U
User Gu
uide
U
User Guuide forr the IDT® Survveyor® PLUS M
MutationD
Detection Kit forf WAVVE® andd WAVEE HS Systems
TThis guide applies to catalog nu
umbers: 70
06035, 7066030, 7060031
w
www.IDTDNA
A.com/Surveyor

R
Revision: 1
FFor Research Use Only

Table of Contents
Step-by-Step Instructions ...................................................................................................................... 11
Detecting Mutations with Surveyor Nuclease ................................................................................... 11
Step 1—PCR Amplification of Reference and Test Samples ........................................................ 11
Step 2—DNA Duplex Formation ............................................................................................... 15
Continue with Step 3—Treatment with Surveyor Nuclease. ......................................................... 16
Step 3—Treatment with Surveyor Nuclease ............................................................................ 17
Step 4—Analysis of DNA Fragments ........................................................................................... 19
Washing Procedure for DNASep and DNASep HT Cartridges used to Analyze Surveyor Nuclease
Digests................................................................................................................................................... 19
Appendix A: Preparing PCR Products using Optimase Polymerase..................................................... 21
Appendix B: Surveyor Nuclease Reaction Conditions .......................................................................... 23
Appendix C: Troubleshooting ............................................................................................................... 26
Problem 1—Low PCR yield or no PCR product ............................................................................... 26
Problem 2—Multiple PCR products ................................................................................................. 26
Problem 3—No cleavage products observed upon analysis after Surveyor Nuclease treatment ofknown heteroduplex .......................................................................................................................... 27
Problem 4—High background after Surveyor Nuclease treatment .................................................. 27
References ............................................................................................................................................. 28

Introduction
IDT Surveyor PLUS Mutation Detection Kits use a mismatch‐specific DNA endonuclease to scan for known andunknown mutations and polymorphisms in heteroduplex DNA. Surveyor Nuclease, the key component of thekit, is an endonuclease that cleaves DNA with high specificity at sites of base‐substitution mismatch and otherdistortions1. These DNA endonucleases cut both strands of a DNA heteroduplex on the 3’‐side of themismatch site. Insertion/deletion mismatches and all base‐substitution mismatches are recognized, but theefficiency of cleavage varies with the sequence of the mismatch1.
Surveyor Nuclease has been shown to be a robust and reproducible tool for mutation detection1, 32, 37. It has
been used to detect accurately a variety of mutations and polymorphisms in the human, mammalian,bacterial, and plant genomes.
Examples of use of Surveyor Nuclease include:•

germline mutations in human genes:

ATRX for X‐linked mental retardation7



ABCC6 associated with pseudoxanthoma elasticum31



HBB gene linked to β‐thalassemia38



genes involved in uracil catabolism8, 9•

mtDNA and nuclear gene mutations associated with respiratory chain defects10‐12, 33•

mutations associated with kidney disease5, 40, 41•

somatic mutations in:•

EGFR gene in human tumors13‐19

JAK2 tyrosine kinase gene in patients with myeloproliferative disorders20, 21

variety of genes in patients undergoing radiotherapy22



p53 gene in squamous‐cell carcinomas23 and hematological malignancies22



KIT tyrosine kinase receptor gene in various malignancies25, 26

PKC412 tyrosine kinase receptor in mast cell leukemia27



LKB1 tumor suppressor in lung cancer36

VHL gene in renal tumours34



hCDC4 gene in patients with AML29

mutations in drug‐resistant genes of Mycobacterium tuberculosis5, 6 and in Group II introns ofbacterial rRNA genes29•

induced point mutations in barley4, 39 and error‐free clones generated from a plant cDNA library by
PCR‐based cloning30

The Surveyor PLUS Mutation Detection Kit for WAVE and WAVE HS Systems has been designed to cleave DNA
fragments at mismatched sites for subsequent analysis by ion‐pairing reverse‐phase HPLC using the WAVE
and WAVE HS Systems. Since the introduction of the original kit, we have made several technical advancesthat improve the performance of Surveyor Nuclease and these are included in this updated version of the kit.
We now provide digestion reaction conditions for DNA amplified by PCR using most commercially available
DNA polymerases; these reaction conditions increase the amounts of mismatch cleavage products generated
by Surveyor Nuclease from DNA containing genetic variations.
The updated kit also contains an Enhancer Cofactor and a new thermostable Enhancer, Surveyor Enhancer
W2 that replaces Enhancer W. Enhancer W2 and its cofactor are superior to Enhancer W in improving thesignal to noise ratio in Surveyor Nuclease reactions, due to improved suppression of background.
Note: Washing procedures specifically recommended and described in this User Guide are for the Surveyor
PLUS Mutation Detection Kit for WAVE and WAVE HS Systems, and the use of DNASep® and DNASep HT
Cartridges are different from those used for standard WAVE DHPLC procedures. Please follow the specificrecommendations in this manual to maintain optimum performance of your WAVE or WAVE HS System.
To use this kit successfully, we strongly recommend that you read this manual thoroughly and carefully
follow the instructions and guidelines provided. First time users should perform all of the control
experiments outlined in the section Control Experiments—Using Control C and Control G Plasmid DNA.

If you have further questions or need assistance, please contact IDT Customer Care at:
USA & Canada
custcare@idtdna.com
1–800–328–2661

Europeeutechsupport@idtdna.com+32 (0) 16 28 22 60

Web Chat at: www.idtdna.com

Asia Pacificasiacustcare@idtdna.com+65 6775 9187

Australia
auscustcare@idtdna.com
1800 092 259

Surveyor PLUS Mutation Detection Kit Components
The kit is available in three sizes as follows:
Component

Surveyor Nuclease W
Surveyor Enhancer W2
Enhancer Cofactor
0.15 M MgCl2
Stop Solution
Control C
Control G

25‐Reaction Kit(706035)
Amount Provided
0.03 mL
0.03 mL
0.25 mL
0.25 mL
0.25 mL
0.01 mL
0.01 mL

100‐Reaction Kit(706030)
Amount Provided
4 x 0.03 mL
4 x 0.03 mL
2 x 0.25 mL
3 x 0.25 mL
3 x 0.25 mL
0.01 mL
0.01 mL

1000‐Reaction Kit(706031)
Amount Provided
1.0 mL
1.0 mL2 x 1.5 mL
2 x 1.5 mL
2 x 1.5 mL
0.01 mL
0.01 mLStore all components at –20°C.
Detecting Mutations with the IDT Surveyor PLUS Mutation Detection Kit
An Overview
Mutation detection and confirmation with Surveyor Nuclease involves 4 steps:
Step 1—Prepare PCR amplicons from mutant (test) and wild‐type (reference) DNA.
Step 2—Mix equal amounts of test and reference DNA; hybridize them by heating and cooling the
mixture to form hetero‐ and homoduplexes.
Step 3—Treat the annealed heteroduplex/homoduplex mixture with Surveyor Nuclease. The
reference DNA alone, treated similarly, serves as a background control.
Step 4—Analyze the DNA fragments with the WAVE or WAVE HS System. The formation of newcleavage products, due to the presence of one or more mismatches, is indicated by the presence ofadditional peaks. The retention time of the cleavage products indicate the size of the fragments andtherefore the location of the mismatch or mismatches.

