Genotyping of Single Nucleotide
Polymorphisms by Minisequencing
Using Tag Arrays
An alteration of one nucleotide in a DNA sequence
gives different phenotypic outcomes depending on its
genomic location. Genomic nucleotide substitutions
present in more than 1% of a population are denoted
single nucleotide polymorphisms (SNPs). SNPs in
protein-coding regions may alter the amino acid
sequence of a protein, introduce stop codons, or
produce new alternative splice sites in mRNA, thereby
affecting the structure and function of the protein.
SNPs located in the regulatory regions of a gene may
alter binding sites for transcription factors and subsequently
the expression level of the gene. The consequences
of SNPs located in noncoding regions of the
genome still remain largely unknown, but with the
increasing interest in the function of noncoding RNA
(Mattick and Gagen, 2001), their impact may soon
Following the completion of the draft sequence of
the human genome (Lander et al.
, 2001; Venter et al.
2001), as well as of an increasing number of other genomes,
the need for large-scale and high-throughput
methods has increased. A promising and today
widely used approach is the microarray format, which
allows highly multiplex analysis of DNA, RNA, and
proteins. This article describes the use of microarrays
of immobilised oligonucleotides for SNP genotyping.
Most SNP genotyping methods used today still
depend on amplification of the region to be interrogated
by the polymerase chain reaction (PCR) to
provide the required sensitivity and specificity. Even
though PCR was the technique that first enabled effective
SNP genotyping, it is now the major factor limiting
the high throughput of the methods due to the
difficulties of designing robust multiplex PCR (Shuber et al.
Significant advantages of performing assays in the
microarray format are the reduced genotyping costs
due to the simultaneous analysis of many SNPs in each
sample and the small reaction volumes employed.
Three major reaction principles are currently used for
SNP genotyping on microarrays; hybridisation with
allele-specific oligonucleotide probes, oligonucleotide
ligation, and DNA polymerase-assisted primer extension
(for a review on genotyping techniques, Syvanen,
2001). Due to their high specificity, the enzymeassisted
methods are gaining acceptance as the reaction
principle of choice for multiplex SNP detection.
In the minisequencing reaction, a DNA polymerase
is used to extend detection primers that anneal immediately
adjacent and upstream of the sites of the SNPs.
The primers are extended with differently labelled
nucleotide analogues that are complementary to the
nucleotides at the SNP sites. The method was initially
devised with microtiter plates as the solid-phase
support and has later been adapted to multiple assay
formats (Syvanen, 1999), including microarrays with
detection primers covalently attached to the microarray
(Kurg et al.
, 2000; Lindroos et al.
, 2001; Pastinen et al.
II. PRINCIPLE OF THE METHOD
A flexible alternative minisequencing system is
based on generic oligonucleotides ("cTags") immobilised
on the microarray instead of specific detection
primers. Cyclic minisequencing reactions with fluorescently
labelled dideoxynucleotides (ddNTPs) are
performed in solution using detection primers with 5'
tag sequences complementary to one of the cTags
included in the array. Each SNP is then interrogated by
hybridizing the extended detection primers to their
corresponding cTags with known locations in the array
and the genotypes are deduced (Fig. 1A). The concept
of using tagged PCR primers was first described
for analysis of gene expression in yeast by PCR
(Shoemaker et al.
, 1996) and was later been applied
to SNP genotyping by primer extension and capture
on fluorescent microparticles (Cai et al.
, 2000), highdensity
oligonucleotide arrays [Affymetrix, GenFlex
arrays (Fan et al.
, 2000)], and medium-density, custommade
oligonucleotide arrays in different formats
(Hirschhorn et al.
, 2000; Lindroos et al.
|FIGURE 1 Schematic illustration of the microarray result for an individual that is heterozygote (A/T) for
SNP 1 and homozygote (G/G) for SNP 2. The SNPs have been interrogated by hybridizing detection primers
with 5' tag sequences, extended with fluorescently labelled ddNTPs, to their complementary immobilised
cTags (A). The format is an "array of arrays" with identical subarrays, allowing 80 samples to be analysed
simultaneously for up to 200 SNPs on the same microscope slide (B).
The format presented here uses an "array of arrays"
formed by a silicon rubber grid (Pastinen et al.
giving separate reaction chambers, each covering a
subarray, that allow 80 samples to be analysed simultaneously
for up to 200 SNPs on the same microscope
slide (Fig. 1B). In contrast to the conventionally used
format for mRNA expression, where a large number of
genes are analysed in a relatively low number of
samples, the "array of arrays" format allows for a large
number of samples to be analysed simultaneously for
an intermediate number of SNPs. The main steps of the
assay are illustrated in Fig. 2.
