N6O9P 838.25
ABI G dye C36H31N7O16P3 910.10 C36H27N7O9P 732.16
ABI T dye C37H35N5O17P3 914.12 C37H31N5O10P 736.18
Fluorescein-12-ddATP C41H41N6O18P3 998.17 C41H37N6O11P 820.23
Fluorescein-12-ddCTP C39H40N5O19P3 975.15 C39H36N5O12P 797.21
Fluorescein-12-ddGTP C41H41N6O19P3 1014.16 C41H37N6O12P 837.22
Fluorescein-12-ddUTP C39H39N4O20P3 976.14 C39H35N4O13P 798.19
Lissamine-5-ddGTP C41H49N7O18P3S2 1084.19 C41H45N7O11PS2 906.24
Coumarine-5-ddUTP C23H24N3O16P3 691.38 C23H20N3O9P 513.09
Tetramethylrhodamine-6- C37H40N6O16P3S 948.29 C37H35N6O9PS 770.19
ddUTP
FAM-ddATP C35H30N5O17P3 885.08 C35H26N5O10P 707.14
FAM-ddCTP C33H29N4O18P3 862.09 C33H25N4O11P 682.17
FAM-ddGTP C35H30N5O18P3 901.08 C35H26N5O11P 723.16
FAM-ddUTP C33H28N3O19P3 863.05 C33H24N3O12P 685.11
a
Exact mass was calculated from the most stable isotopes.
b
Mass shift is mass increase of the extension primer from incorporating a modified ddNMP residue in a single nucleotide primer extension reaction.

yielded two peaks with a relative intensity of 5:1,
confirming the hypothesis. A five-fold excess of the
modified oligonucleotide produced approximately equal
peak intensities for both compounds. The reason for this
difference in MALDI detection efficiency is not known. It is
readily compensated for, however, by simply employing an
excess of the mass-tagged ddNTP relative to unmodified
ddNTP as was done in the present studies.

Genotyping PCR amplicons Human tyrosinase gene

exon 4
SNuPE primer design. Tyrosinase is an enzyme that controls
the first two steps in the melanin biosynthetic pathway. The
human tyrosinase gene is encoded by five exons located on
the long arm of human chromosome 11, spanning a region
over 50 kb. More than 50 pathologic mutations have been
identified among patients with type I oculocutaneous
albinism.45,46 Five of the single-base mutations located in
the 182-bp exon IV of human tyrosinase gene have been
studied previously.40 Primers HTGE406, HTGE422 and
HTGE448 are used for analysis of the three SNPs located in
the sense strand and primers HTGE419-b and HTGE446-b
are positioned in the antisense strand. The sequence and
calculated masses (in singly charged, protonated form,
[M ‡ H]‡) of the primers are summarized in Table 1.

Analysis of the five single-base mutations on exon IV. Exon

Figure 1. Analysis of all six biallelic ‘heterozygotes’ from an
equimolar mixture of two synthetic oligonucleotide templates. Each
reaction mixture (20 mL) contained 1 mM each of the corresponding
template, 2 mM Primer WI-867, Taq FS DNA polymerase. (a) C/T,
1 mM ddGTP and 20 mM fluorescein-12ddATP; (b) C/G, 1 mM ddGTP
and 20 mM fluorescein-12ddCTP; (c) A/T, 1 mM ddTTP and 20 mM
fluorescein-12ddATP; (d) A/G, 1 mM ddTTP and 20 mM fluorescein-
12ddCTP; (e) A/C, 1 mM ddGTP and 20 mM fluorescein-12ddUTP;
and (f) G/T, 1 mM ddCTP and 20 mM fluorescein-12ddATP; 2 mM
Primer WI-867 was added to the mixture employed for Fig. 1(f) after
the primer extension reaction to provide an internal mass reference for
the extension products. All peaks are labeled according to the
genotypes of SNPs found in target DNAs.
IV was amplified by PCR with a forward primer, 5'-biotin-
TATTTTTGAGCAGTGGCTCC-3', and a rear primer, 5'-
CTGAATCTTGTAGATAGCTA-3'. The PCR products
were analyzed by agarose gel electrophoresis. The PCR
amplicons were purified with streptavidin-coated magnetic
beads and the strands were separated by alkali denaturation.
The biotinylated strand bound to the beads was annealed
with primer HTGE406, HTGE422 and HTGE448 separately
and extended with ThermoSequenase and ddNTPs. The 5'-
biotin modification on templates was used to purify the
extension products by biotin-streptavidin affinity capture as
discussed above (Scheme 2). The extension products
captured on beads were released by heating the beads in
pure water. Since the unextended primer can compete with

