Surveyor KIT

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www.transgenomic.com User Guide for the Transgenomic SURVEYOR™ Check-It Kit for Clone Sequence Validation

Transcript of Surveyor KIT

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www.transgenomic.com

User Guide for the �

Transgenomic SURVEYOR™ Check-It Kit �

for Clone Sequence Validation

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For technical support and/or technical tips, please contact Transgenomic Technical �Support:

US – Toll Free (888) 233-9283 or 402-452-5400UK – +44 1670-732-992Europe – +33 1-30-68-90-00

E-mail: [email protected]

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Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

SURVEYOR Check-It Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Detecting Mutations with the Transgenomic SURVEYOR Check-It Kit . . . . . . . . . . . . . 2An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Factors Affecting the Quality of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Noise (Background) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Step-by-Step Instructions Detecting Mutations with the SURVEYOR Check-It Kit . . . . . . 5Step 1 – PCR Amplification of Reference and Test Samples . . . . . . . . . . . . . . . . . . 5

Amplification of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Preparing PCR Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Step 2 – DNA Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Performing Heteroduplex Formation using a Thermocycler . . . . . . . . . . . . . . . . . 9Performing Heteroduplex Formation without a Thermocycler . . . . . . . . . . . . . . . 9

Step 3 – Treatment with SURVEYOR Nuclease . . . . . . . . . . . . . . . . . . . . . . . 10Step 4 – Analysis of DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Control Experiments – Using Control G and Control C Plasmid DNA . . . . . . . . . . . . . 11

Confirmation of Mutations Introduced by Site-Directed Mutagenesis . . . . . . . . . . . . . . 15

Appendix A: Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Preparing Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Running Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Photographing Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Appendix B: Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Problem 1 – Low PCR yield or no PCR product . . . . . . . . . . . . . . . . . . . . . . . . 19Problem 2 – Multiple PCR products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Problem 3 – No cleavage products observed upon analysis after SURVEYOR Nuclease treatment of known heteroduplex . . . . . . . . . . . . . . . . 20Problem 4 – High background after SURVEYOR Nuclease treatment . . . . . . . . . . . . 21

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

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IntroductionThe Transgenomic SURVEYOR™ Check-It Kit for Clone Sequence Validation has beendesigned to detect mutations in cloned DNA using a new mismatch-specific plantendonuclease.

Key applications and benefits of using the SURVEYOR Check-It Kit include:

• Identification of error-free cDNA or genomic DNA clones that have been generated usingPCR-based cloning methods

• Verification that clones modified specifically by site-directed mutagenesis contain thedesired mutation and lack undesired mutations

• Elimination of the need to isolate and sequence DNA from multiple colonies

PCR-based cloning and site-directed mutagenesis are established methods that requireconfirmation that the cloned insert possesses the desired sequence. This confirmation andidentification step usually requires direct DNA sequencing of the insert DNA in multipleclones. Sequencing is necessary either to confirm the presence of the desired sequence and theabsence of PCR or cloning errors, or to verify that the desired mutation has been properlyintroduced and that unwanted mutations are absent.

Colonies generated by cDNA or genomic DNA cloning, or derived from site-directedmutagenesis experiments can be easily checked directly to determine whether unwantedmutations are absent, or whether desired mutations are present. This eliminates the need toisolate and sequence DNA from multiple single colonies. The kit is designed to digestunlabeled DNA with SURVEYOR Nuclease for subsequent analysis by agarose gel electro-phoresis.

SURVEYOR Nuclease, the key component of the kit, is a member of a new family ofmismatch-specific plant endonucleases that cleaves DNA heteroduplexes at mismatch sites1,2.These DNA endonucleases cut both strands of a DNA heteroduplex on the 3’-side of themismatch site3,4. Insertion/deletion mismatches and all base-substitution mismatches arerecognized, but the efficiency of cleavage varies with the sequence of the mismatch1,4.SURVEYOR Nuclease has been used to verify the presence of known mutations in a numberof genes in human peripheral blood DNA5, to carry out screening for induced point mutationsin barley6, and to screen for error-free clones generated from a plant cDNA library by PCR-based cloning7.