Factors Affecting the Quality of Results
The following factors influence the quality of results when using Surveyor Nuclease.
•

The quality of the genomic DNA to be amplified.
High quality DNA (from fresh or frozen cells or tissue) should be used. The DNA should have aconcentration of >5 ng/μL as determined by absorbance at 260 nm, have an absorbance ratio at
260/280 nm of >1.7 and be >90% DNA (i.e. free of most tRNA and rRNA contamination as judged byappearance on an agarose gel). Store DNA samples at –20°C.
If the DNA template is extracted from paraffin‐embedded tissue, several additional precautions canbe taken. The extracted DNA can be treated with uracil DNA glycosylase to prevent amplification of
DNA fragments containing deaminated C residues46. Often a high percentage of the A260 adsorbing
material extracted from paraffin‐embedded tissue is not amplified well during PCR. Using a larger
amount of starting DNA, e.g. ~50 ng versus 10 ng, will often help to produce a reasonableamplification product.

•

The quality of the PCR amplified DNA.
PCR should produce a sufficiently high yield (>15 ng/μL) of a SINGLE amplified species of the correct
size. We strongly recommend the use of a proofreading DNA polymerase (such as Transgenomic
Optimase® Polymerase) to reduce the amount of base misincorporation during PCR (which leads to
the generation of ‘false’ mutations and spurious Surveyor Nuclease cleavage fragments). Similarly,
non‐specific PCR fragments can be interpreted as mutations and can mask Surveyor Nucleasemismatch cleavage products. If possible, a reference DNA should be digested with Surveyor Nuclease
and run to exclude spurious background by visual comparison of chromatogram profiles (see forexample Figures 3 and 5). Primer‐dimers should be strenuously avoided as their presence
dramatically inhibits Surveyor Nuclease cleavage at mismatch sites. Examine each amplified DNA
product before digestion by gel electrophoresis or WAVE HPLC to be sure it is a single species of the
expected size.

•

The relative proportion of mutant (test) to wild‐type (reference) DNA in the hybridized sample.
Whenever possible, test and reference PCR products should be hybridized in equal proportion to
maximize the amount of heteroduplex DNA available for digestion.

•

Suppression of DNA nicking.
Surveyor Nuclease nicks double‐stranded DNA at random matched sites, which produces background
during extended incubations32. This activity is suppressed by Surveyor Enhancer W2 and its cofactor
without otherwise negatively affecting the reaction. Surveyor Nuclease Enhancer W2 and cofactor
are included in this kit.

•

The composition of the PCR buffer.
Commercially available PCR buffers vary dramatically in content and the contents are often notdefined by suppliers. A few buffers are NOT compatible with Surveyor Nuclease due to pH or thepresence of additives, surfactants or other proprietary ingredients. Surveyor Nuclease reaction
conditions have been defined for a large number of different PCR buffers that improve signal
intensity significantly (1.5–4 fold, depending upon the buffer) while maintaining low background.

These new reaction conditions are included in the protocols described in this User Guide (see Step
3—Treatment with Surveyor Nuclease and Appendix B: Surveyor Nuclease Reaction Conditions).
•

Signal to Noise ratio.
The signal to noise ratio is generally high enough to detect mutations present at a low percentage ofthe total DNA template; it is possible to detect 1% to 20% mutant DNA depending upon the particular
DNA amplicon, its size, the number and type(s) of mutation(s) and the analysis platform. Figures 2
and 3 show detection of heteroduplex present in homoduplex at a ratio of 1 in 128 (0.8%heteroduplex) with a WAVE HS system and Figures 4 and 5 show detection at a ratio of 1 in 64 (1.8%
heteroduplex) with a WAVE UV system. Figures 6 and 7 show the digestion products generated withhomoduplex and heteroduplex Control DNA (included in this kit) fractionated by ion pairing reverse
phase HPLC under non‐denaturing conditions using the WAVE and WAVE HS System, respectively.
The mutation‐specific cleavage products are clearly seen as two new peaks eluting with the expected
sizes that can be estimated relative to the DNA size marker.

Note: If you are a first time user, perform the experiments in the section Control Experiments—Using Control
G and Control C Plasmid DNA after reading and understanding the section Step‐by‐Step Instructions.
Examples of Results
Examples of results obtained using the Surveyor PLUS Mutation Detection Kit for WAVE and WAVE HS
Systems are shown in Figures 1 through 5 below. In these examples, the four step process outlined in the
Overview section was followed carefully. Surveyor Nuclease W in combination with a WAVE or WAVE HS
system can be used to identify single and multiple mutations in fragments <1,000 bp long. Mutations can also
be found in amplified, pooled genomic DNAs.
Figure 1 shows Surveyor Nuclease digestion products from a 405 bp DNA fragment amplified fromheterozygous mutant and normal human genomic DNA. The mutant fragment contains a G>A mutation 116
bp from one end. Digestion products were analyzed on a WAVE HS system.
Figure 2 shows WAVE HS System analysis of Surveyor Nuclease digestion products from mixtures ofheteroduplex and homoduplex DNA representing the level of mutation detection in pooled genomic DNAs.
Figure 3 shows the WAVE HS digestion product profiles from Figure 2 for homoduplex and the 1:64 and 1:128
heteroduplex/homoduplex ratios superimposed with the fluorescence signal axis expanded. Surveyor
Nuclease digestion products derived from heteroduplex mixed with homoduplex at a ratio of 1:128 canclearly be seen in the chromatograms.
Figures 4 and 5 show the sensitivity of WAVE UV analysis of the same Surveyor Nuclease‐digested
heteroduplex/homoduplex mixtures analyzed in Figures 3 and 4, respectively. The mismatch could be
detected down to a ratio of heteroduplex to homoduplex of 1:64 with UV detection.

Figure 1. Surveyor Nuclease digestion productts of an amplico
on derived from exon 28 in the ABCC6 gene ofhuman genomiic DNA. The 405 bp amplicon waas PCR amplified
d with Optimasee Polymerase. A
Approximately 6000
ng of unpurified amplicon DNA
A were self‐hybrridized, digested
d with Surveyorr Nuclease and aanalyzed by WAV
VE
HS HPLC as described in the Ste
ep‐by‐Step Instru
uctions. The mu
utant was heteroozygous for a siingle mutation(G>A; fragmentts were 116 + 289
2 bp long). Digestion produccts from self‐hy bridized mutantt (green, top) aandnormal control DNA (red, midddle) are shown. Marker
M(100 bp DNA
D Ladder) is sshown at the botttom.

FFigure 2. Surveyo
or Nuclease dige
estion of hetero
oduplex DNA preesent in decreassing amounts inn homoduplex DNA.
H
Human target DN
NA shown in Figure 1 was ampliffied from both mutantmand norm
mal DNA with O
Optimase
P
Polymerase. Wildd‐type and mutaant allele ampliccons were mixed
d in the ratios indicated (HTD)) and 600 ng off
eeach mixture weere hybridized, digesteddwith Surveyor Nucleasse and analyzedd by WAVE HS HPLC asddescribed in thee Step‐by‐Step Instructions.
I
Digestion products 116 + 289 bp loong formed by ccutting at the sinngle‐bbase mismatch are visible down to a ratio of heteroduplexhto homoduplex off 1:128. Homodduplex (HMD) iss
sshown near the bottom.
bMarker (100 bp DNA Laddder) is shown ata the bottom.