Rapid Commun. Mass Spectrom. 14, 950–959 (2000) Copyright # 2000 John Wiley & Sons, Ltd.
 SNPS BY PRIMER EXTENSION AND MALDI-TOF
Scheme 2. Solid-phase purification of single nucleotide primer extension products.
the extended primer for binding to beads, multiple extension
cycles and excess beads were used. For the non-biotinylated
strand, 5'-biotinylated primer 419 and primer 446 were used
to allow solid-phase purification.
Representative results from a plasmid DNA sample and a
genomic DNA sample are shown in Fig. 2. All the results
Figure 2. Mass spectra obtained from genotyping single-base
substitutions in exon IV of the human tyrosinase gene. The purified
PCR product (5 mL) from exon IV was extended using Thermo-
Sequenase (4 U) with one of the following primers (2 mM) for 35
cycles. (a) Primer HTGE406, a plasmid pcTyr406L, ddGTP (1 mM),
and fluorescein-12-ddATP (10 mM); (b) Primer HTGE446-b, genomic
DNA from a single individual, ddGTP (0.5 mM), and fluorescein-12-
ddATP (10 mM).
Copyright # 2000 John Wiley & Sons, Ltd.
955
obtained by this approach are 100% concordant with
previous determinations by DNA sequencing. Figure 2(a)
shows a single peak obtained from plasmid pcTyr406L (the
last letter L is the single-letter amino acid code for leucine,
the amino acid substitution at that position; 406 refers to
codon 406 of tyrosinase gene exon IV). Figure 2(b) depicts
results obtained from an analysis of codon 446. The
template for SNPuE at codon 446 was PCR amplified from
a single individual. Since the priming site for the SNP at
codon 446 is in the antisense strand, a ddG instead of ddC
was incorporated. In all cases, the masses of the peaks that
represent the extension products are in excellent agreement
with predicted m/z values. Figure 2(b) illustrates the large
mass separation created by using the mass-tag strategy. The
large mass difference greatly facilitates heterozygote
typing, providing an avenue for circumvention of the
limitation of small mass differences between natural bases.
Typing of human STSs
SNPs in human sequence-tagged sites (STSs)47 were also
analyzed. Figure 3 shows representative results from three
SNPs known to be heterozygous.31 The mass spectra show
two peaks corresponding to the alternative alleles of a SNP
at three STSs.
Ten single nucleotide variations were analyzed in known
plasmid and human genomic DNAs using these methods,
and the results are shown in Table 3 (representative mass
spectra are shown in Figs 2 and 3). Five of these were
selected from exon IV of the human tyrosinase gene and the
others were located in sequence-tagged sites (STSs) of the
human genome. In all but one case, the genotyping results
obtained by this approach produced extension products with
the expected masses. For the single-base mutation shown in
Table 3 as HTGE422, a genomic DNA target was extended
with tetramethylrhodamine-6-ddUTP. The mass spectrum
shows a peak which has a mass lower than expected (mass
observed—5160 u; mass expected—5321 u). The reason for
this discrepancy is presently unknown–a likely possibility is
that this particular dye is unstable to the MALDI conditions
and undergoes fragmentation leading to the observed mass
difference. The discrepancy between the expected and
Rapid Commun. Mass Spectrom. 14, 950–959 (2000)
956 SNPS BY PRIMER EXTENSION AND MALDI-TOF