FOR SUCCESSFUL USE OF THIS KIT, WE RECOMMEND STRONGLY THAT YOUREAD THIS MANUAL THOROUGHLY AND FOLLOW THE INSTRUCTIONS ANDGUIDELINES PROVIDED.

If you have further questions or need assistance, please contact Transgenomic TechnicalSupport Hotline/Help Desk:

US – Toll Free (888) 233-WAVE (9283) or 402-452-5400

UK - +44 1670-732-992

Europe - +33 1-30-68-90-00

Technical Support E-mail: [email protected]

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SURVEYOR Check-It Kit ComponentsThe SURVEYOR Check-It Kit (Catalog No. 706040) has been designed for 100 reactions.

Component Amount Provided

100 Reactions

SURVEYOR Nuclease S 4 x 0.03 mLStop Solution 0.25 mLControl C 0.02 mLControl G 0.02 mL

Store all components at –20 ºC.

Detecting Mutations with the Transgenomic SURVEYOR Check-It Kit

An OverviewMutation detection and confirmation with SURVEYOR Nuclease involves four steps:

Step 1 – Preparation of PCR amplicons from mutant (test) and wild-type (reference) DNA.

Step 2 – Mixing equal amounts of test and reference DNA; hybridizing them by heating andcooling the mixture to form hetero- and homoduplexes.

Step 3 –Treatment of the annealed heteroduplex/homoduplex mixture with SURVEYORNuclease. The reference DNA alone, treated similarly, serves as a background control.

Step 4 – Analysis of the DNA fragments by agarose gel electrophoresis. The formation of newcleavage products indicates the presence of a mutation, while their size indicates the locationof the mutation.

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The combination of these four steps is outlined in the flow chart in Figure 1.

Figure 1. A schematic representation of mutation detection using SURVEYOR Nuclease.

Factors Affecting the Quality of Results The two key factors that influence the quality of results are signal and noise (background).

SignalThe magnitude of the signal depends upon

• The amount of DNA analyzed on a gel.

The amount of DNA used as substrate for SURVEYOR Nuclease digestion should be atleast 200 ng and as much as 400 ng. All of the digested DNA should be analyzed duringgel electrophoresis.

• The intensity of the fluorescent signal emitted during transillumination of the dye-intercalated DNA.

We recommend the use of ethidium bromide (EtBr) to stain DNA, but alternativefluorescent dyes such as SYBR Gold can also be used with the appropriate detectionsystem. For EtBr-stained DNA a transilluminator platform emitting light at theappropriate wavelength, i.e. 250 to 300 nm, must be used.

• The sensitivity of the photography system used to capture the image of the gel.

The sensitivities of commercial digital photography systems vary.

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• The quality of the PCR amplified DNA.

The presence of high concentrations of primer-dimers in PCR products dramaticallyinhibits SURVEYOR Nuclease cleavage at mismatch sites. Examine each amplified DNAproduct before digestion by gel electrophoresis to be sure it is a single sharp band of theexpected size.

• The relative proportion of mutant (test) to wild-type (reference) DNA in thehybridized sample.

Whenever possible test and reference PCR products should be hybridized in equalproportion to maximize the amount of heteroduplex DNA available for digestion. Afterhybridization of equal amounts of test and reference DNA, on average half of the resultingDNA molecules will reanneal as homoduplexes and the other half as heteroduplexes; theheteroduplex population will contain two distinct heteroduplexes, each representingapproximately 25% of the total population.

• The efficiency of heteroduplex cleavage.

SURVEYOR Nuclease cleaves heteroduplexes with efficiencies that can vary over a broadrange. However, each substrate with a mismatch will have at least one heteroduplexspecies that will be efficiently cleaved at an ideal rate for analysis as described here.

