Application Note
SNP Genotype Detection With HEFP™
Table of Contents
Page
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Equipment and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Terminator Base Extension Using Synthetic Templates . . . . . . . . . . . . . . . . . 5
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Experimental Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Instrument Parameter Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Sample Results and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Four-Dye Genotyping Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Overview of the Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Primer and dNTP Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
SNP Genotyping Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Sample Results and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Two-Dye Genotyping Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Appendix A: Dimensions of PCR Microplates . . . . . . . . . . . . . . . . . . . . . . . . 17
Appendix B: Frequently Asked Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
SNP Genotype Detection With HEFP™
2
Overview
Single nucleotide polymorphisms (SNPs) are the most frequent and stable
sequence variations when comparing two chromosomes. With the
availability of a confirmed SNP map in human and other organisms in the
very near future, high throughput SNP genotyping is becoming the method
of choice for novel gene discovery, drug target identification, and
pharmacogenomic studies. Researchers can now quickly and accurately call
Single Nucleotide Polymorphisms (SNPs) by combining commercially
available single base extension technology with Molecular Devices' proven
HEFP™ detection technology and convenient AlleleCaller™ data analysis
software. In summary, genomic DNA containing the SNP of interest is PCR
amplified. Fluorescently labeled ddNTPs are then added with a primer
upstream of the SNP site in the terminator base extension reaction.
Identification of the specific fluorescent ddNTP incorporated at the
polymorphic site is determined in a fluorescence polarization assay.
Covalent linkage of the fluorophore onto the primer increases the molecular
weight of ddNTP labels by at least 10 fold resulting in increased
polarization of fluorescence. Detection by fluorescence polarization is made
possible by the high efficiency detection provided in the Molecular Devices
Criterion™ platform of high throughput screening instruments.
This application note describes a detailed protocol that optimizes the SNP
genotyping assay for use with HEFP. It is a modification of the method
described by Chen et al. Two systems are presented; one system uses four
labeled dyes and the other uses two labeled dyes. The choice of which
system to use will depend on your specific requirements.
Equipment and Materials
Recommended
Equipment and
Materials
Table 1. The following materials were used in the SNP assay:
Item
Source
Criterion™ System with Release
2.0 Software—
one of the following:
• Analyst™ AD or HT
• Acquest™
• ScreenStation™
Please contact your local Molecular
Devices Corporation representative
for the instrument best suited to
meet your needs.
The following ddNTP labeled dyes
at 100 µM for the four-dye assay:
• R6G-ddUTP
• ROX-ddGTP
• TAMRA-ddCTP
• BODIPY FL-14-ddATP
New England Nuclear P/N NEL-488
New England Nuclear P/N NEL-479
New England Nuclear P/N NEL-473
New England Nuclear P/N NEL-574
SNP Genotype Detection With HEFP™
3
Table 1. The following materials were used in the SNP assay: (Continued)
Item
Source
The following ddNTP labeled dyes
at 100 µM for the two-dye assay:
• TAMRA-ddCTP
• TAMRA-ddATP
• TAMRA-ddGTP
• R110-ddUTP
• R110-ddCTP
• R110-ddATP
The following unlabeled ddNTPs
for the two-dye assay:
• ddNTP Kit
New England Nuclear P/N NEL-473
New England Nuclear P/N NEL-474
New England Nuclear P/N NEL-475
New England Nuclear P/N NEL-492
New England Nuclear P/N NEL-493
New England Nuclear P/N NEL-494
Amersham Pharmacia Biotech (APB)
P/N US77118
AmpliTaq Gold, 5 U/µL
Applied Biosystems P/N N808-0247
GeneAmp 10X PCR Gold Buffer
Applied Biosystems P/N 4306894
Alkaline Phosphatase, 1000 U
Roche P/N 1 758 250
Exonuclease I, 2500 U
Amersham Pharmacia Biotech
P/N E70073Z
One of the following:
• ThermoSequenase DNA
polymerase, 32 U/µL
• AmpliTaq FS, 8 U/µL
Amersham Pharmacia Biotech P/N
E79000Y
Applied Biosystems P/N 402118
DNA from 24 unrelated Individuals
Coriell Cell Repositories
P/N M24PDR
Synthetic oligos, EconoPURE
BioSource International
96-well microplates—
at least one of the following:a b
• Microseal skirted, black
• Microseal skirted, clear
• High Efficiency HE™
microplate
MJ Research P/N MSP-9661
MJ Research P/N MSP-9601
Molecular Devices P/N 042-000-0089
a. If methanol is added before reading, black Costar microplates can be
used and the dimensions are included as manufacturing default on all
Criterion instruments.
b. 384-well microplates can also be used.