100

200

300

400

500

600

Figure 3. Supeerimposed WAV
VE profiles show differences in sm
mall proportion of heteroduplexx: homoduplex D
DNA. The
1:64 (green) anda 1:128 (blue) heteroduplex/h
homoduplex dige
estion profiles inn Figure 2 are sshown superimp
posed onthe homodup
plex digestion prrofile (red). The fluorescence siggnal axis has beeen expanded com
mpared to Figuree 2.

Figure 4. Survveyor Nuclease digestiondof hetteroduplex DNA present in decrreasing amountss in homodupleex DNA. The ddigested DNAs
analyzed by WAVE HS HPLC in Figure 2 were analyzed by HPLC on a W
WAVE UV system
m. Digestion pro
oducts 116 + 2899 bp long,
formed by cu
utting at the singgle‐base mismattch, are visible downdto a ratio of heteroduplexx to homoduplexx of 1: 64. Hommoduplex(HMD) is sho
own near the bottom.
bMarkerr (100 bp DNA Ladder) is show
wn at the bottomm.

Figure 5. Sensitivity of WAVEW
UV analyysis of Surveyo
or Nuclease‐diggested heteroduplex/homoduplex
mixtures. The
T 1:64 (green) and 1:128 (bblue) heterodu
uplex/homodupplex digestion profiles in Figgure 4 areshown supperimposed onn the homodup
plex digestion profile (red). TThe absorbancce signal axis hhas been
expanded comparedcto Figure 4.

Step‐by‐Step Instructions
Detecting Mutations with Surveyor Nuclease
This section provides detailed instructions for the detection of mutations using the Surveyor
PLUS Mutation Detection Kit for WAVE and WAVE HS Systems.
In general, processing of samples should be carried out from start to finish as described in thisUser Guide. If processing of a sample is stopped before completion of all steps, the DNA should
be stored at –20°C until the next step is carried out. However, exposure of any frozen sample torepeated freeze‐thaw cycles should be avoided and storage at –20°C of PCR amplified DNA
or Surveyor Nuclease digestion products for extended periods (>1 week) should be avoided.

Step 1—PCR Amplification of Reference and Test Samples
THIS STEP IS CRITICALTO THE SUCCESS OF THE Surveyor NUCLEASE DIGESTION. DO NOTPROCEED UNTIL:
Your PCR yield is sufficiently high (>15 ng/μL).

Your PCR product has low background (preferably a single species of the correctsize).

Your PCR product is essentially free of primer‐dimer artifacts.

The first step in the process is to prepare the PCR amplified DNA samples.
Several factors must be considered carefully in preparing PCR amplified DNA to be used as substrate for
Surveyor Nuclease analysis. Primer placement and amplified product quality and yield are crucial toobtaining good results. The following should be considered:Amplified DNA fragments in the size range of 200 to 1,000 bp are most effectively resolved
from potential digestion products on the WAVE system. Place primers at least 50 bp outside theregion of interest to ensure cleavage products are longer than 70 bp, since cleavage too close to theend of a large substrate produces a cleavage product not easily resolved from undigested substrate.

Optimize your PCR conditions carefully. The PCR amplicon should appear as a single sharp peak orband of the expected size when analyzed by WAVE HPLC or agarose gel electrophoresis. Mispriming
during PCR amplification can result in the formation of spurious DNA fragments that produce
increased background during Surveyor Nuclease digestion. Use primers that are at least 20
nucleotides long (oligomers 25–35 nucleotides in length are preferred), and have a G‐C content of
45–60%. If possible, use a high fidelity DNA polymerase to minimize the introduction of errors thatwill result in higher background. If after careful design of primers and optimization of PCR
conditions nonspecific PCR products persist, consider using a hot‐start DNA polymerase, a touchdown
PCR protocol and/or a second amplification with nested primers.

Both the amount and concentration of DNA in a Surveyor Nuclease reaction mixture influence theefficiency and specificity of Surveyor Nuclease digestion. For the amount of enzyme recommended
for use in a reaction mixture (1 μL of Surveyor Nuclease W), 600 ng of substrate at 50 ng/μL is
optimal. If DNA product yield is <15 ng/μL, consider a second amplification with nested primers.
Alternatively concentrate the DNA by ethanol precipitation and dissolve the DNA pellet in a smaller