Noise (Background)

• The digestion pattern obtained by digesting heteroduplex DNA can also reveal thepresence of fragments derived from PCR artifacts e.g., primer-dimers, products fromprimer mis-annealing, and DNA polymerase errors. A nearly identical background shouldbe present in the digestion pattern of reference DNA, and this background can beidentified by visual comparison of test and reference digestion patterns (see Figure 4 and6). When the quality of the PCR product is poor, the background after SURVEYORNuclease digestion can reach a magnitude sufficient to obscure the signal. Examine eachamplified DNA product before digestion by agarose gel electrophoresis to be sure it is asingle sharp band. If it is not, optimize the PCR conditions until you obtain a single-bandquality PCR product.

• SURVEYOR Nuclease has 5’-exonuclease activity that gives rise to non-specificbackground products as digestion time is increased, and as the ratio of enzyme to DNA isincreased. The SURVEYOR Check-It Kit has been designed to set up optimal reactionconditions that keep this background to a minimum.

• Reaction conditions for SURVEYOR Nuclease recommended in published literatureand in an early version of this kit required dilution of DNA substrate into a reactionmixture containing a 1X reaction buffer. Subsequent research has shown thatdigestion of substrate DNA directly in PCR reaction buffer without dilution reducesbackground resulting in improved signal to noise.

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Step-by-Step InstructionsDetecting Mutations with theSURVEYOR Check-It KitThis section provides detailed instructions for the four-step process used for detection ofmutations using the SURVEYOR Check-It Kit.

The step-by-step process described can be stopped subsequent to completion of any step. DNAshould be stored at –20 ºC until the next step is carried out.

Step 1 – PCR Amplification of Reference and Test SamplesTHIS STEP IS CRITICAL TO THE SUCCESS OF THE SURVEYOR NUCLEASEDIGESTION. DO NOT PROCEED UNTIL:

• Your PCR yield is sufficiently high (>25 ng/µL).

• Your PCR product has low background (preferably a single band species of thecorrect size).

The first step in the process involves the preparation of the amplified DNA samples.

Amplification of DNA PCR product from a test sample must be hybridized with PCR product from a reference samplein order to generate potential mismatches for SURVEYOR Nuclease cleavage. Both testsample and reference DNA are amplified with the same primer pair. Test sample and referencePCR products are mixed in a 1:1 ratio to maximize the formation of heteroduplexes duringhybridization.

• For genomic or cDNA gene cloning, amplify the gene or gene segment from genomic orcDNA to generate reference DNA and amplify the gene of interest from 5 to 10 individualcolonies to generate a set of test DNAs.

• For site-directed mutagenesis applications, amplify the gene of interest from theoriginal non-mutated plasmid to generate reference DNA and from 5 to 10 individualcolonies to generate a set of test DNAs.

Several factors must be considered carefully in preparing PCR amplified DNA to be used assubstrate for SURVEYOR Nuclease analysis. Primer placement and amplified productquality and yield are crucial to obtaining good results. The following should be considered:

• Amplified DNA fragments in the size range of 200 to 4,000 bp can be analyzed formutations with SURVEYOR Nuclease on agarose gels. With smaller substrates (<1,000bp), place primers at least 50 bp outside the region of interest to ensure that the cleavageproducts are at least 70 bp, since shorter cleavage products are not resolved easily frompotential PCR product artifacts (e.g. primer dimers) and are not visualized efficiently onagarose gels. For larger substrates (>1,000 bp), it becomes important to place primers at adistance from a potential mutation equivalent to >10% of the length of the substrate, sothat large digestion products can be separated from undigested substrate and the lengthand amount of small digestion products are sufficient to give visible bands.

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• For a new PCR amplicon, the PCR parameters must be optimized carefully. The PCRamplicon should appear as a single sharp band of the expected size when analyzed byagarose gel electrophoresis. Mispriming during PCR amplification can result in theformation of spurious DNA fragments that produce increased background duringSURVEYOR Nuclease digestion. This places a premium on the careful design of primersand optimization of PCR conditions. Use primers that are at least 20 nucleotides long (25to 35 oligomers are preferred), and have a G-C content of 45-60%. If after careful designof primers and optimization of PCR conditions non-specific PCR products persist,consider using a hot-start DNA polymerase, a touchdown PCR protocol, and/or a secondamplification with nested primers.