Excitation
Emission Filters,
and Beamsplitters
Table 2. The filters for the fluorescent labels for the four-dye assay:
Dye
Excitation
BODIPY-FL
BDF/R110 Ex
R6G
Dichroic
Filter Source
BDF/R110
Em
50/50
R6G Ex
R6G Em
50/50
Molecular
Devices
P/N 42-000-0135
TAMRA
TAMRA Ex
TAMRA Em
50/50
ROX
ROX Ex
ROX Em
50/50
SNP Genotype Detection With HEFP™
Emission
4
Table 3. The filters for the fluorescent labels for the two-dye assay:
Dye
Excitation
Emission
Dichroic
Filter Source
Molecular
Devices
P/N 42-000-0144
R110
BDF/R110 Ex
BDF/R110
Em
R110/
TAMRA
TAMRA
TAMRA Ex
TAMRA Em
R110/
TAMRA
Terminator Base Extension Using Synthetic
Templates
Purpose
The purpose of this preliminary assay is to enable you to understand the
following.
•
•
•
Materials
Principles of HEFP
Proper controls
Operation of the instrument.
Molecular Devices has been using the ApoE polymorphism as a model
system. Synthetic templates were designed to correspond to the common
sequence variations occurring at codon 112. The sequences are as follows
with the brackets enclosing the polymorphic site. Four templates were made
for all base variations at this position.
•
ApoE-112 templates: 5'-gctggcgcggacatggaggacgtg [C/T/A/G] gcggc cgcctggtgcagtaccgcgg-3'
Specific SNP primer lies just upstream from the polymorphic site and is
complementary to the templates. Since the annealing and extension occur at
60˚C, the primer was designed to have an annealing temperature of 70˚C or
higher. The sequence is as follows:
•
ApoE-112 primer: 5'-ccgcggtactgcaccaggcggccgc-3'
Note Similar experiments with other synthetic SNPs will require you to
design these primers to work with the desired annealing temperature and be
at least 20 bases in length.
Experimental
Procedure
Terminator base extension procedure using synthetic templates:
Step
Action
1
Assemble reactions one of two ways:
a. Using MJ Research 96-well PCR microplates, which can be
read directly on the instrument at the end of the terminator
base extension reaction
b Using PCR tubes or other PCR microplates and then transferring 10–20 µL of the reaction mix to HE or Costar microplates for HEFP measurements
SNP Genotype Detection With HEFP™
5
Terminator base extension procedure using synthetic templates: (Continued)
Step
Action
2
Assemble label-ddNTP mix. Use the following guidelines:
• If all four labels are used, mix equal volumes of four
ddNTPs to a final concentration of 25 µM for each dye.
• If two labels are used, supplement the mix with unlabeled
ddNTPs for the other two nucleotides to prevent
misincorporation.
Store aliquotted 100 µM stock solutions and the mixed working
solution at –20˚C. Avoid repeated freeze-thaw cycles.
3
Include buffer-only wells i.e., without dye-ddNTPs in your assay.
These will serve as background readings in data interpretation
and troubleshooting.
Note It is highly recommended that you run replicate samples of
three or more for all the samples in the initial studies.
4
Make up a “master mix” (all components except template DNA) if
you are running many samples. This will avoid pipetting imprecision. Vortex the master mix and spin it briefly. Dispense 19 µL of
the master mix to sample wells that have 1 µL of the template
DNA.
If you are running only a few reactions, it is more convenient to
dilute the dyes in 10 mM Tris, pH 9.0 and the Thermosequenase
in its dilution buffer prior to use.
The final reaction mix is 20 µL and should contain the following:
Quantity
Final
Concentration
1 µM template DNA
1.0 µL
50 nM
10 µM internal detection primer
1.0 µL
500 nM
10X Thermosequenase buffer
2.0 µL
1X
Four-dye ddNTPs, 25 µM each
dye label
0.05 µL
62.5 nM
Thermosequenase or
AmpliTaq FS
0.025 µL
0.1 µL
0.8 U
Deionized water
To a final volume of 20 µL
Component
5
Start the terminator base extension reaction using the following
recommended conditions established for ApoE-112.
a. One cycle of 95˚C for 2 min
b. 35 cycles of 94˚C for 15 sec and 55˚C or 60˚C for 30 sec
Note The annealing temperature depends on the primer’s TM.
The polymerase is active at both temperatures for extension.
Successful annealing may require temperatures as low as 45˚C
for some primers with low TM.
6
Take a reading by either transferring 10–20 µL into an HE microplate or placing the reaction microplates directly on the Analyst
detection system.
Use the settings outlined on the following table.
SNP Genotype Detection With HEFP™
6
Instrument
Parameter
Settings
The following instrument settings are recommended when using 96-well
PCR microplates with a 10–30 µL volume in Analyst instruments using
Release 2.0 software or 10–20 µL in HE microplates:
Table 4. Terminator base extension assay settings:
Parameter
Setting
Mode
Fluorescence Polarization
Excitation and emission filters
See Table 2, “The filters for the
fluorescent labels for the fourdye assay:,” on page 4
Dichroic mirror
50/50 beamsplitter
Z-height
3 mm
Attenuator
Out
Integration time
100,000 µsec
Lamp
Continuous
Readings per well
1
Raw data units
Counts/sec
Switch polarization
By well
Plate settling time
0 msec
PMT setup
SmartRead, sensitivity 2
Dynamic polarizer
Emission
Static polarizer
S
G-factora
1
a. G-factor is set by default as 1. Individual G-factors for each dye can
be calculated and then entered into the software. Please refer to the
User’s Manual for instructions on calculating the G-factor.