volume of 1X PCR buffer to increase DNA concentration.
Amplification of Homogeneous DNA Populations
In order to detect a homogeneous mutation in a test sample the PCR product must be hybridized with a wild‐
type reference PCR product to generate mismatches for Surveyor Nuclease cleavage. Both test sample andwild‐type reference DNA are amplified with the same primers. Test sample and wild‐type reference PCR
products are then mixed in a 1:1 ratio to maximize the formation of heteroduplexes during hybridization.
Amplification of Heterogeneous DNA Populations
A DNA sample can be heterogeneous either because it is derived from a heterozygous source or because it
contains a pool of fragments derived from genetically different homozygous or heterozygous sources.
Such heterogeneous samples can be PCR amplified and hybridized without mixing them with a wild‐typereference DNA. The proportion of mutant to wild‐type DNA in the population should be above 1% and 2%
for analysis on the WAVE HS and WAVE System, respectively. After hybridization, retain some of thehybridized PCR product as an undigested reference (control).
Preparing PCR ProductsThis protocol was developed using Optimase Polymerase. Optimase is a proof‐reading enzyme that has beendeveloped specifically for SNP and mutation discovery applications using the WAVE and WAVE HS
Systems. Other proofreading enzymes should work as long as the PCR is optimized according to this guide.
Optimase Polymerase is not included in the Surveyor PLUS Mutation Detection Kits, but can be purchased
separately from Transgenomic.
If you are using Optimase Polymerase, please refer to Appendix A: Preparing PCR Products using Optimase
Polymerase. If you are not using Optimase Polymerase, perform the PCR amplification of test sample
and wild‐type (reference) DNA using a high‐fidelity thermostable DNA polymerase according to the
manufacturer’s instructions, and follow the steps below.
1. To ensure compatibility of the PCR components with a WAVE or WAVE HS System, consult the
PCR Considerations for Successful Mutation Detection chapter in Volume I— Getting Started on the WAVE System of the WAVE System Operator’s Guide.
2. Analyze 2–5 μL aliquots of each product by electrophoresis in a 2% (w/v) agarose gel, prepared withhigh‐resolution agarose and cast in 1X TBE [89 mM Tris‐Borate (pH 8.3), 1 mM EDTA] + 0.2 μg/mL
ethidium bromide. Add 1/6 volume of a 6X loading dye buffer [10 mM Tris‐HCl (pH 8.0), 10 mM EDTA
(pH 8.0), 50% (w/v) sucrose, 0.15% (w/v) bromophenol blue] or your loading dye buffer of choice tothe aliquot and mix. Run the gel in 1X TBE at 5 V/cm until the bromophenol blue has run 2/3 of thelength of the gel. Run several different amounts of a mass DNA ladder, such as a 100 bp DNA Ladder(New England BioLabs, Beverly, MA), as a reference.
3. Visualize the DNA bands using a UV transilluminator at 250 to 300 nm and photograph the gel.
4. Use the ladder to estimate the concentration of the amplified DNA by visual inspection. If a single
band is visible in each sample, proceed; if not, consider optimizing the PCR further as alreadydescribed in this section. The DNA concentration is ideally ~50 ng/μL, but should be in the range of 15
to 80 ng/μL.
5. As long as the DNA polymerase buffer is compatible with Surveyor Nuclease, the amplified DNA canbe used without further purification. Alternatively, the DNA can be concentrated by ethanol
precipitation as follows. Transfer the reaction mixtures to suitable microcentrifuge tubes, add 2.5
volumes of ethanol and store the tubes at –20°C for 30 min. Centrifuge the tubes at 13,000 rpm for
10 min in a microcentrifuge. Carefully remove the ethanol with a micropipetter, being sure not todisturb the invisible pellet on the tube sidewall and bottom. Concentrated PCR products aresuspended in 1X PCR buffer. Estimate the DNA concentration on an agarose gel as described above.
Alternatively, evaluate the quality and concentration of each DNA product by WAVE HPLC analysis at
50°C. The PCR product should appear as a single peak. A DNA mass ladder run in parallel in different
amounts can be used to establish a relationship between peak area and DNA mass during UV WAVE
analysis. Estimate the DNA concentration in each sample.
Control Experiments—Using Control G and Control C Plasmid DNA
Two plasmid DNAs, Control G and Control C, are provided in the Surveyor Mutation Detection Kit. These twocontrol DNAs are plasmids with inserts that differ at a single base pair. They are provided in separate vials,
each at a concentration of 5 ng/µl; forward and reverse primers needed for PCR amplification are alreadycombined with the plasmid templates. The sequence of the PCR product for Control G is shown below.
Control C differs from Control G because it has a Cytosine base in lieu of the Guanine base (underlined).
Primer sequences are underlined at the 5′ and 3′ end of the amplicon sequence.
ACACCTGATCAAGCCTGTTCATTTGATTACCAGAGAGACTGTCATGATCCACATGGAGGGAAGGACATGTGTG
TTGCTGGAGCCATTCAAAATTTCACATCTCAGCTTGGCCATTTCCGCCATGGAACATCTGATCGTCGATATAATATGACAGAGGCTTTGTTATTTTTATCCCACTTCATGGGAGATATTCATCAGCCTATGCATGTTGGATTTACAAGTGATATGGGAGGAAACAGTATAGATTTGCGCTGGTTTCGCCACAAATCCAACCTGCACCATGTTTGGGATAGAGAGATTATTCTTACAGCTGCAGCAGATTACCATGGTAAGGATATGCACTCTCTCCTACAAGACATACAGAGGAACTTTACAGAGGGTAGTTGGTTGCAAGATGTTGAATCCTGGAAGGAATGTGATGATATCTCTACTAGCGCCAATAAGTATGCTAAGGAGAGTATAAAACTAGCCTGTAACTGGGGTTACAAAGATGTTGAATCTGGCGAAACTCTGTCAGATAAATACTTCAACACAAGAATGCCAATTGTCATGAAACGGATAGCTCAGGGTGGAATCCGTTTATCCATGATTTTGAACCGAGTTCTTGGAAGCTCCGCAGATCATTCTTTGGCG

PCR amplification of 2 µL of each DNA solution in a 50 µL reaction should produce >25 ng/µL of a 633 bp PCR
product. Sufficient DNA is provided to perform ten PCR reactions with each control.
Control G and Control C plasmids can be used to troubleshoot the PCR amplification, hybridization andSurveyor Nuclease digestion steps of the Surveyor Nuclease Kit.
WE STRONGLY RECOMMEND THAT FIRST TIME USERS PERFORM THE CONTROL EXPERIMENTS OUTLINEDBELOW.
Successful completion of the control experiments will give the user an appreciation for: the yield and qualityof PCR product obtained with their amplification system, the amount of the user’s amplified DNA that
optimally matches the recommended amount of Surveyor Nuclease S in Appendix A, and the expectedamount and appearance of digestion products on an agarose or polyacrylamide gel.
Use Control G and Control C as follows.
1. Amplify 2 µL of Control G and 2 x 2 µL of Control C DNA in separate 50 µL reactions using a
proofreading DNA polymerase and the PCR program described in Appendix A: Preparing PCR Products
using Optimase Polymerase (Step 4) where Ta = 65°C and the 72°C extension time is appropriate forthe polymerase used, e.g. 1 min for Optimase Polymerase.
2. After amplification, analyze a 2 µL aliquot of each amplified DNA and different amounts of a DNA massladder [e.g., 100 bp DNA Ladder from New England BioLabs] on a 2% high resolution agarose gel or a
10% polyacrylamide gel and compare the band intensities with those of the DNA ladder to establish
the DNA concentration. See Appendix B: Agarose Gel Electrophoresis or Appendix C: Polyacrylamide
Gel Electrophoresis for details. The yield with Optimase Polymerase is in the range of 25–80 ng/µL.

3. Hybridize Control G‐ and Control C‐amplified DNA in equal amounts (15 µL of each if the amplified
DNA concentrations are ≥40 ng/µL) as described in Step 2—DNA Hybridization. This produces a
population of molecules containing 50% homoduplex, 25% heteroduplex with a C/C mismatch, and
25% heteroduplex with a G/G mismatch. Also self‐anneal 30 µL of Control C homoduplex in a separate
tube.
4. Digest hybridized Control G/C and Control C homoduplex with Surveyor Nuclease S. Follow the
instructions in Step 3—Treatment with Surveyor Nuclease to identify the Surveyor Nuclease reaction
parameters, i.e. requirements for additional MgCl2 and/or enzyme, that match the PCR DNA
polymerase/buffer formulation used to prepare the DNA substrate. Further optimization can beachieved by carrying out a pilot study matching amount of Control C and Control G/C to therecommended amount of Surveyor Nuclease. An example of this for Optimase Polymerase PCR
products is shown in Table 1. Set up 6, 0.5 mL reaction tubes on ice with the indicated amounts of PCR
product in Table 1 and the amount of Surveyor Nuclease and additional MgCl2 determined as
described in Step 3. Incubate the tubes at 42°C for 60 min. Add 1/10th volume of Stop Solution.
5. Analyze the digested DNA in each reaction mixture as described in Step 4—Analysis of DNA
Fragments. Load all the samples on the gel. Program the autosampler to inject 6, 12, and 24 μL fromtube number 1 and 4, 2 and 5, and 3 and 6, respectively.
Once you have prepared the PCR products, continue with Step 2—DNA Hybridization.

Step 2—DNA Duplex Formation
In this step, sample and reference DNA are mixed and hybridized to form heteroduplexes. Make sure to
take along sample and/or reference DNA alone in separate tubes to serve as controls. The use of a heated‐lid
thermocycler is recommended—it is important to carefully follow the protocol shown here.
If your thermocycler cannot be programmed appropriately for hybridization or if it lacks a heated lid, go to the
Performing Heteroduplex Formation without a Thermocycler section below.

Performing Heteroduplex Formation using a Thermocycler
1. Mix equal amounts of test sample and reference PCR products in a 0.2 mL tube. Place reference DNA
alone in a separate 0.2 mL tube. For efficient annealing the final volume should be at least 10 µL.
Note the following:

The concentration of test sample DNA and wild‐type reference DNA should be in the range
25–80 ng/µL (ideally 50 ng/µL). About 200–400 ng of hybridized DNA is recommended fortreatment with Surveyor Nuclease S, so that each tube should contain at ≥200 ng total DNA.