• Both the amount and concentration of DNA in a SURVEYOR Nuclease reaction mixtureinfluence the efficiency and specificity of SURVEYOR Nuclease digestion. For theamount of enzyme recommended for use in a reaction mixture (1 µL of SURVEYORNuclease S), 200 - 400 ng of substrate at 50 ng/µL is optimal. If DNA product yield is <25ng/µL, consider a second amplification with nested primers or concentrating the DNA byethanol precipitation to increase the DNA concentration.

• For a new PCR amplicon derived directly from cDNA or genomic DNA, the primersequences must be optimized carefully. Primers that do not prime in pseudogenes orsequence repeats for example, can be designed using Primer3 (http://www.broad.mit.edu/cgibin/primer/primer3_www.cgi).

Continue with the Preparing PCR Products section.

Preparing PCR ProductsTo prepare smaller PCR products (<2,000 bp) from E. coli colonies, we recommend T-TaqDNA polymerase (Transgenomic Catalog Number 703105 or 703110). For amplification oflarger fragments in good yield, use a DNA polymerase blend (Taq DNA polymerase plus aproofreading DNA polymerase). We recommend Maximase™ Polymerase (TransgenomicCatalog Number 703205 and 703245):

1 Suspend 5 to 10 individual E. coli colonies from an overnight plate in separate tubes in 10µL of TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) by gentle agitation. After removing therequired amount (about 2 µL) of the suspension for the PCR amplification, add 10volumes of culture medium with antibiotic and store the cell suspension at 4 ºC. Thebacteria are stable for several weeks for replating.

2 Add the following components to each of the required number of 0.2-mL tubes (kept onice). One tube will be used for reference DNA and the others for individual colony DNAs:

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

• 5 µL 10X Taq DNA polymerase reaction buffer (includes MgCl2)

• 2 µL colony suspension or reference DNA (10 ng plasmid DNA or 100 ng genomicDNA or cDNA)

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

• 10 picomoles sense primer (~80 ng of a 25-mer)

• 10 picomoles antisense primer

• 0.5 µL Taq DNA polymerase (2.5 units)

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Note the following:

• The PCR amplified reference DNA must be prepared in sufficient quantity (800-1,500 ngtotal) to hybridize to each colony’s amplified DNA and to itself. Depending upon PCRproduct yield, it may be necessary to set up several PCR amplifications of reference DNA.

• Determine the annealing temperature (Ta) by calculating the Tm for each primer using thefollowing equation:

Tm = 63.728 + (0.41 x %GC) – (600/length)%GC = percentage GC of the primerlength = length of the primer in nucleotidesTa is the average of the two primer Tms + 3 ºC

3 Set up the PCR amplification using the following program for a heated-lid thermocycler:

94 ºC 2 min x 1 cycle

94 ºC 30 sTa ºC 30 s x 35 cycles72 ºC 30 s per 500 bp

72 ºC 5 min4 ºC Hold

4 Analyze 2- to 5-µL aliquots of each product by electrophoresis in the appropriatepercentage agarose gel (see Appendix A: Agarose Gel Electrophoresis). Add 1/6 volumeof 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 own loading dye buffer of choice andmix. Also run several different amounts of a DNA mass ladder, such as a 100-bp DNALadder (New England Biolabs, Beverly, MA), as a reference.

5 Visualize the DNA bands using a UV transilluminator at 254 or 302 nm and photographthe gel.

6 Use the DNA ladder to estimate the concentration of the amplified DNAs by visualinspection. If a single band is visible in each sample, proceed; if not, consider optimizingthe PCR procedure further. The DNA concentration of the PCR product should be >25 µg/µL and is usually in the 40–50 ng/µL range.

7 The amplified DNA can be used without further purification. Alternatively, the DNA canbe concentrated by ethanol precipitation. To precipitate DNA, transfer the reactionmixtures to microcentrifuge tubes that can be centrifuged at high speed. Add 2.5 volumesof ethanol and store the tubes at –20 ºC for 30 min. Centrifuge the tubes at 13,000 rpm for10 min in a microcentrifuge. Carefully remove the ethanol with a micro-pipetter, beingsure not to disturb the invisible pellet on the tube sidewall and bottom. Concentrated PCRproducts are resuspended in a reduced volume of 1X PCR buffer. Estimate the DNAconcentration on an agarose gel as described above.