Sample Results
and Analysis
Four replicas for each template were measured for incorporation of each dye
with the filter combinations described in Table 2 on page 4. Average mP
(milli polarization, output of the instrument) and standard deviation of the
replicates were calculated and displayed in Table 5 on page 8.
Please refer to the Analyst User’s Manual for detailed explanation on the
calculation of the mP values from parallel and perpendicular intensities.
When complementary templates are present, for example, template T, we
expect incorporation of ddATP. As a result, BODIPY-FL dye shows an
increase in polarization (increased mP values), whereas the other three dyes
have mP values comparable to the samples without any template DNA (see
the data below).
SNP Genotype Detection With HEFP™
7
Table 5. Primer extension data using synthetic templates for ApoE-112.
BODIPY-FL-ddATP (mP)
Template
TAMRA-ddCTP (mP)
No
DNA
C
A
T
G
67
80
71
124
73
82
68
85
79
79
Mean
Std Dev
Template
No
DNA
C
A
T
G
88
40
60
75
51
121
151
84
47
55
76
56
111
78
129
90
50
57
71
54
129
78
75
133
95
48
53
71
51
124
76
80
73
134
89
Mean
46
56
73
53
121
8
2
4
12
5
Std Dev
4
3
3
2
8
R6G-ddUTP
Template
Guidelines
ROX-ddGTP
No
DNA
C
A
T
G
39
50
96
48
43
37
92
45
46
46
Mean
Std Dev
Template
No
DNA
C
A
T
G
44
73
179
82
92
93
50
44
68
182
86
104
94
101
47
43
73
172
87
93
101
38
94
43
49
71
175
83
77
102
43
43
96
47
45
Mean
71
177
85
92
98
3
6
4
3
3
Std Dev
2
4
2
11
5
Collecting Reliable Data
•
•
•
•
To ensure reliable data, the average mP for extended primers should be
at least 50 mP greater than the mP of the free fluorophores.
The standard deviation of replicate samples should be less than 10 mP.
Accurate measurement is obtained only if the incorporation of the dyelabeled ddNTPs is efficient. Chen et al. estimated that the efficiency
should be >50%.
For initial evaluation and assay optimization, it is helpful to estimate the
incorporation efficiency by running the reaction mixture on a sequencer
in parallel.
Analyzing the Raw Signal Intensities
•
Take advantage of the fact that Analyst provides raw parallel and perpendicular signal intensities in addition to the calculated mP values.
Using the above protocol, expect to obtain a parallel intensity of
300,000–1,000,000 counts/sec (cps) for buffer-only wells (no label
added). The four dyes have varying intensity:
• BODIPY-FL at 1,500,000 to 3,000,000 cps
• ROX and TAMRA at 4,000,000 to 6,000,000 cps
• R6G at over 8,000,000 cps in the parallel channel
•
•
It is important to inspect the raw signal intensities since it helps to diagnose any mistakes that might have been made assembling the reaction
mixture. If the signals are less than three times the background, the
instrument will not be able to detect polarization with accuracy.
High raw signal values may indicate a low percentage of incorporation
as a result of excess free dye-ddNTP relative to the incorporated label.
This condition will mask the capability of the mP value to reflect incorporation.
SNP Genotype Detection With HEFP™
8
Monitoring the Background
Background subtraction improves data quality only when the parallel raw
intensity signal to background ratio is low (<5). This applies in particular to
BODIPY-FL. However, background subtraction has been found not to be
helpful for R6G when quenching of intensity occurs upon incorporation. In
addition, background subtraction may not be practical in high throughput
settings. Therefore, we routinely do not recommend background
subtraction. However, it is highly useful to include buffer wells without any
labels in assay development stage to monitor the background signals.
Assessing Signal Spillover
There is reproducible signal spillover among the four labels: BODIPY-FL
into R6G; R6G into TAMRA; TAMRA into ROX. However, with a
clustering algorithm for base-calling, this signal spillover should not
interfere with data interpretation.
Optimizing the Primers
The length of the primer may not contribute to the magnitude of mP change
significantly as long as the primer is 20-nucleotides or longer. For example,
20-, 22-, and 25- mers work comparably in the ApoE-112 system. Instead,
appropriate cycling conditions should be chosen to ensure efficient
annealing of the primer onto the template, thus affording higher
incorporation.
Theoretically a “no DNA control” should give the same mP value as using
the “wrong” template. However, differences in mP (sometimes as large as 20
mP) between the two measurements have been observed. The small
difference in mP values is attributed to non-specific incorporation resulting
from a hairpin structure formed by the primer. Regardless, this difference is
not significant enough to mistake for a “true” positive since changes in
polarization for the positive controls are routinely >50 mP. This problem
can also be solved by using a primer designed from the reverse direction.
Assessing the Polymerases
A difference in performance between the two polymerases used in the
terminator base extension reaction has not been observed in our model
system.