Figure 3. Analysis of heterozygous human SNPs. PCR product (1 mL) amplified from an individual genomic DNA was
extended with one of the following primers (2 mM), and Thermosequenase DNA polymerase (4 U). (a) Primer STSWI-
921, ddCTP (0.5 mM), and fluorescein-12-ddUTP (10 mM); (b–c) Primer STSWI-2032 or Primer STSWIAT-2057,
ddGTP (0.5 mM), and fluorescein-12-ddCTP (10 mM). The extension products were purified by float dialysis with MF-
Millipore membrane filters. P stands for unextended primers; A, C, G and T indicate the genotypes of human genomic
DNA; peaks labeled with * in Figs 3(b)-(c) were generated from depurination of G.

observed mass is of little practical consequence as long as
the product is consistently observed and distinguishable
from the unextended primer or dye-tagged extension
products. Minor depurination peaks (labeled with * in Fig.
3) are also evident in the mass spectra.
Sample preparation for MALDI-TOFMS
Sample preparation is an important step in analysis by
MALDI-TOFMS. Prior to analysis, PCR and/or other
enzymatic procedures are usually required to amplify target
or signal molecules. The presence of contaminants such as
salts, detergents, and glycerol in corresponding biological
buffers can severely interfere with MALDI sample
preparation.48
We examined two different sample purification methods;
biotin-avidin affinity capture and float dialysis by using
PCR products amplified from human tyrosinase gene exon
IV. Solid-phase purification methods using streptavidin-
coated magnetic beads for MALDI-TOFMS DNA sequen-
cing have been reported previously.49 The immobilization
of DNA to a solid support greatly facilitates the removal of
contaminants by washing. In some cases, a 5'-biotin group
was introduced in synthetic templates during oligonucleo-
tide synthesis (Scheme 1, synthetic targets) or incorporated
into PCR amplicons during PCR amplification by using a 5'-
biotinylated primer (Scheme 2, PCR-generated targets).
Binding these biotinylated DNAs to streptavidin-coated
beads allows the primer extension products to be captured
on the beads by DNA hybridization. After washing with the
‘no salt’ (substituted with volatile ammonium acetate)
B&W buffer, the purified extension products may be eluted
by thermal denaturation in water. A small volume (1–2 mL)
of water was used for elution to maintain a high sample
concentration suitable for MALDI-TOF analysis.
Figure 4(a) shows the mass spectrum obtained from an
unpurified SNuPE reaction mixture. A number of small
peaks observed between 4800 and 4900 Da represent a

Rapid Commun. Mass Spectrom. 14, 950–959 (2000) Copyright # 2000 John Wiley & Sons, Ltd.
 SNPS BY PRIMER EXTENSION AND MALDI-TOF 957

Table 3. Calculated masses of extension products

DNA samplea Sequence (5'-3@) Chromosome Genotyping results
pcTyr406L GTC[C/T]TCT 11 T
G419wt GTC[T/C]AAT 11 C
pcTyr422mt ACC[A/G]GGA 11 G
Genomic mixed ACC[A/G]GGA 11 A/G
G446h AGC[T/C]CAG 11 C/T
G448mt TAT[A/G]ACT 11 A
Genomic (mixed) ATT[A/G]TTA 2 A/G
Genomic (individual) TCT[A/G]GGG 11 A/G
Genomic (individual) TCT[C/T]ACC 20 C/T
Genomic (individual) GAT[A/G]AGT 4 A/G
Genomic (individual) TCA[G/C]CTT 9 G/C
Genomic (individual) ATT[G/C]AAT 22 G/C
a
pc stands for plasmid DNA and G stands for genomic DNA.