III. MATERIALS AND
|FIGURE 2 Steps of the procedure for genotyping of SNPs by minisequencing using tag arrays.
The microarray slides are CodeLink-activated slides
(reference number 25-6700-01) from Amersham Biosciences.
The oligonucleotides are synthesized by Integrated
DNA Technologies, and the tag sequences are
obtained from Affymetrix. Elastosil (RT601) A (Cat.No.
60003804) and B (Cat.No. 60003815) are from Wacker-
Chemie GmbH. In the multiplex PCR AmpliTaq
DNA polymerase, 5 U/µl, and GeneAmp 10x PCR
gold buffer [100mM
Tris-HCl, pH 8.3, 500mM
, and 0.01% (w/v gelatin)] (Part.No.
N808-0245) from Applied Biosystems are used
together with 10mM
dNTPs (Cat.No. 10297-018) from
Invitrogen Life Technologies.
Exonuclease I (Ref.No. E70073Z), 10 U/µl, shrimp
alkaline phosphatase (Ref.No. E70072Z), 1U/µl, and
ThermoSequenase (Ref.No. E79000Y), 32U/µl, are
from Amersham Biosciences. The fluorescent dideoxynucleotides
used are Texas red-ddATP 85,000M-1
(Prod.No. NEL 411), TAMRA-ddCTP 91,000M-1
(Prod.No. NEL 473), R110-ddGTP 78,000M-1
No. NEL 495), and Cy5-ddUTP 250,000M-1
No. NEL 589), all from PerkinElmer Life Sciences.
Reagents of the highest purity grade from various
sources are used for preparation of buffers and other
A ProSys 5510A instrument from Cartesian Technologies
Inc., with four Stealth Micro Spotting Pins
(Cat.No. SMP3), from TeleChem International Inc., are
used for microarray printing. A Tetrad programmable
thermal controller from MJ Research is used for
thermo cycling. For scanning of arrays, a ScanArray
5000 instrument from PerkinElmer Life Sciences is
used. Additionally, a centrifuge, an incubation oven, a
heat block at 42°C, and optimally a multichannel
pipette and a pipetting robot are needed. For all instrumentation, other equivalent equipment could be used
A. SNP Selection
SNPs can be identified either experimentally or in
databases. Database searches may be aimed at genes
of interest, candidate chromosomal regions, or randomly
distributed SNPs with known allele frequencies
Be aware that many of the SNPs are not validated
and that the fraction of "real" SNPs in the databases is
still unknown. Validation may be done in a particular
population by analysing pooled DNA samples using
quantitative minisequencing in microtiter plates or
directly on the microarray as has been described
(Lindroos et al.
B. Primer Design
Design PCR primers flanking the SNPs of interest
using available software. Primer 3, http://www.
genome.wi.mit, edu/cgi-bin/primer/primer3_www.cgi, is freely available online or commercial
software such as OLIGO: http://www.oligo.net/
can be purchased. The sequence of each PCR product
should be "blasted" against the genome sequence
(http://www.ncbi.nlm.nih.gov/BLAST/) and give a
single hit only to the intended region.
Minisequencing primers anneal immediately adjacent
and upstream of the SNP position. Minisequencing
primers from both forward and reverse strands are
helpful as internal controls for the genotyping results.
Design the minisequencing primers to have a specific
length of 18-22bp and to have a common melting temperature of 55-60°C to ensure specificity in the
cyclic primer extension reaction. At the 5' end of the
primer add the tag sequences, complementary to
the cTags, that will be spotted onto the microarray. The
tags should be 20bp long, have similar melting temperature,
and not be complementary to either each
other or the human genome. The Affymetrix GeneChip
tag collection can be used as source for tag sequences.
The complementary tag sequences (cTags) should have
15T residues as a spacer located 3' of the specific
sequence and a final 3'-amino group to enable covalent
attachment of the cTags to the slides.
To avoid strong hairpin-loop structures, evaluate
the final minisequencing primer, including the tag
sequence, with primer design software that predicts
secondary structures (mfold: http://www.bioinfo.
rpi.edu/applications/mfold/old/dna/ or NetPrimer
http://www.premierbiosoft.com/netprimer/netprimer.html). Secondary structures that involve the
3' end of a primer may lead to misincorporation of
C. Microarray Preparation
- 2× print buffer: 300mM phosphate buffer, pH 8.5.