Determining the Optimal Microplates
No significant differences in detection between the MJ Research PCR
microplates and the HE microplates have been observed in the ApoE model
system. Both the black and clear 96-well MJ Research microplates can be
used without significant compromise in signal intensity. The clear
microplates can be stacked in the stacker and registered by the stacker’s
microplate sensor. The black PCR microplates are stackable in the
magazines. However, to be detected by the microplate sensor, a 1cm x 0.5cm
area has to be painted with whiteout near the center of the microplate’s
short edge facing the instrument. Alternatively, a reflective white piece of
barcode sticker can be used. Unfortunately, the footprint of the current MJ
Research PCR microplates after thermocycling is slightly larger than HTS
standards. As a result, users have experienced dropping of microplates when
using MJ Research microplates on the stacker. We are exploring other
vendors’ PCR microplates for ease of robotic handling and appropriate
dimensions. Currently, we recommend using the MJ Research microplates
for detection if you can feed them one by one directly into the gripper. If a
SNP Genotype Detection With HEFP™
9
stack of microplates will be read, it is more reliable to transfer samples to
HE microplates for detection using the stacker.
Four-Dye Genotyping Procedure
Overview of the
Assay
The outline of the complete genotyping assay is presented below.
Overview of the assay
Step
Action
1
PCR Amplification of Template DNA
a. Mix PCR primers, dNTPs, source DNA (usually
genomic) and polymerase.
b. Obtain sufficient PCR product with the minimal
amount of PCR primers and dNTPs.
PCR Amplification
2
Degradation of PCR Primers and dNTPs
a. Treat with alkaline phosphatase (SAP) and exonuclease I.
3
Incorporation of ddNTPs
a. Mix internal primer, fluorescent dye-labeled ddNTPs, and
thermo-stable DNA polymerase.
b Allow efficient incorporation of free ddNTP-label.
4
Detect HEFP in Analyst.
5
Analyze data and interpret results.
The reagent mixture for the PCR and the cycling conditions are provided
below. The volume for each PCR is 10 µL. This mixture works well for
amplicons of 150–350 bp and yields approximately 100 ng/reaction as
judged by ethidium bromide staining.
For the best genotyping results:
•
Determine the minimal concentration of primers and dNTPs required to
obtain a visible signal on a gel when stained with ethidium bromide.
This should be at least 50 ng/reaction.
Note The optimal conditions for PCR amplification will depend on the characteristics of the specific reagents. If the product is not visible upon ethidium
bromide staining of a gel, you will probably not obtain satisfactory HEFP
results.
Table 6. PCR mixture
Component
Final Concentration
10X PCR Gold Buffer
1X
MgCl2
2.5 mM
dNTPs
5–25 µM
Primer 1
25–100 nM
Primer 2
25–100 nM
AmpliTaq Gold (5 U/µL)
1 U/10 µL
Genomic DNA
20 ng
SNP Genotype Detection With HEFP™
10
Table 6. PCR mixture (Continued)
Component
Final Concentration
Deionized water
to a final volume of 10 µL
PCR Cycling Conditions
Table 7. PCR cycling conditions
Conditions
Cycles
Primer and dNTP
Degradation
Temperature
Time
Hot start
95˚C
12 min
15
Touch down
95˚C
66˚C (step
down 1˚C/cycle)
72˚C
30 sec
30 sec
30–35
95˚C
50˚C
72˚C
30 sec
30 sec
30 sec
1
72˚C
6 min
End
4˚C
storage conditions
30 sec
When you have completed the PCR amplification and are ready to continue
with the SNP assay, follow the procedure outlined below.
Note The following procedure can be carried out in a thermocycler.
Procedure for primer and dNTP degradation
Step
Action
1
To each PCR tube or well add the following (total addition = 10 µL/
reaction).
Component
volume (µL)
10X SAP buffer
1 µL
Alkaline phosphatase
2 µL
Exonuclease I
Deionized water
0.1 µL
to a final volume of 10 µL
Note Each tube/well should contain 20 µL.
2
Place samples at 37˚C for 45–90 min.
3
Inactivate the enzymes by heating the samples at 95˚C for 15 min.
SNP Genotype Detection With HEFP™
11
SNP Genotyping
Assay
After you have enzymatically treated the PCR samples, the genotyping
portion of the assay can be performed.
Procedure for genotyping
Step
1
Action
To each enzymatically treated PCR product, add the following
(total addition = 10 µL/sample):
Component
Volume (µL)
10X ThermoSequenase buffer
1 µL
10 µM internal detection primer
1 µL
25 µM dye-labeled ddNTP mix
AmpliTaq FS or ThermoSequenase
Deionized water
0.05 µL
0.8 U/reaction
to a final volume
of 10 µL
Note Each tube/well should now contain 30 µL.
2
Perform terminator base addition according to the following
cycling conditions:
Condition
Cycles
3
Sample Results
and Analysis
Temperature
Time
Hot start
94˚C
1 min
35
94˚C
55˚C
10 sec
30 sec
End
4˚C
storage conditions
Read samples using the conditions outlined in “Instrument
Parameter Settings” on page 7.