Heterogeneous test sample DNA that does not require the addition of wild‐type, or reference
DNA should still undergo the following hybridization procedure to ensure the formation ofcleavable mismatches.
2. Place the tube in a thermocycler and run the following program:Temperature
95°C
95°C to 85°C
85°C
85°C to 75°C
75°C
75°C to 65°C
65°C
65°C to 55°C
55°C
55°C to 45°C
45°C
45°C to 35°C
35°C
35°C to 25°C
25°C
4°C

Time
10 minTemperature ramp(–2.0°C/s)

1 min(–0.3°C/s)
1 min(–0.3°C/s)
1 min(–0.3°C/s)
1 min(–0.3°C/s)
1 min(–0.3°C/s)
1 min(–0.3°C/s)
1 min
Hold ∞The product is now ready to be treated with Surveyor Nuclease for heteroduplex analysis. Continue with Step
3—Treatment with Surveyor Nuclease.

Performing Heteroduplex Formation without a Thermocycler
To perform heteroduplex formation without a thermocycler:
1. Mix in equal quantities of the two PCR products to generate the heteroduplex. Set up reference DNA

in a separate tube as above.
2. Incubate the mixture at 95°C for 5 min in a 1‐liter beaker filled with 800 mL of water and then allow
the water to come to <30°C.
Note:
Because of evaporation of liquid at the tube bottom and condensation under the tube lid, the volume in a tube
should be ≥20 µL so that sufficient volume is present to prevent the concentrations of constituents in the
mixture from changing substantially during the hybridization step.
3. Spin the tube contents to the bottom of the tube and mix.
The product is now ready to be treated with Surveyor Nuclease for heteroduplex analysis.
Continue with Step 3—Treatment with Surveyor Nuclease.

Step 3—Treatment with Surveyor Nuclease
In this step, the heteroduplex test sample DNA from Step 2 is cleaved by the Surveyor Nuclease, along withnecessary reference DNA and controls.
Optimized Mg2+ concentration is essential to Surveyor Nuclease function. If a different PCR DNA
polymerase is used to amplify the DNA, consult the table in Appendix A for appropriate Surveyor Nuclease
reaction conditions to digest the DNA. If the DNA polymerase you are using is not listed in the Appendix A
table, consult the instructions in Appendix A to select the appropriate reaction conditions.
Digest the hetero/homoduplex DNA experimental samples and any reference DNA in separate tubes.
Set up Surveyor Nuclease reactions for Control G and Control C duplexes as follows:
Volume Required (µL)
Tube
No.
1
2
3
4
5
6

Hybridized
Control
G/C
‐
‐
‐
6
12
18

Hybridized
Control C
6
12
18
‐
‐
‐

0.15 M
MgCl2
0.6
1.2
1.8
0.6
1.2
1.8

Surveyor
Enhancer S

1
1
1
1
1
1

Stop Solution(Add after
incubation)

Surveyor
Nuclease S

1
1
1
1
1
1

0.9
1.5
2.2
0.9
1.5
2.2

Set up Surveyor Nuclease reactions for experimental sample DNA as follows:
1. The volumes of 0.15 M MgCl2 Solution required are calculated based upon the volume (V) of the PCR
product used.
2. For each digestion, add the following components in the order shown to a nuclease‐free 0.2 mL tube
(kept on ice):
Component

AmountHybridized DNA1

200–400 ng

0.15 M MgCl2 Solution2

1/10th volume

Surveyor Enhancer S

1 µL

Surveyor Nuclease S

1 µL

1
2

DNA volume should be 8–40 µL
This additional magnesium is required even if PCR buffer also contains some magnesium

3. Mix by vortexing gently, by agitation or by aspiration/expulsion in a pipette tip using a micro pipetter.

4. Incubate at 42°C for 60 min.


We recommend staring with a 60 min incubation time at 42C. In some cases, reaction time
can be reduced to 20 minutes. Increasing reaction time beyond 60 min will increase DNHA
degradation due to exonuclease activity of the Surveyor enzyme

Add 1/10th volume of Stop Solution and mix. Store the digestion products at –20°C if not analyzed immediately.
Note:DNA prepared with some DNA polymerase/buffer formulations requires double the amount ofSurveyor Nuclease S (2 µL; see APPENDIX A table). The amount of Enhancer S used should be
kept at 1 µL in all cases.

To reduce the number of manipulations, MgCl2, Surveyor Enhancer S and Nuclease S can bemixed and a single pipetting can be done. This mixture should be used immediately after
preparation and should not be stored, since reducing agent in the Enhancer storage buffer willinactivate Surveyor Nuclease over time.

When a heterogeneous DNA sample is analyzed, a portion of the hybridized heterogeneous DNA
is NOT digested with Surveyor Nuclease and is run as a control in Step 4—Analysis of DNA
Fragments.

Continue with Step 4—Analysis of DNA Fragments

Step 4—Analysis of DNA Fragments
To analyze Surveyor Nuclease digestion products on the WAVE or WAVE HS System:
1. Transfer the 0.2 mL tube with the Surveyor Nuclease digestion mixture to the Autosampler of a WAVE
or WAVE HS System. Digests of control or undigested control DNA and digests of mixtures ofheteroduplexes and homoduplexes need to be analyzed in parallel. A sizing standard such as a 100 bp
DNA ladder should also be run in parallel.
2. Set up methods to inject 300 to 500 ng of Surveyor Nuclease digested DNA into a DNASep or
DNASep HT Cartridge. Injection volumes exceeding 22 μL may require a 500 μL syringe in the
Autosampler.
3. Run the following program
Application Type:DS Multiple Fragments

Oven Temperature:

50°C

Min/100 bp:

2.5

Number of Segments:

5

Base Pair Minimum:

30 bp

Base Pair Maximum:

Amplicon Size + 50 bp

Clean Duration:

0.5

Equilibrium Duration:

0.9

Flow rate:

0.9 mL/min

Dyes:

WAVE‐HS 1

Clean Type:

ActiveInjection Type:All

Sequence Context:

None

Amplicon:

None

Eluent A:

WAVE Optimized® Buffer A

Eluent B:

WAVE Optimized Buffer B

Detection:

260 nm for UV; 492 nm Ex and 526 nm Em for
Fluorescence Detection (using High Sensitivity
Detection with the WAVE HS system)

4. Run the samples.
5. Analyze the chromatograms.

Washing Procedure for DNASep and DNASep HT Cartridges used toAnalyze Surveyor Nuclease Digests
When injecting Surveyor Nuclease digests onto DNASep and DNASep HT Cartridges in a WAVE System, therecommended cleaning option is either ACTIVE CLEAN or FAST CLEAN. A NORMAL CLEAN is not sufficient.

Note the following:Every 100 injections of Surveyor Nuclease digests should be followed by a HOT WASH. To perform a
HOT WASH, at 80°C pump 100% Solution D (75% ACN) for 15 minutes, followed by 50%/50% buffer
A/B for 30 minutes through the system. This HOT WASH should also be performed at the end of eachbatch of Surveyor Nuclease digest runs irrespective of whether 100 injections have been reached ornot, e.g. when temporary cessation of cartridge use or a change to DHPLC analysis is anticipated.