Store at room temperature up to 1 month.
- Oligonucleotides: Dissolve the cTags in 1× print
buffer and dilute with dH2O to a final concentration of
25µM. Store at -20°C but limit freeze-thaw cycles.
- 1M Tris-HCl, pH 9.0: Dissolve 121.1g Tris in
800ml H2O, adjust the pH to 9.0 with HCl, and adjust
to a total volume of 1 litre.
- Blocking solution: 50mM ethanolamine, 100mM Tris-HCl, pH 9.0, and 0.1% SDS. Prepare directly
before use and preheat to 50°C. To make 500ml, use
1.6g ethanolamine, 50ml 1M Tris-HCl, 5ml 10%
SDS, and add water to 500ml. Take care that
the ethanolamine is highly corrosive and should be
handled according to safety instructions.
- 20×SSC: 3M NaCl and 300mM sodium citrate,
pH 7.0. Dissolve 175.3 g NaCl and 88.2 g sodium citrate
in 800ml H2O. Adjust the pH to 7.0 with NaOH and
adjust the volume to 1 litre. Autoclave and store at
- 10% SDS: 10% (w/v) sodium dodecyl sulphate.
Dissolve 100g of SDS in 900ml H2O. Adjust the pH to
7.2 with HCl and adjust the volume to 1 litre. Store at
- Washing solution: 4xSSC and 0.1% SDS preheated
Prepare the arrays by contact printing the cTag
oligonucleotides on CodeLink-activated slides (previously
3DLink slides). This may be done using a ProSys
5510A instrument with SMP3 pins that delivers i nl of
the cTag solution to the slides as spots with a diameter
of 125-150µm and with a center-to-center distance
of, for example, 200µM
. For the possibility of using the
"array of arrays" format, print spots in a subarray
pattern of either a 384- or 96-well format (see Fig. 1).
Mark the position of some subarrays on the back side
of the slides using a diamond pen.
Postprocess the slides according to the instructions
of the manufacturer. The following protocol for
CodeLink-activated slides is given.
- Prepare a saturated NaCl chamber with 75% relative
humidity. In the bottom of a plastic container
with an airtight lid, add as much solid NaCl to
water as needed to form a 1-cm-deep slurry.
- After printing, keep the arrays in the NaCl chamber
between 4 and 72h.
- Deactivate the excess of amine-reactive groups by
immersing the slides for 15 min in the blocking solution
- Wash with dH2O, washing solution at 50°C for
15-60min, and dH2O subsequently.
- Spin dry the slides for 5 min at 900rpm. Store the
slides desiccated at 20°C until use.
A fluorescently labelled cTag may be included in the
array as a spotting control. For each batch of printed
slides it is useful to analyse a few subarrays by hybridisation
as quality control of the spots. After deactivation
of the slides, hybridize a 3'-fluorescently labelled
oligonucleotide designed to hybridize to all cTags (5'-
AAA AAA AAA ANN NNN NNN NN-3') to some
subarrays at 300nM
concentration in 6xSSC for
10min with subsequent washing and scanning as
D. Preparation of Silicon Rubber Grid
|FIGURE 3 The arrayed slide is covered with a silicon
to give separate reaction chambers and is
placed in a custom-made
heat -conductive aluminium rack.
A Plexiglas cover with a drilled
hole through which the
reaction chambers are accessed is tightly
screwed on top
the assembly, thus securing the silicon grid position
Miniaturized silicon rubber (polydimethyl siloxan)
reaction chambers are made using inverted microtiter
plates with V-shaped wells as mould (Fig. 3).
- Mix the two Elastosil RT 601 components in a
50-ml Falcon tube in a mass ratio of 9:1, i.e., 46.8 g of
A and 5.2 g of B for 30min (Elastosil RT625 may be
used instead of RT601, thus giving a slightly softer
- Pour the mixture onto an inverted 384-well
microtiter plate, leaving about 1-2 mm of the tip of the
wells uncovered. Allow to harden overnight at room
- Remove the silicon from the plate and use a
scalpel to cut the silicon rubber into pieces of the size
of microscope slides with the wells matching the
The silicon is reusable, wash it with water and allow
it to dry after each use.
E. Multiplex PCR
Primers for multiplex PCR should have as similar
melting temperature and G/C content as possible. Different
design programs, see earlier discussion, may be
used to minimize primer-primer interactions. Complementary
3' sequences in the primers can be avoided
by designing primers with the same 3' terminal
nucleotides. Another possibility is the introduction of
common tails on the 5' ends of all PCR primers and
subsequent amplification with one common primer for
all the fragments at an elevated temperature (Brownie et al.