In our example we examined SNP 100113 from the public database dbSNP.
The PCR primers were 5'-actgatgattcctgaggaggcac-3' for the forward and
5'-tttgggctcattctttgatgt-3' for the reverse primer. The product is 220 bp long.
The internal terminator base extension primer is
5'-gggttttgaggctgttttatgttc-3'.
For data analysis, the mP values of the two dyes are plotted on the two axes
of a scatter plot (see Figure 1).
•
•
•
Homozygotes are expected to have an increase in mP upon incorporation of one dye-labeled ddNTP.
Heterozygotes will incorporate both labels and therefore form a cluster
at the diagonal corner opposite to the origin.
The no DNA samples and failed PCR samples will have no incorporation and therefore low mP values in both directions, clustering towards
the origin.
Note In order for genotyping to be successful in a high-throughput mode,
tight clustering is desirable when plotting large numbers of samples.
SNP Genotype Detection With HEFP™
12
TAMRA-ddCTP
160
C/C homozygotes
C/T heterozygotes
120
80
T/T homozygote
40
No DNA controls
0
0
40
80
R6G-ddUTP
120
Figure 1. HEFP-SNP Genotyping results for SNP 100113 from the public database
dbSNP.
Guidelines
Examining the Raw Data
It is crucial to examine the raw parallel and perpendicular intensity of each
dye. Please refer to “Guidelines” on page 8 for the expected values of each
dye.
Optimizing the PCR Amplification
Successful PCR from genomic DNA is crucial. Although experiments that
vary synthetic template concentrations indicate this method can tolerate
some variability in PCR yield, the quality of PCR in a high throughput
environment will impact the results. The conditions described here are best
for PCR products of 150–350 bp in length. Longer product may have
compromised results due to reduced PCR efficiency.
Evaluating the Dye-ddNTP incorporation
To evaluate the dye-ddNTP incorporation efficiency, run parallel samples on
a sequencer to optimize assay conditions.
Ensuring the dNTPs and PCR Primers are Removed
Complete removal of dNTPs and PCR primers from the PCR reaction is also
very important. To monitor enzymatic digestion, a control may be added
where primer is omitted in the last terminator base extension step. Without
the primer, any increase in fluorescent mP will indicate residual PCR primers
from incomplete enzymatic digestion.
SNP Genotype Detection With HEFP™
13
Two-Dye Genotyping Procedure
Introduction
In addition to genotyping with four labeled dyes, the HEFP genotyping
application has been developed using two labeled dyes. A two-dye system
has the following advantages over the four-dye system:
•
•
•
Dye Preparation
Reduction in dye concentration required
Reduction of microplate read time
Avoidance of inconsistent results with the BDF-ddATP dye
The following procedure describes how to prepare the two dyes for use in
the incorporation portion of the assay.
With optimized optics to detect R110 and TAMRA, the dyes can be
significantly diluted and still achieve uncompromising sensitivity. The
following experiment demonstrates the feasibility of using 1/64 of the dye
concentration used in the four-dye system.
To prepare the dyes:
Step
Action
1
Dilute unlabeled ddNTPs to 100 µM in deionized water.
Keep stock aliquots of ddNTPs at –20˚ C.
2
Choose a dye combination using the following list which illustrates each of the six possible nucleotide variations:
SNP
SNP Genotype Detection With HEFP™
Dyes Used
A/G
R110-ddA/TAMRA-ddG
C/T
R110-ddU/TAMRA-ddC
C/G
R110-ddC/TAMRA-ddG
A/C
R110-ddA/TAMRA-ddC
G/T
R110-ddU/TAMRA-ddG
A/T
R110-ddU/TAMRA-ddA
14
To prepare the dyes: (Continued)
Step
3
Action
Prepare a 25 µM ddNTP mix containing two labeled and two
unlabeled dideoxynucleotides. Examples are given below.
Note Store mixed dyes in aliquots at –20˚ C.
Dye Dilution Relative to the
Four-Dye Mix
1:8
Dideoxynucleotide
SNP Assay
Procedure
1:16
1:32
1:64
Volume (µL)
ATP
8
16
32
64
GTP
8
16
32
64
CTP
7
15
31
63
TTP
7
15
31
63
R110-UTP
1
1
1
1
TAMRA-CTP
1
1
1
1
Carry out the thermocycling and post-PCR clean-up steps as described in the
four-dye genotyping procedure, using the two-dye mixture instead of the
four-dye mix in the labeled ddNTP incorporation step.
Measure fluorescence polarization in a Criterion instrument using the
settings provided in Table 4 .