After every 500 injections of Surveyor Nuclease digests the in‐line filter must be changed. The in‐line filter should also be changed if the pressure becomes too high (>2300 PSI for DNASep and>1500 PSI for DNASep HT Cartridge).

After every 500 injections a REVERSE HOT WASH should be performed. Refer to Performing a
REVERSE HOT WASH on a DNASep or DNASep HT Cartridge section below.
WARNING! Failure to follow these procedures will lead to high column pressure and deterioratingcolumn performance.

Performing a REVERSE HOT WASH on a DNASep or DNASep HT Cartridge
To perform a REVERSE HOT WASH:
1. Remove the DNASep Cartridge or DNASep HT Cartridge and reinsert it in the reverse orientation in
the flow path.
2. Remove the in‐line filter and substitute it with a union during the REVERSE HOT WASH.
3. Pump 100% Solution D (75% ACN) for 30 minutes at 80°C through the system at a flow rate of 0.9
mL/min.
4. Pump 50%/50% buffer A/B for 1 hour at 80°C through the system at a flow rate of 0.9 mL/min.
5. Remove the union and insert a new in‐line filter.
This REVERSE HOT WASH should be performed without the in‐line filter. Insert a new in‐line filter after the
REVERSE HOT WASH and run the cartridge in the reverse direction for the next 500 injections.

Surveyor Nuclease digestion of hybridized Control G/C PCR products gives rise to two cleavage products,
217 and 416 bp in size, which are clearly distinguishable by WAVE analysis as shown in Figures 6 and 7.
Analysis of different amounts of substrate digested with the recommended amount of Surveyor Nuclease
W provides an opportunity to establish the optimal ratio of enzyme to DNA with DNA amplified with your
PCR enzyme and the optimal amount of DNA to inject. The optimal conditions produce the maximum
amount of cleavage products while maintaining low background. In Figures 6 and 7, the digestion andinjection conditions for tube 6 gave the best results. The amounts of digestion products produced in the 6,
12 and 24 µL digestion of Control G/C substrate were approximately proportional to the amount of
substrate present. Under the reaction conditions used, 1 µL of Surveyor Nuclease W contained sufficientmismatch cutting activity to continue to cut the mismatches in 1200 ng substrate during a 60 minuteincubation. Keep in mind that PCR amplification of a plasmid template such as Control G or C will result inhigher yields of DNA product than amplification of a comparable amount of genomic DNA template. Using
5–10 times more genomic DNA (50–100 ng) will give comparable yields of PCR product. This should betaken into consideration in preparing and digesting DNA amplified from genomic DNA.

Figure 6. Survveyor Nuclease digestiond
produccts of self‐anneaaled Control C hoomoduplex (HMMD) and Controll G/C
homoduplex//heteroduplex (HHTD). The 633 bp amplicons werre PCR amplifiedd with Optimasee Polymerase froom 2 μL of Contro
ol G and Control
C. Control G/C
C homoduplex/heteroduplex waas formed by hyb
bridizing equal aamounts of Control G and Contro
ol C homodupleex PCR productand contains homoduplexeshanda C/C and G/G mismatched heeteroduplexes. D
DNA (300 ng, 600 ng, and 1200 nng) was digested
d with 1 µL oftth
Surveyor Nucclease W, 1 μL off Surveyor Enhanncer W2, 1/10th volume 0.15 M M
ncer Cofactor forr 60 min at
MgCl2 and 1/10 volume Enhan
42°C. Surveyo
or Nuclease digestion products [1180 (6 µL), 400 (12( µL), and 850 ng (24 µL)] weree analyzed usingg the WAVE Systeem run undernon‐denaturing conditions att 50°C with UV deetection. Transgenomic 100 bp LLadder DNA wass run as a marker. The 217 and 4416 bp cleavage
products expe
ected from the Control
C
G/C heteroduplex are cle
early visible. Alsoo visible in all three chromatograams is the full‐leength 633 bp
homoduplex.

Figure 6. Surveyor Nucleasee digestion products of self‐anne
ealed Control C hhomoduplex (HHMD) and Contro
ol G/C
homoduplex/heteroduplexx (HTD). The 633 bp amplicons were PCR amplifi ed with Optimasse Polymerase frrom 2 μL of Conttrol G
and Control C. Control G/C homoduplex/heeteroduplex was formed by hybr idizing equal am
mounts of Contro
ol G and Control C
homoduplexx PCR product an
nd contains homoduplexes and C/C
C and G/G mis matched heterroduplexes. DNA
A (300 ng, 600 ngg, and1200 ng) waas digested with 1 µL of Surveyorr Nuclease W, 1 μL of Surveyor EEnhancer W2,1//10th volume of 00.15 M MgCl2 annd
1/10th volum
me of Enhancer CofactorC
for 60 min
m at 42°C. Survveyor Nuclease ddigestion produccts [180 (6 µL), 4400 (12 µL) and 8850 ng(24 µL)] werre analyzed usingg the WAVE Systtem run under non‐denaturingncconditions at 50°°C and equippedd with a Fluorescence
Detector and a High‐Sensitivvity Accessory foor post‐column DNA
D intercalationn with fluorescent dye. Transgen
nomic 100 bp Laadder
DNA was run as a marker. Th
he 217 and 416 bp cleavage prod
ducts expected from the Controol G/C heterodup
plex are clearly vvisible.
Also visible in all three chrom
matograms is th
he full‐length 633
3 bp homodupleex. Flat‐top peaks were produced
d by injection ofamounts of DNA that saturaated the instrum
ment detector.

20

Appendix A: Preparing PCR Products using Optimase Polymerase
To prepare PCR products using Optimase Polymerase:
1. Isolate test sample and wild‐type (reference) DNA by standard methods.
2. Add the following components to each of the two 0.2 mL tubes (kept on ice). One tube will be
used for test sample DNA and the other for reference DNA:

Sterile, deionized water sufficient to bring the final volume to 50 μL



5 μL 10X Optimase Polymerase Buffer

Test sample or reference DNA (10 ng plasmid DNA or 100 ng genomic DNA)

4 μL dNTPs (2.5 mM each of dTTP, dATP, dCTP and dGTP; final concentration of eachdNTP is 0.2 mM)

15 picomoles sense primer (~120 ng of a 25‐mer)

15 picomoles antisense primer



1 μL Optimase Polymerase (2.5 units)

3. Set up PCR amplification using one of the following methods:
IF
You want to use
Optimase
ProtocolWriter™ todesign a PCR
protocolTHEN
1. Access www.mutationdiscovery.com.
2. On MutationDiscovery.com, click on
ProtocolWriter under Optimase. The
ProtocolWriter entry window appears.
3. Enter the appropriate information and click theDevelop PCR Protocol button. The PCR protocol
appears for a heated‐lid thermocycler. Perform PCR.
4. Go to step 5.