, 1997). An example of a protocol with the common
tail approach that has been used for a 20-plex PCR
reaction in 384-well format in our laboratory is given.
Primers with common tails for multiplex PCR
should have 18-25bp of specific sequence, have Tm
60-65°C, and give fragments about 100-200bp long.
On the 5' end of both primers include a 26-bp-long
common tail with Tm
~80°C (5'-GCG TAC TAG CGT
ACC ACG TGT CGA CT-3'). In the PCR mixture use
the tailed primers at 5 to 20nM
depending on priming efficiency (trial and error), and
use the primer complementary to the tail at 1µM
Amplify the genomic DNA using 1 ng/µl DNA,
0.04 U/µl AmpliTaq
Gold DNA polymerase, and 200µM
of dNTPs in 4mM
Tris-HCl, pH 8.3,
KCl, and 0.001% (w/v) gelatin and the primers
as described earlier in a final volume of 10µl. Amplify
at 94°C for 5 min followed by four cycles of 94°C 60°C and 72°C for 1 min each. Then do 35 cycles of 94°C for
1 min and 74°C for 2 minutes and finally do an extension
at 72°C for 10min. The success of the reactions
may be verified on a 1% agarose gel for a subset of the
F. PCR Cleanup
- MgCl2 (50mM): To make a 1M stock solution,
dissolve 203.3 g MgCl3·6H2O in 800ml of H2O and
adjust the volume to 1 litre. Autoclave and store at
- Tris-HCl, pH 9.5: Dissolve 121.1 g Tris in 800ml H2O,
adjust the pH to 9.5 with HCl, and adjust to a total
volume of 1 litre.
- Enzymes: Keep the ExoI and sAP enzymes on ice
during handling and store at -20°C.
- For each sample, pool all multiplex PCR products.
- Prepare a master mix of the PCR cleanup reagents
- To 7 µl of the pool, add 3.5 µl of the cleanup mixture
to a total volume of 10.5 µl.
- Incubate at 37°C for 30-60min and inactivate the
enzymes by heating to 95°C for 15 min.
Alkaline phosphatase (sAP) inactivates the remaining
dNTPs and exonuclease I (ExoI) degrades the
single-stranded PCR primers, thus limiting extension
of them in the subsequent minisequencing reaction.
Include negative PCR controls at this step.
G. Cyclic Minisequencing
- Triton X-100 (0.5%, v/v): Store at room temperature
for up to 1 month.
- Tris-HCl, pH 9.5 (500mM): Dilute from 1M Tris-HCl, pH 9.5.
- ThermoSequenase: Keep on ice during handling, store
at -20°C. Dilute the enzyme to required concentration
directly prior to use.
- Fluorescent ddNTPs: Keep light-protected aliquots in
4°C and store stock solutions at -20°C.
- Prepare a master mix of the minisequencing reaction
reagents (Table III).
- After the cleanup step, add 4.5 µl of minisequencing
reaction mixture to give a total volume of 15 µl.
- Perform the minisequencing reaction using an
initial 3 min at 95°C followed by 33 to 99 cycles of
20 s at 95°C and 20 s of 55°C in a thermocycler.
Additionally, an internal reaction control should
be used. For that purpose, four synthesized singlestranded
oligonucleotide templates differing only in
one position mimicking the four possible alleles of
a SNP are useful. Add the control template to the
minisequencing reaction at a final concentration of
. A complementary-tagged minisequencing primer
should be included with the other minisequencing
primers and its cTag should be included in the array.
Because fluorophores are light sensitive, protect all
reaction mixtures containing fluorophores from light.
H. Capture by Hybridization
- 20×SSC: See Section IV, C.
- Hybridisation control: Oligonucleotide labelled with,
for example, TAMRA, that hybridises to a corresponding
cTag included in the array. Keep light-protected aliquots at 4°C and store stock solutions
- Position a silicon rubber grid over the arrayed
slide with the aid of the diamond pen markings (see
Fig. 3). Place the arrayed slides into the custom-made
aluminium reaction rack and tighten the Plexiglas
cover. Preheat the assembly on the side of a heat block
- To the products from the minisequencing step,
add 6.85µl 20xSSC and 0.15µl hybridisation control
oligonucleotide at 45nM concentration.
- Transfer 20µl of each sample to a separate reaction
chamber on the microscope slide. A multichannel
pipette is feasible for this step.