SNP Genotype Detection With HEFP™
15
Two-Dye Results
Detection Using an R110/TAMRA Dichroic (Top Panel) vs. 50/50
Beamsplitter (Bottom Panel)
TAMRA-ddATP
240
220
220
180
180
Labels diluted 1:8
Labels diluted 1:16
140
100
150
200
140
300 100
250
200
300
400
TAMRA-ddATP
270
270
230
230
190
190
Labels diluted 1:64
Labels diluted 1:32
150
150
250
350
R110-ddCTP
450
TAMRA-ddATP
300
400
R110-ddCTP
500
370
280
330
240
290
200
250
Labels diluted 1:8
160
150
460
TAMRA-ddATP
150
200
250
Labels dilluted 1:16
210
450 200
350
400
600
550
420
510
380
470
340
430
Labels diluted 1:32
300
250
450
R110-ddCTP
650
Labels diluted 1:64
390
400
600
R110-ddCTP
800
Figure 2. Genotyping results for 101304 from the public database.
Twenty-four individuals from the human diversity panel (Coriell cell repositories) were analyzed on the polymorphism on SNP STSG-101304. The PCR primers used are as follows:
forward PCR primer, 5' agggtattctgctgggtgatttt 3'; reverse PCR primer, 5' accatgacatagaac
cgcaaact 3'. The PCR amplification and SAP/Exo digestion were carried out as recommended in the application note. Four sets of replicates are processed identically in the PCR
and SAP/Exo reactions. The terminal base extension reaction was performed with either 1/8,
1/16, 1/32, or 1/64 of the dye concentration as described in this application note for using
four labeled dyes. The internal extension primer has the sequence of 5' agagagtaccagcattatgtt gagc 3' and either R110-ddCTP or TAMRA-ddATP was incorporated. The samples were
read in the presence of either R110/TAMRA dichroic mirror, or a 50/50 beamsplitter.
As seen in Figure 2, in the presence of an R110/TAMRA dichroic mirror,
R110 and TAMRA dyes can be reduced to 1/64 of the original
concentration and still maintain the integrity of the three clusters. This
reduces reagent cost dramatically. Using the more generic 50/50
beamsplitter allows more flexibility with dye selection. However, lower dye
concentrations cannot be used because of the decreased sensitivity.
Reference
Chen, X., Levine, L., and Kwok, P.Y. 1999. Fluorescence polarization in
homogeneous nucleic acid analysis. Genome Research 9:492–498.
SNP Genotype Detection With HEFP™
16
Appendix A: Dimensions of PCR Microplates
Table 8. Microplate dimensions:
MJ Research
96-Well
MJ Research
384-Well
Robbins
384-Well
Length
127.43
127.43
127.77
Width
85.34
85.48
85.44
Height
16.51
10.12
9.95
Column offset
14.66
11.85
12.12
Row offset
11.4
8.85
9
Column spacing
8.92
4.52
4.5
Row spacing
8.92
4.52
4.5
Well depth
14.69
8.62
8.66
Parameter (mm)
SNP Genotype Detection With HEFP™
17
Appendix B: Frequently Asked Questions
Primers
Does the length of the terminator base extension primers matter?
We have tested terminator base extension primers of 20–30 nucleotides long
with molecular weights of 6–8 kDa with very similar magnitude of mP
increase. Therefore, increasing the length of the terminator base extension
primer may not contribute to better mP change. What is important is to
ensure that the primer anneals to the PCR product/template at the annealing
temperature of your thermocycling program.
Are there any tricks in designing the third, internal terminator base
extension detection primer?
Not really. Since there are only two choices of the location of the third,
internal detection primer (forward or reverse), we are quite limited to the
design of the primer sequence. All rules used in picking a good sequencing
primer should apply, including appropriate length (20–25 bases) to offer
better specificity, ideally 40–60% GC content, and avoid the primer that
forms stable secondary structures. Keep in mind, however, as with any
primers (PCR or sequencing), we may not be able to predict precisely how
successful each SNP primer is going to be.
PCR Assay
How much PCR product do I need to obtain good HEFP results?
In our experience, obtaining sufficient PCR product is essential for
successful SNP assays. A visible band on the agarose gel is the minimal
requirement for products 150–350 bp in length. We estimate that to be 50–
100 ng of DNA in 10 µL of reaction.
Is there any restriction on the length and GC content of the PCR
product for this assay?
We have tried about two dozens of amplicons 150–350 bp in length with
varying GC contents and have not observed any bias at this time. Longer
products may not work as well due to the compromised efficiency in PCR.
We can speculate that one restriction may be that the third internal detection
primer itself does not contain a stable hairpin structure resulting in nonspecific incorporation of ddNTP labels. This can be tested using a negative
control with no template DNA added.
Why are the concentration of the PCR primers and dNTPs in the initial PCR reaction crucial for the SNP results?
Reducing the amount of PCR primers and dNTPs will ensure complete
removal of the excess material in the SAP/Exo reaction, therefore lowering
the nonspecific incorporation of the ddNTP labels in the terminator base
extension reaction. Optimization should be performed, however, in order
not to compromise the PCR yield.
Does the SNP reaction work if I get more than one band in the PCR?
The addition of the third, internal SNP primer just upstream from the
polymorphic site theoretically increases the entire assay’s specificity even in
the presence of other nonspecific PCR products. We have tested these
SNP Genotype Detection With HEFP™
18
situations in a couple of assays mixing two different template PCR products
and have obtained the same specificity as a single template.