series of adducts formed with Na‡ and K‡. Substantial
sample purification is necessary to remove these adducts
and thus obtain high quality mass spectra. Figure 4(b) shows
the effect of solid-phase purification. The spectrum is
greatly simplified and easy to interpret after solid-phase
purification. The two singly charged, protonated molecular
ion peaks represent two alternative alleles at the 422 locus.
The different peak intensities observed for the two alleles is
because the template DNA employed is a mixture from five
individuals, and the ratio of the two peak intensities may
reflect the actual allele frequency in this particular
population. However, a disadvantage of this particular
purification approach is that it does not permit efficient
recovery of the biotinylated extension products—since
multiple extension cycles are employed to give a high yield
of extension products (ideally, n thermal cycles will produce

Figure 4. Purification methods were evaluated using the human tyrosinase exon IV system. The purified PCR
products (5 mL) amplified from a mixture of five individuals’ DNAs were extended with 2 mM primer HTGE422,
10 mM ddNTP, and 4 U ThermoSequenase for 30 cycles. (a) Before dialysis; b) after solid-phase purification and (c)
after dialysis; the full scale of this plot is 8-fold greater than that in panels a and b.

Copyright # 2000 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 14, 950–959 (2000)
958 SNPS BY PRIMER EXTENSION AND MALDI-TOF
Figure 5. Detecting rare-type mutations in the presence of a large
excess of wild-type DNA. Primer extensions (20 mL) were performed
using 2 mM template A, 2 mM primer WI-687, 10 mM fluorescein-12-
ddATP, 0.5 ml Taq FS DNA polymerase and Template T at (a) 0.2 nM;
(b) 2 nM and (c) 20 nM. The templates used here are 44-mer synthetic
oligonucleotides, which are the same as those used in Fig. 1.
n copies of extension product per copy of biotinylated
template), there are excess extension products compared
with the biotinylated template, and only a small fraction of
these can be captured on the beads. Thus, the majority of the
extension products in solution were lost during this solid-
phase purification process.
To address this issue we investigated float dialysis as a
simple alternative for sample purification.50 Low molecular
weight contaminants such as sodium ions in the reaction
mixture are removed through diffusion across a MF-
Millipore membrane floated on deionized water. No special
chemical modification or special reagents are needed except
ammonium acetate is added before dialysis to displace Na‡
and K‡. It is particularly convenient for purification of
SNuPE products from dsDNA templates. This is an
advantage since using dsDNA as template is simpler and
more straightforward than using ssDNA as template, as it
obviates the need for strand separation and purification
procedures. The dialysis takes half an hour to several hours
to complete. A fully automated, 96-well microtiter plate
format can be envisioned since the sample handling
involves only transferring samples to and from the dialysis
membrane. Figure 4(c) is the mass spectrum obtained after
float dialysis treatment of the unpurified extension products
for 2 h. The peak intensity of adducts was greatly reduced
compared with the unpurified sample analyzed in Fig. 4(a),