- Hybridize for 2.5-3h at 42°C in a humid environment
accomplished, for example, by placing a wet
tissue on the Plexiglas lid and cover it with plastic film
and aluminium foil.
- 4×SSC: Dilute from 20xSSC and store at room
- 2×SSC, 0.1% SDS: Store at room temperature. Before
use, preheat to 42°C.
- 0.2×SSC: Dilute from 20xSSC and store at room
- After hybridization, take the slides from the reaction
rack and rinse briefly with 4xSSCat 20-25°C.
- Wash the slides twice for 5min with 2xSSCand
0.1% SDS preheated to 42°C and twice for 1min
with 0.2xSSC at 20-25°C in 50-ml falcon tubes.
- Finally, spin dry the slides for 5 min at 900rpm and
store them protected from light.
If allowed by the scanner used, balance the signal
intensity from each laser channel so that no signals are
saturated and the signals from the four fluorophores
are equally strong. Balancing is easy if a reaction
control with signals from all four fluorophores is
included on the array, as described earlier. An example
of a scanning result is given in Fig. 4.
K. Data Analysis
|FIGURE 4 Scanning results for 45 SNPs in one subarray, i.e., one sample, after cyclic minisequencing with ddATP, ddGTP, and ddUTP labelled with Texas red, TAMRA, R110, and Cy5, respectively. Each cTag was spotted as horizontal duplicates, and both strands of all SNPs were analysed.
A quantification program such as QuantArray
handles the scanning images and quantitates the signals from each spot. Raw data are collected as an
Excel sheet. From the signals from each channel, subtract
the background measured either around the spots
or at negative control spots, i.e., spotted cTags without
corresponding tagged primers. Assign the genotypes
of the SNPs in each sample by calculating the ratios
between the signals from one of the alleles and the sum
of the signals from both the alleles: signalAllele 1
+ signalAllele 2
)" A scatter plot with this
ratio on the X
axis and the sum of the signals from
both alleles on the Y
axis is used for assigning the
genotypes (Fig. 5). This scatter plot should give
three distinct clusters with the homozygote samples
clustering at each side and the heterozygotes in
the middle. The ratios may vary between SNPs
depending on the sequence surrounding it, the type of
nucleotide incorporated, and the light intensity of the
|FIGURE 5 Scatter plot for one SNP with a C/T variation in 56 samples. The logarithm of the sum of the fluorescent signal from both alleles. C + T, on the Y axis has been plotted against the ratio, C/(C + T), on the X axix. The three distinct clusters represent the three different genotypes, where two negative controls and three failed samples fall outside the clusters.
- Hairpin and loop secondary structures in the
primers can give rise to false signals. Some primers
may occasionally fail to give signals, probably due to
secondary structures either in the primer itself or in the
- Cy5-ddUTP can be used in higher concentrations
than the other ddNTPs to compensate for its lower
- Insufficient amounts of the PCR products resulting
in low signals may be compensated for by increasing
the amount of cycles in the minisequencing step.
- All solutions containing fluorophores are light
sensitive and so are the slides after hybridization. To
avoid bleaching, cover all reaction tubes and slides
with aluminium foil.
- Background problems can arise if the hybridisation
chamber is not kept humid and the sample dries
out on the slide.
- If the Plexiglas cover is not tightened enough or
if the silicon-rubber grid is damaged, leakage between
reaction wells may occur. This can be controlled for by
using differentially labelled hybridisation controls in
adjacent reaction wells.
Depending on the available laboratory facilities or
specific requirements of a project, this technique may
be altered. Instead of multiplex PCR, single fragment
PCR can be used with subsequent pooling of the
amplified fragments, possibly after concentration
using ethanol precipitation or spin dialysis. Different
slides and attachment chemistries for the oligonucleotides
have been tested, and new ones are continuously
being developed (Lindroos et al.
Depending on the number of SNPs to be interrogated,
an inverted 96-well microtiter plate may be used as
well for as silicon rubber mould to allow larger subarrays.
When using the QuantArray program for signal
analysing, the genotyping results can be visualised
using the SNPSnapper software that has been custom
made for this method (http://www.bioinfo.helsinki.
fi/snpsnapper). Instead of using four differently
labelled nucleotides in the same reaction, depending
on the available microarray scanner, a single label or
two labels may be used in four or two separate reactions,
respectively (Liljedahl et al.
, 2003). It has been
shown that the method described is quantitative and
well suited to determine allele frequencies of SNPs in
pooled DNA samples and is therefore a useful tool for
rapid SNP validation (Lindroos et al.
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