If I need to use mineral oil for my PCR, will that interfere with the
HEFP detection?
We would recommend that you carefully remove 10–20 µL of the reaction
(avoid transfer of mineral oil) and transfer that to an HE microplate prior to
reading in the instrument.
Labels
Can I try different combinations of dyes and ddNTPs?
With the current four dyes, we have tried some of the twelve other
combinations of dyes and ddNTPs without any significant change in
performance of the assay except ROX-ddUTP. Some customers prefer to
work with only two colors and substitute the nucleotides. This is outlined in
the two-dye genotyping protocol.
Can I use less ddNTP labels?
Yes, but with caution, and not with all fluorophores. Since BODIPY-FL
signal tends to be lower and barely 5-fold above the buffer background, we
made a dye mix maintaining the original BODIPY-FL-ddATP concentration,
while reducing the other three labels by half. This label mix was prepared by
diluting the concentrated labels in 10 mM Tris, pH 9.0. This mix effectively
normalized the intensity of all four labels per well. Remember that there is
lot-to-lot variation of the labels; therefore, the ratios for mixing the dyes
may vary. In addition, the final concentration of each ddNTP cannot fall
below the Km of the polymerase. It is beneficial to titrate in unlabeled
ddNTPs to keep the final concentration of ddNTP constant as described in
the two-dye protocol.
Can I use the new dye, R110, to replace BODIPY-FL in the four-dye system?
No. Although R110 has spectral properties very similar to BODIPY-FL and
we use the same 490 excitation and 520 emission filters for both
fluorophores, R110 has much wider emission spectrum than BODIPY-FL.
As a result, the signal spillover of R110 into the spectrally neighboring R6G
dye will interfere with detection of R6G polarization detection under the
current filter settings. Therefore, R110 is recommended to be used only in
combination with TAMRA or ROX, (for e.g., in the 2-dye method) but not
in combination with R6G.
In the four-dye system, BODIPY-FL that used to work well started
giving me low signals in the samples that were supposed to have no
incorporation and low mP values. What do I do?
In the original Chen et al.’s protocol, 100 µL of the Thermosequenase buffer
and 50 µL of MeOH was added after the reactions were complete and prior
to reading the microplate. We have found that the addition of MeOH dilutes
the signal intensity and may not be beneficial. However, BODIPY-FL is
somewhat prone to aggregation, and the addition of MeOH was to increase
BODIPY-FL’s solubility. We don’t yet understand fully the mechanism. We
do notice that when aggregation or coating onto the microplates of
BODIPY-FL occurs, addition of MeOH provides accurate base-calling.
However, since the addition of solvent dilutes the signal of other dyes, we
recommend that under those circumstances, you read the other three colors
first, then add 50 µL of 1x Thermosequenase buffer and 25 µL of MeOH
directly into the PCR microplates and then proceed to read BODIPY-FL.
Utilization of a different dye may be the ultimate solution to this potential
SNP Genotype Detection With HEFP™
19
problem. Inappropriate storage of the BODIPY-FL dye may also contribute
to the stickiness of the free ddNTP label. Storing the dyes at –20˚C can
minimize the adverse effect.
Enzymes
What alkaline phosphatase can I use?
We have been using the shrimp alkaline phosphatase (SAP) from Roche
(previously Boehringer Mannheim) with very consistent results. In one
experiment with 24 individuals, when SAP from Amersham Pharmacia was
tested side-by-side, Roche’s SAP appeared to give tighter clusters.
Are there alternative enzymes I can use to replace the expensive
Amersham Pharmacia Biotech’s Thermosequenase?
An alternative source of Thermosequenase, other than APB’s
Thermosequenase and Applied Biosystems AmpliTaq FS, is from USB
(www.usbweb.com, 1-800-321-9322). Their sequencing kit (US78500)
contains a Thermosequenase that has similar performance to the APB’s
Thermosequenase. Be aware that USB packages their Thermosequenase
enzyme as 4 U/µL whereas the enzyme from APB is packaged as 32 U/µL.
General Assay
What is the optimal volume of the SNP reactions with HEFP?
We have been using the volumes used by Chen et al. in their initial
publication, i.e., 10 µL of PCR reaction, 10 µL of SAP/Exo reaction, and
10 µL of SNP reaction with 30 µL of final assay volume. These assays are
routinely performed in 96-well PCR microplates. However, 5 µL + 5 µL +
5 µL reactions have also been used with satisfactory results which reduce the
cost of the assay by 50%. Other ratios of these three reactions have also
been reported by some of our customers.
I’d like to run the PCR, the extension and the detection in the same
microplate. What kind of microplate can I use?