and the peak intensity of the protonated molecular ion is
roughly ten times more than that obtained by the solid-phase
method (note the compressed scale of Fig. 4c compared with
4(a) and 4(b) - figure legend).
Detecting mutations in the presence of a large excess of
wild-type DNA
One important analytical capability in mutation detection is
how well a rare mutant can be identified in a large excess
background of wild-type.51 The high accuracy and sensi-
tivity of SNuPE were demonstrated. Three concentrations of
template T (0.2, 2 and 20 fmol) in the presence of 2 pmol of
template A were used in a 10 mL SNuPE reaction. We were
Rapid Commun. Mass Spectrom. 14, 950–959 (2000)
able to detect 0.2 fmol synthetic target in 25 cycles (Fig. 5).
For analysis of samples such as human genomic DNA
containing only a small amount of DNA target, multiple
SNuPE cycles are needed to increase the assay sensitivity.
The large amount of template A (2 pmol) added here
simulates a large background excess of wild-type allele.
We can accurately detect as little as 0.2 fmol of the T
allele in the presence of a 104-fold excess of A allele (Fig.
5(a)). This is two or three orders of magnitude higher than
has previously been reported using a non-MS format.10
CONCLUSIONS
We have demonstrated here that the typing of heterozygotes
by single nucleotide primer extension-coupled MALDI-
TOF mass spectrometric analysis can be significantly
improved and simplified by use of modified dideoxynucleo-
tides as base-specific mass-tags. The high sensitivity and
specificity of the approach permits detection of as little as
0.2 fmol of a rare allele in the presence of a 104-fold excess
of an alternative allele. The versatility of using modified
nucleotides to manipulate mass separation was demon-
strated in the analysis of PCR-amplified DNAs containing
SNPs at STS sites or heterozygous single-base substitutional
mutations in the human DNA.
Acknowledgements
We acknowledge Tetsuyoshi Ono and Timothy J. Griffin for their
assistance with this work and valuable discussion. This work was
supported in part by Grant DE-FG02-91ER from the US Department of
Energy and Grant 952068 from the National Science Foundation.
REFERENCES
1. Collins FS, Patrinos A, Jordan E, et al. Science 1998; 282: 682.
2. Venter JC, Adams MD, Sutton GG, Kerlavage AR, Smith HO,
Hunkapiller M. Science 1998; 280: 1540.
3. Landegren U, Nilsson M, Kwok PY. Genome Research 1998; 8:
769.
4. Wang D, Fan JB, Siao C-J, et al. Science 1998; 280: 1077.
5. Schafer AJ, Hawkins JR. Nat. Biotechnol. 1998; 16: 33.
6. Bailey DS, Bondar A, Furness LM. Curr. Opin. Biotechnol. 1998;
9: 595.
7. Kirpekar F, Nordhoff E, Larsen LK, Kristiansen K, Roepstorff P,
Hillenkamp F. Nucleic Acids Res. 1998; 26: 2554.
8. Chee M, Yang R, Hubbell E, et al. Science 1996; 274: 610.
9. Hacia JG, Brody LC, Chee MS, Fodor SPA, Collins FS. Nature
Genet. 1996; 14: 441.
10. Syvanen A-C. Hum. Mutat. 1999; 13: 1.
11. Werntges H, Steger G, Riesner D, Fritz H-J. Nucleic Acids Res.
1986; 14: 3773.
12. Tibanyenda N, De Bruin SH, Haasnoot CAG, Van der Marel GA,
Van Boom JH, Hilbers CW. Eur. J. Biochem. 1984; 139: 19.
13. Guo Z, Liu Q, Smith LM. Nat. Biotechnol. 1997; 15: 331.
14. Crain PF, McCloskey JA. Curr. Opin. Biotechnol. 1998; 9: 25.
15. Gross J, Strupat K. Trac-Trend Anal. Chem. 1998; 17: 470.