Once you have prepared the PCR products, continue with Step 2 – DNA Hybridization.

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Step 2 – DNA Hybridization Hybridize each test sample DNA with reference DNA to form potential hetero- andhomoduplexes. Hybridize the reference DNA alone to form a reference control. The use of aheated-lid thermocycler is recommended.

Because amplified PCR products are hybridized and digested with SURVEYOR Nucleasedirectly in 1X PCR buffer, careful consideration must be given to the 1X PCR buffercomposition.

Read the manufacturer’s literature to determine the salt concentration of the 1X PCRbuffer before carrying out the hybridization step. The salt concentration in the PCR productsolution should be in the range of 50 to 75 mM to ensure that complete annealing ofcomplementary DNA strands takes place. Reaction buffers used for most PCR DNApolymerases do not require additional salt. For example a PCR buffer for a Taq polymerase istypically 10 mM Tris-HCl, 50 mM KCl and 1.5 mM MgCl2; for Optimase Polymerase the 1XPCR buffer consists of 10 mM Tris-HCl, 75 mM KCl and 1.5 mM MgCl2. Therefore PCRproducts amplified with these buffers can be annealed directly without the addition of KCl.

However, if the PCR product was prepared with a low salt PCR buffer, sufficient 0.5 M KClshould 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 PCRbuffer before carrying out the SURVEYOR Nuclease digestion step. The constituents ofmost 1X PCR buffers support efficient digestion of heteroduplex DNA by SURVEYORNuclease. These include 10 to 20 mM Tris-HCl or Tris-SO4 (pH 8.3 to 9.3), 50 to 75 mM KCl,1 to 3 mM MgCl2 or MgSO4, 0.1% to 1% nonionic detergent, and BSA or gelatin. (NH4)2SO4at 10 to 20 mM is also acceptable as long as the KCl concentration is <50 mM. PCR additivessuch as DMSO (>5%), glycerol (>10%), betaine (>1 M), and 1X PCRx Enhancer (Invitrogen)inhibit SURVEYOR Nuclease above the concentrations listed. If any of these or otheradditives are present in the PCR reaction mixture, they must be removed before the PCRproduct is treated with SURVEYOR Nuclease.

If additives are present, we suggest using ethanol precipitation to clean up the PCR productbefore the hybridization step (see Preparing PCR Products section). The precipitated DNAshould be dissolved in a 1X PCR buffer compatible with the hybridization and SURVEYORNuclease digestion steps, such as 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, and 50 mM KCl.If the manufacturer does not reveal the contents of the PCR buffer, precipitate the DNAproduct and dissolve it in a compatible PCR buffer.

If you are using a thermocycler, go to the Performing Heteroduplex Formation using aThermocycler section below. If your thermocycler cannot be programmed appropriately forhybridization or if it lacks a heated lid, go to the Performing Heteroduplex Formation withouta Thermocycler section below.

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Performing Heteroduplex Formation using a ThermocyclerTo perform heteroduplex formation using a thermocycler:

1 Mix equal amounts of test sample and reference PCR products in a 0.2-mL tube. Placereference DNA alone in a separate 0.2-mL tube. For efficient annealing the final volumeshould be at least 10 µL.

NOTE the following: The concentration of test sample DNA and reference DNA should be at least 25 ng/µL. About 200 ng of hybridized DNA is treated with SURVEYOR Nuclease S, so that each tube should contain >200 ng total DNA.

2 Place the tubes in a thermocycler and run the following program:

95 ºC 2 min95 ºC to 85 ºC (-2 ºC/s)85 ºC to 25 ºC (-0.1 ºC/s)4 ºC 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 ThermocyclerTo perform heteroduplex formation without a thermocycler:

1 Mix equal masses of the two PCR products intended to generate the heteroduplex asdescribed above. As a control, set up reference DNA in a separate tube as above.