You want tocalculate theannealing
temperature

1. Calculate the Tm for your primers using the IDT

OligoAnalyzer® tool at www.idtdna.com/SciTools.
Enter the primer sequences and the exact PCR
conditions into the tool.
2. The Ta is estimated as 5°C below the lowest Tm of

the PCR primer pair. Optimization of primer Tm may
be necessary.
3. Proceed to step 4

TIP: Please use the Optimase MasterMix Calculator at http://www.mutationdiscovery.com toprepare a master mix for your PCR reaction.
4 . Use the following program for a heated‐lid thermocycler:
94°C

2 min

1 Cycle

94°C

30 s

30 Cycles

Ta°C

30 s

72°C

30 s per 250 bp

72°C

5 min

1 Cycle

4°C

∞

Hold

5. Analyze 2–5 μL aliquots of each product by electrophoresis in a 2% (w/v) agarose gel,prepared with high‐resolution agarose and cast in 1X TBE [89 mM Tris‐Borate (pH 8.3), 1 mM
EDTA] + 0.2 μg/mL ethidium bromide. Add 1/6 volume of a 6X loading dye buffer [10 mM Tris‐
HCl (pH 8.0), 10 mM EDTA (pH 8.0), 50% (w/v) sucrose, 0.15% (w/v) bromophenol blue] or
your loading dye buffer of choice to the aliquot and mix. Run the gel in 1X TBE at 5 V/cm until
the bromophenol blue has run 2/3rd of the length of the gel. Run several different amounts ofa mass DNA ladder, such as a 100 bp DNA Ladder (New England BioLabs, Beverly, MA), as areference.
6. Visualize the DNA bands using a UV transilluminator at 250–300 nm and photograph the gel.
7. Use the ladder to estimate the concentration of the amplified DNA by visual inspection. If asingle band is visible in each sample, proceed; if not, consider optimizing the PCR further as
already described in Step 1—PCR Amplification of Reference and Test Samples. The DNA
concentration is ideally ~50 ng/μL, but should be in the range of 15–80 ng/μL.
The amplified DNA can be used without further purification. Alternatively, the DNA can beconcentrated by ethanol precipitation. To precipitate DNA, transfer the reaction mixtures tomicrocentrifuge tubes that can be centrifuged at high speed. Add 2.5 volumes of ethanol and store
the tubes at –20° C for 30 min. Centrifuge the tubes at 13,000 rpm for 10 min in a
microcentrifuge. Carefully remove the ethanol with a micropipetter, being sure not to disturb theinvisible pellet on the tube sidewall and bottom. Concentrated PCR products are suspended in 1X PCR
buffer. Estimate the DNA concentration on an agarose gel as described above.

Appendix B: Surveyor Nuclease Reaction Conditions
Because amplified PCR products are hybridized and digested with Surveyor Nuclease directly in 1X PCR
buffer, careful consideration must be given to the 1X PCR buffer composition. Consult Table B1 in thissection for the Surveyor Nuclease reaction conditions recommended for use with several commercial
DNA polymerase/buffer formulations. If your DNA polymerase/buffer formulation is not in the table
use the following recommendations.
Read the manufacturer’s literature to determine the salt concentration of the 1X PCR buffer
before carrying out the hybridization step. The salt concentration in the PCR product solution should
be in the range of 50 to 75 mM to ensure that complete annealing of complementary DNA strands
takes place. Reaction buffers used for most PCR DNA polymerases do not require additional salt. For
example a PCR buffer for a Taq polymerase is typically 10 mM Tris‐HCl, 50 mM KCl and 1.5 mM MgCl2;
for Optimase Polymerase the 1X PCR buffer consists of 10 mM Tris‐HCl, 75 mM KCl and 1.5 mM
MgCl2. Therefore PCR products amplified with these buffers can be annealed directly without theaddition of KCl.
However, if the PCR product was prepared with a low salt PCR buffer, sufficient 0.5 M KCl should be
added to adjust the final KCl concentration to be within the range of 50 to 75 mM. Please note that KCl
concentrations above 75 mM inhibit the Surveyor Nuclease; therefore it is critical that KCl be added
only when it is required.
Read the manufacturer’s literature to determine the other constituents of the 1X PCR buffer before
carrying out the Surveyor Nuclease digestion step. The constituents of most 1X PCR buffers support
efficient digestion of heteroduplex DNA by Surveyor Nuclease. These include 10–20 mM Tris‐HCl or Tris‐
SO4 (pH 8.8 to 9.3), 50–75 mM KCl, 1–3 mM MgCl2 or MgSO4, 0.1%–1% (v/v) nonionic detergent and BSAor gelatin. (NH4)2SO4 at 10–20 mM is also acceptable as long as the KCl concentration is ≤50 mM. PCR
additives such as DMSO (5% v/v), glycerol (10% v/v), betaine (1 M) and 1X PCRx Enhancer (Invitrogen)are acceptable at or below the concentrations listed. If any of these additives are present in the PCR
reaction mixture at higher concentrations than those listed, Surveyor Nuclease mismatch cutting
activity will be inhibited.
If an additive is present at an inhibitory concentration, we suggest cleaning up the PCR product. Use
ethanol precipitation (see Preparing PCR Products section) or a commercial PCR clean‐up column before
the hybridization step. The DNA should then be placed in a buffer compatible with the hybridizationand Surveyor Nuclease digestion steps, such as 10 mM Tris‐HCl (pH 8.8), 15 mM MgCl2, and 50 mM
KCl. After hybridization in this optimal buffer, digest the DNA with 1 µL of Surveyor Nuclease W, 1 µL
of Surveyor Enhancer W2 and 1/10th volume of Enhancer Cofactor at 42°C for 60 min.
If the DNA polymerase/buffer formulation is not listed in the table and the manufacturer does not reveal
the contents of the PCR buffer, there are several options. Clean up the DNA and proceed as described in
the paragraph immediately above. Alternatively, carry out pilot Surveyor Nuclease digestion studies
with Control G/C or genomic DNA containing a known mismatch amplified with your DNA
polymerase to establish empirically the best digestion conditions. Digest the hybridized PCR productin 1X PCR buffer with and without additional Mg2+ with 1 or 2 µL of Surveyor Nuclease, 1 µL of Enhancer
W2 and 1/10th volume of Enhancer Cofactor for 60 min at 42°C (see Step 3—Treatment with Surveyor
Nuclease and Control Experiments—Using Control G and Control C Plasmid DNA).

Note: Injection of 1X PCR buffers other than those used with Optimase, Maximase™, or T‐Taq DNA
Polymerases can rapidly degrade the performance of DNASep and DNASep HT cartridges. The
cartridges are not warranted for injection of such samples.
NOTE: Information about competitor products is included for guidance only and should in no way beconstrued or implied as a endorsement or recommendation.
Table B1 Surveyor Nuclease Reaction Conditions Recommended for use with Various PCR DNA
Polymerase/Buffer Formulations
EnzymeOptimal Surveyor Nuclease
Reaction Conditionsa

Transgenomic T‐Taq DNA Polymerase
Transgenomic Optimase Polymerase
Transgenomic Maximase DNA Polymerase
ABI AmpliTaq® DNA Polymerase
ABI GeneAmp® Fast PCR Master Mix
Bio‐Rad iProof™ HF DNA Polymerase + HF Buffer
Denville Hot Start Taq
Epicentre FailSafe™ PCR 2X PreMix A through L
GeneChoice® AccuPol™ DNA Polymerase
Invitrogen Platinum® Taq DNA Polymerase
NEB Taq DNA Polymerase
NEB Phusion™ High‐Fidelity DNA Polymerase + HF Buffer
Novagen NovaTaq™ DNA Polymerase
Roche Taq DNA Polymerase
Roche FastStart Taq DNA Polymerase
Roche FastStart Taq DNA Polymerase + Q Solution
Roche Expand High FidelityPLUS PCR System
Sigma JumpStart™ Taq DNA Polymerase
Stratagene SureStart™ Taq DNA Polymerase
Stratagene PfuTurbo® Hotstart DNA Pol.
Stratagene PfuUltra™ Hotstart DNA Pol.
Takara Taq™ DNA Polymerase
Takara Ex Taq™ DNA Polymerase