16. Karas M, Bahr U, Hillenkamp F. Int. J. Mass Spectrom. Ion
Processes 1988; 92: 231.
17. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CW. Science
1989; 264: 64.
18. Nordhoff E, Kirpekar F, Roepstorff P. Mass Spectrom. Rev. 1996;
15: 67.
19. Monforte JA, Becker CH. Nat. Med. 1997; 3: 360.
20. Berkenamp S, Kirpekar F, Hillenkamp F. Science 1998; 281: 260.
21. Pieles U, Zurcher W, Schar M, Moser HE. Nucleic Acids Res.
1993; 21: 3191.
22. Nordhoff E, Ingedoh A, Cramer R, Overberg A, Stahl B, et al.
Rapid Commun. Mass Spectrom. 1992; 6: 771.
23. Wu KJ, Shaler TA, Becker CH. Anal. Chem. 1994; 66: 1637.
24. Nordhoff E, Kirpekar F, Karas M, Cramer R, Hahner S,
Hillenkamp F, Kristiansen K. Nucleic Acids Res. 1994; 22: 2460.
Copyright # 2000 John Wiley & Sons, Ltd.
 SNPS BY PRIMER EXTENSION AND MALDI-TOF
25. Zhu L, Parr GR, Fitzgerald MC, Nelson CM, Smith LM. J. Am.
Chem. Soc. 1995; 117: 6048.
26. Ch’ang LY, Tang K, Schell M, Ringelberg C, Matteson KJ,
Allman SL, et al. Rapid Commun. Mass Spectrom. 1995; 9: 772.
27. Liu YH, Bai J, Zhu Y, Liang X, Siemieniak D, Venta PJ, et al.
Rapid Commun. Mass Spectrom. 1995; 9: 735.
28. Tsuneyosh T, Ishikawa K, Koga Y, Naito Y, Baba S, Terunuma H,
et al. Rapid Commun. Mass Spectrom. 1997; 11: 719.
29. Laken S, Jackson PE, Kinzler KW, Vogelstein B, Strickland PT,
Groopman JD, Friesen MD. Nat. Biotechnol. 1998; 16: 1352.
30. Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova
T, et al. Nat. Biotechnol. 1999; 17: 292.
31. Griffin TJ, Hall JG, Prudent JR, Smith LM. Proc. Natl. Acad. Sci.
USA 1999; 96: 6301.
32. Kuppuswami MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce
SL, Bajaj SP. Proc. Natl. Acad. Sci. USA 1991; 88: 1143.
33. Syvanen A-C, Ikonen E, Maninen T, Soderlund H, Aula P,
Peltonen L. Genomics 1992; 12: 590.
34. Nikiforov TT, Rendle RB, Goeler P, Rogers YH, Kotewicz ML,
Anderson S, Trainor GL, Knapp MR. Nucleic Acids Res. 1994; 22:
4167.
35. Shumaker JM, Metspalu A, Caskey CT. Hum. Mutat. 1996; 7: 346.
36. Chen X, Kwok PY. Nucleic Acids Res. 1997; 15: 347.
37. Haff LA, Smirnov IP. Genome Res. 1997; 7: 378.
Copyright # 2000 John Wiley & Sons, Ltd.
959
38. Ross P, Smirnov IP, Haff LA. Nat. Biotechnol. 1998; 16: 1347.
39. Fei Z, Ono T, Smith LM. Nucleic Acids Res. 1998; 26: 2827.
40. Guo Z, Guilfoyle A, Thiel AJ, Wang R, Smith LM. Nucleic Acids
Res. 1994; 22: 5456.
41. Hultman T, Stahl S, Hornes E, Uhlen M. Nucleic Acids Res. 1989;
17: 4937.
42. Kwok PY, Deng Q, Zakeri H, Taylor SL, Nickerson DA. Genomics
1996; 31: 123.
43. Chen D, Johnson AF, Severin JM, Rank DR, Smith LM, Guilfoyle
RA. Gene 1996; 172: 53.
44. Ono T, Scalf M, Smith LM. Nucleic Acids Res. 1997; 25: 4581.
45. Giebel LB, Strunk KM, Spritz RA. Genomics 1991; 9: 435.
46. Tripathi RK, Strunk KM, Giebel LB, Weleber R, Spritz RA. Am. J.
Med. Genet. 1992; 43: 865.
47. Hudson TJ, Stein LD, Gerety SS, Ma J, Castle AB, Silva J, et al.
Science 1995; 270: 1945.
48. Bleicher K, Bayer E. Biol. Mass Spectrom. 1994; 23: 320.
49. Chou CW, Bingham S, Williams P. Rapid Commun. Mass
Spectrom. 1996; 10: 1410.
50. Kallweit U, Bornsen KO, Kresbach GM, Widmer HM. Rapid
Commun. Mass Spectrom. 1996; 10: 845.
51. Gonzalgo ML, Bender CM, You EH, Glendening JM, Flores JF,
Walker JG, et al. Cancer Res. 1997; 57: 5336.
Rapid Commun. Mass Spectrom. 14, 950–959 (2000)