2 Place the tubes in a 1-liter beaker filled with 800 mL of water, preheated to 95 ºC.

3 Incubate the tubes at 95 °C for 5 min and then allow the water to cool to <30 ºC.

NOTE the following: Evaporation of liquid at the tube bottom and condensation inside the tube lid can change the concentrations of constituents in the mixture at the bottom of the tube. To minimize this effect, the volume at the bottom of the tube should be >20 µL.

4 Spin the tube contents to the bottom of the tube. Store the DNA at –20 ºC if it is notdigested immediately.

The products are now ready to be treated with SURVEYOR Nuclease for heteroduplexanalysis. Continue with Step 3 — Treatment with SURVEYOR Nuclease.

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Step 3 – Treatment with SURVEYOR NucleaseOnce you have prepared the test sample/reference hetero/homoduplex mixtures and hybridizedreference control, you must treat them separately with SURVEYOR Nuclease directly inappropriate 1X PCR buffer (for details see Step 2 – DNA Hybridization).

To treat the test/reference mixtures and reference control with SURVEYOR Nuclease:

1 Digest the test/reference DNA samples and reference control DNA in separate tubes.

2 For each digestion, add the following components in the order shown to a nuclease-free0.2-mL tube (kept on ice):

• 200 - 400 ng (4 to 16 µL) hybridized DNA

• 1 µL SURVEYOR Nuclease S

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

4 Incubate at 42 ºC for 20 min.

5 Add 1/10 volume of Stop Solution and mix. Store the digestion products at –20 ºC if theyare not going to be analyzed immediately.

Continue with Step 4 – Analysis of DNA Fragment.

Step 4 – Analysis of DNA FragmentsTo analyze the SURVEYOR Nuclease digestion products by agarose gel electrophoresis:

1 Add 1/6 volume of a 6X loading dye buffer [10 mM Tris-HCl (pH 8.0), 10 mM EDTA,50% (w/v) sucrose, 0.15% (w/v) bromophenol blue] or your own loading dye buffer ofchoice to each SURVEYOR Nuclease reaction mixture and mix.

2 For each digestion/loading dye mixture load all the mixture into the well of an appropriatepercentage agarose gel (see Appendix A: Agarose Gel Electrophoresis) run in appropriaterunning buffer. Also load a DNA ladder as a size reference marker in one of the adjacentwells. Run the gel at 5 V/cm until the bromophenol blue has migrated 2/3 of the length ofthe gel.

3 Illuminate the gel using a UV transilluminator (254 or 302 nm) to visualize the bands andtake a photograph.

Note: Small SURVEYOR Nuclease digestion products (<100 bp) are more easily detected early during a gel electrophoresis run. We recommend that the run be stopped after the dye has migrated about 1/3 of the length of the gel for the purpose of photographing the gel early in the run. To resolve the larger fragments, continue with the electrophoresis process until the dye has run 2/3 of the length of the gel, and photograph the gel a second time.

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Control Experiments – Using Control G and Control C Plasmid DNATwo DNAs, Control G and Control C, are provided in the SURVEYOR Check-It Kit. Thesetwo control DNAs are plasmids with inserts that differ at a single base pair. They are providedin separate vials each at a concentration of 5 ng/µL; forward and reverse primers needed forPCR amplification are already combined with the plasmid templates. The sequence of the PCRproduct for Control G is shown below. Control C differs from Control G because it has a C inlieu of the G (underlined). Primer sequences are underlined at the 5’ and 3’ end of theamplicon sequence.