1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+b
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Less Mg2+c
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+
1 µL SNW/Add Mg2+

EnzymeABI GeneAmp® High Fidelity PCR Kit
Invitrogen Platinum Taq DNA Polymerase High
Fidelity
Novagen KOD XL DNA Polymerase
Promega GoTaq® DNA Polymerase
Qiagen HotStarTaq® DNA Polymerase
Qiagen HotStarTaq DNA Polymerase + Q‐Solution
Qiagen ProofStart® DNA Polymerase
Qiagen ProofStart DNA Polymerase + Q‐Solution
Roche Expand™ High Fidelity PCR System
Stratagene PfuUltra™ II Fusion HS Polymerase
Takara E2TAK™ DNA Polymerase
Takara PrimeSTAR® HS DNA Polymerase

2 µL SNW/Add Mg2+
2 µL SNW/Add Mg2+
2 µL SNW/Add Mg2+
2 µL SNW/Add Mg2+
2 µL SNW/Add Mg2+ (Not
Recommended)d2 µL SNW/Add Mg2+ (Not
Recommended)
2 µL SNW/Add Mg2+
2 µL SNW/Add Mg2+
2 µL SNW/Add Less Mg2+
2 µL SNW/Add Mg2+ (Not
Recommended)

Invitrogen Platinum Pfx DNA Polymerase + PCRx
Enhancer
NEB Phusion High‐Fidelity DNA Polymerase + GC Buffer

1 µL SNW/Add No Mg2+f
1 µL SNW/Add No Mg2+
1 µL SNW/Add No Mg2+ (Not
Recommended)
1 µL SNW/Add No Mg2+ (Not
Recommended)
1 µL SNW/Add No Mg2+

Sigma AccuTaq™ LA DNA Polymerase
Takara LA Taq™ DNA Polymerase

1 µL SNW/Add No Mg2+
1 µL SNW/Add No Mg2+

Bio‐Rad iProof HF DNA Polymerase + GC Buffer
Eppendorf HotMaster™ Taq DNA Polymerase
Invitrogen Platinum Pfx DNA PolymeraseaOptimal Surveyor Nuclease
Reaction Conditionsa
2 µL SNW/Add Mg2+
2 µL SNW/Add Mg2+

All reactions should contain the indicated volume of Surveyor Nuclease W (SNW), 1/10th volume
Enhancer Cofactor and 1 µL Surveyor Enhancer W2b
Add Mg2+ unless indicated otherwise add 1/10th volume of 0.15 M MgCl2 to reactions as
described in Step 3
c
FailSafe PCR PreMixes contain high Mg2+ concentrations; add only 1/20th volume of 0.15 M
MgCl2 to reactionsd
Not Recommended—reaction conditions could not be found that produced acceptable amounts ofmismatch digestion products with acceptable background; the reaction conditions specified were thebest identifiede
E2TAK DNA Polymerase 1X PCR reaction buffer appears to contain a high Mg2+
concentration; add only 1/40th volume of 0.15 M MgCl2 to reactionsf
Add No Mg2+ do not add any 0.15 M MgCl2 to these reactions

Appendix C: Troubleshooting
Effective use of the Surveyor PLUS Mutation Detection Kit depends upon successful completion of anumber of steps. One of the most critical is PCR amplification that must result in the production of
specific, uniform‐sized DNAs in sufficient quantity to be detected after hybridization and cleavage. Also
critical is matching the amount of DNA and Surveyor Nuclease used. If you are a first‐time user, you
should process the control DNAs provided through all the steps as described in Control Experiments—
Using Control G and Control C Plasmid DNA.
The Control DNAs should be used also to troubleshoot various steps in the procedure.
This appendix section covers a list of issues that you might encounter when using the Surveyor PLUS
Mutation Detection Kit and how to resolve them.

Problem 1—Low PCR yield or no PCR product
POSSIBLE CAUSE
Not enough template
and/or too few cycles
Suboptimal PCRparameters

Suboptimal DNA
polymerase for target

SOLUTION
Increase the template concentration and/or add more
PCR cycles.
Do one or more of the following: Decrease the annealing temperature inincrements of 2°C.
 Increase the extension time. For Optimase
Polymerase, use 30 sec per 250 bp.
 Adjust the Mg2+ concentration
Use a “hot‐start” PCR DNA polymerase.
Increase the amount of DNA polymerase.

Problem 2—Multiple PCR products
POSSIBLE CAUSE
Poor primer design
Annealing temperature
too low
Extension time too long
Cycle number too highSuboptimal PCR
conditions

SOLUTION
Redesign primers to improve specificity, Tm, and GC
content.
Increase the annealing temperature in increments of 2°C.
Reduce the extension time. For Optimase
Polymerase, use 30 sec per 250 bp.
Reduce cycle number in increments of 2.
Use “hot‐start” PCR and/or touchdown PCR or
perform nested PCR.

Problem 3—No cleavage products observed upon analysis after Surveyor
Nuclease treatment of known heteroduplex
POSSIBLE CAUSE
Proportion of mismatchtarget too low
Cleavage site too close to
PCR product end
Inactive Surveyor
Nuclease
Too little enzyme
Too little substrate
Incompatible 1X PCR
buffer

SOLUTION
Mix equal amounts of test and reference DNA before
annealing.
Redesign the primer set to move the target site awayfrom ends.
Perform the Control reaction to verify enzyme
performance.
Increase the amount of Surveyor Nuclease 2‐fold andrepeat digestion.
Concentrate the PCR products by ethanol
precipitation before annealing.
Consult Appendix B for suggested courses of action

Problem 4—High background after Surveyor Nuclease treatment
POSSIBLE CAUSE
Suboptimal hybridization
step

Errors introduced by
PCR enzyme
Incompatible 1X PCR
bufferToo much Surveyor
Nuclease
DNA amount too low
Nonspecific PCR
products
Enhancer W2 has lost
activity

SOLUTION
Do the following:
1. Make sure the DNA concentration is in therange of >25 ng/μL to <50 ng/μL.
2. Repeat the hybridization step, taking care tocool the annealing mixture slowly.
3. Add 1X PCR reaction buffer to precipitatedproducts before annealing.
4. Use sufficient sample volume (>20 μL) duringhybridization in a water bath.
5. Add 1/10 volume 0.5 M KCl to PCR productcontained in low salt 1X PCR reaction buffer
Use a high fidelity PCR enzyme, such as Optimase
Polymerase.
Consult Appendix B for suggested courses of action
Reduce the Surveyor Nuclease 2 fold and repeat
digestion.
Increase the DNA amount to at least 400 ng of substrate
per 1 μL of Surveyor Nuclease W used.
Optimize the PCR parameters to increase specificity.
Always use an appropriate substrate as a control toidentify background.
Increase the amount of Enhancer W2 2‐fold andrepeat digestion.

References
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