There are two things to consider here:
a) Microplate dimensions. Generally speaking, both 96- and 384-well
skirted microplates that fit onto MJ Research’s PCR machines are physically
compatible with Analyst detection. In particular, the dimensions for PCR
microplates manufactured by MJ Research are included as appendix in the
application note. On the other hand, the 96-well microplates for Applied
BioSystems' thermocycler have no skirts and are too tall to be read on the
Analyst. The 384-well format thermocycler from Applied BioSystems can
use MJ Research’s 384-well skirted PCR microplates and, therefore, can be
used.
b) Microplate optical properties. Microplates that are optically uniformed
and have low background fluorescence (for example, black microplates) are
preferred. Black 96- and 384-well PCR microplates are available from MJ
Research and have been the microplates of choice. Clear 96-well MJ
Research microplates are acceptable as well due to the large well-to-well
distance which eliminates cross talk between wells. However, clear 384-well
microplates are not desirable due to the cross talk.
Do I use the Amersham Pharmacia Biotech’s Thermosequenase reaction buffer as 5x or 10x?
Amersham Pharmacia Biotech sells their Thermosequenase originally with a
“concentrated reaction buffer”, which was later labeled as 5x. The
application note indicates to use the buffer as 10x. We have subsequently
SNP Genotype Detection With HEFP™
20
tested the buffer as 10x or 5x on a limited number of SNPs and samples and
obtained comparable results (base-calling as well as relative mP change)
with either buffer.
Interpreting
Results
Can I use the SNP method for quantitative population studies to estimate the frequency of one SNP in mixed samples?
The current protocol has been developed to give a “yes” or “no” answer to
the presence of one allele in base-calling and strive to be insensitive to the
dose of the template present. In the future, protocols may be optimized for
quantitative studies.
Is it important to inspect the raw parallel (s) and perpendicular intensity (p) data in the Analyst™ output?
Yes. It is a good idea to get accustomed to the kind of raw parallel signal
level expected for each fluorophore you use. Too high intensity indicates
that excess ddNTP labels have been added. Since FP measures the
accumulative polarization of all labeled molecules in each sample, excess
ddNTP labels will mask any incorporation you may have. On the other
hand, too low intensity may imply insufficient label resulting in reduced
detection accuracy and increased background contribution. In addition, one
common mistake we have observed in assay development stage of the
genotyping assay is incomplete mixing of the reagent for the incorporation
reaction, therefore uneven distribution of dyes in assay wells. In inspecting
the raw intensity of wells that have received the same reaction mix, if
drastically different values are observed, it may imply uneven reagent
addition. However, BODIPY-FL, R110, and R6G have varied parallel
intensities upon incorporation. TAMRA and ROX intensities are more
reliable to troubleshoot for uneven reagent addition.
What do I do if my clusters are not very tight?
There are a few things to check for:
a) Good reagent distribution among assay wells. Look for raw intensity of
TAMRA or ROX to make sure that all wells have received approximately
the same amount of label.
b) Sufficient PCR products. Running the samples on agarose gel will be
helpful after the FP detection. Compare the band’s brightness to a SNP that
has good clustering results. Increasing the number of PCR cycles or
redesigning PCR primers may help.
c) Sufficient SAP/EXO clean-up. You can consider increasing the incubation
time with SAP/EXO.
d) Ensure good annealing of the internal detection primer in the last step and
no hairpin structure formation of the primer by itself.
e) Kwok et al., have recently developed a protocol in which single strand
binding protein (SSB) is added prior to reading on the Analyst instrument.
They reported that the SSB helped to tighten the clusters with troublesome
SNPs.
What went wrong when all of the samples seem to contain unincorporated dye (low mP values)?
You can start out by running the already read SNP samples on an agarose
gel to determine if the PCR reactions had failed. Check to see if the correct
SNP Genotype Detection With HEFP™
21
PCR product is obtained and if the correct SNP primer has been added.
Failed SNP reactions may be due to the following:
•
•
•
too much dye (check to see if the raw intensity signal is much higher
than expected)
problematic SNP primer (try genotyping with the reverse SNP primer)
enzyme-buffer incompatibility
You can test these other possibilities by running the SNP reaction alone with
synthetic templates.
What went wrong when all labels had nonspecifically high mP in all
samples?
Frequently, incomplete digestion of the PCR product and dNTPs also
introduce high background mP values. Also check to see if your SNP primer
forms a hairpin structure on itself resulting in nonspecific incorporation.
Author: Yan Zhang-Klompus, Ph.D., Senior Application Scientist
Analyst™ and AlleleCaller™ from Molecular Devices Corporation may be useful for the detection of genetic polymorphisms
(genotyping) by the method of SNP-IT™. Orchid BioSciences Inc. of Princeton, NJ asserts that the SNP-IT method is covererd by
U.S. Patent #5,888,819; 6,004,744 and their foreign counterparts. These Molecular Devices products may not be used genotypng
by the method of SNP-IT unless a separate license for such use is Obtained from Orchid BioSciences, Inc. or one of its authorized
sub-licensees. No license to practice SNP-IT is granted through the sales of this product to the purchased or the end-user.
SNP Genotype Detection With HEFP™
22
SNP Genotype Detection With HEFP™
23
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SNP Genotype Detection With
HEFP™
are trademarks
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©2001 Molecular Devices Corporation. Printed in USA. 7/01 2500