ACACCTGATCAAGCCTGTTCATTTGATTACCAGAGAGACTGTCATTGATCCACATGGAGGGAAGGACATGTGTGTTGCTGGAGCCATTCAAAATTTCACATCTCAGCTTGGCCATTTCCGCCATGGAACATCTGATCGTCGATATAATATGACAGAGGCTTTGTTATTTTTATCCCACTTCATGGGAGATATTCATCAGCCTATGCATGTTGGATTTACAAGTGATATGGGAGGAAACAGTATAGATTTGCGCTGGTTTCGCCACAAATCCAACCTGCACCATGTTTGGGATAGAGAGATTATTCTTACAGCTGCAGCAGATTACCATGGTAAGGATATGCACTCTCTCCTACAAGACATACAGAGGAACTTTACAGAGGGTAGTTGGTTGCAAGATGTTGAATCCTGGAAGGAATGTGATGATATCTCTACTAGCGCCAATAAGTATGCTAAGGAGAGTATAAAACTAGCCTGTAACTGGGGTTACAAAGATGTTGAATCTGGCGAAACTCTGTCAGATAAATACTTCAACACAAGAATGCCAATTGTCATGAAACGGATAGCTCAGGGTGGAATCCGTTTATCCATGATTTTGAACCGAGTTCTTGGAAGCTCCGCAGATCATTCTTTGGCG

PCR amplification of 2 µL of each DNA solution in a 50-µL reaction should produce 20-50µg/µL of a 632-bp amplicon. Sufficient DNA is provided to perform ten PCR reactions witheach control.

Control G and Control C can be used to troubleshoot the PCR amplification, hybridization, andSURVEYOR Nuclease digestion steps of the SURVEYOR Check-It Kit.

Use the control plasmids Control G and Control C as follows:

1 Amplify Control G and Control C DNA using Taq DNA polymerase and the reactionconditions and the PCR program described in Step 1 – PCR Amplification of Referenceand Test Samples where Ta = 65 ºC and the 72 ºC extension time is 1 min.

2 After amplification, analyze a 2-µL aliquot of each amplified DNA and a DNA ladder ona 2% high-resolution agarose gel and compare the band intensities with those of the DNAladder to estimate the DNA concentration.

3 Hybridize Control G- and Control C-amplified DNA in equal amounts as described in Step2 – DNA Hybridization. This produces a population of molecules containing 50%homoduplex, 25% heteroduplex with a C/C mismatch, and 25% heteroduplex with a G/Gmismatch.

4 Digest 200 ng of hybridized Control G/C DNA and 200 ng of Control G or C withSURVEYOR Nuclease as described in Step 3 – Treatment with SURVEYOR Nuclease.

5 Analyze the digested Control G/C DNA and Control G or C DNA as described in Step 4 –Analysis of DNA Fragments.

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SURVEYOR Nuclease digestion of hybridized Control G/C PCR products gives rise to twocleavage products, 217 and 415 bp in size, which are clearly distinguishable by agarose gelelectrophoresis as shown in Figure 2. Since SURVEYOR Nuclease cleaves the mismatchedDNA substrate strands on the 3’ side of the mismatched base, the two resulting fragments havea single base 3’ overhang.

Figure 2. SURVEYOR Nuclease digestion products derived from 200 ng of Control G/Cheteroduplex (lane 1), Control G homoduplex (lane 2), and Control C homoduplex (lane 3)analyzed by electrophoresis on a 1.5 % standard agarose gel run in 1X TBE. The Control G/Cheteroduplex was formed by hybridizing equal amounts of Control G and C homoduplex PCRproduct DNA and contains C/C and G/G mismatched heteroduplexes and the two originalhomoduplexes. Lane M shows a 100-bp Ladder (New England BioLabs, Beverly, MA). DNAsubstrates were digested with 1 µL of SURVEYOR Nuclease S for 20 min at 42 ºC.

M 1 2 3 M

217 bp Product 2

415 bp Product 1

632 bp Substrate

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cDNA Cloning and Verification of Clone FidelityInserts in cDNA clones (and also genomic clones) that have been generated using a PCRcloning method can be analyzed very effectively for errors using SURVEYOR Nuclease. Anexample of how the SURVEYOR Check-It Kit can be used to identify error-free cDNA clonesgenerated by RT-PCR cloning is presented below. The procedures for cDNA generation andclone verification are summarized in the flowchart in Figure 3, and specific clone verificationresults for three different cDNA constructs are presented in Figure 4.

Generation of cDNA Clones by RT-PCR Cloning

Isolation of total RNA

↓RNA template

↓First Strand cDNA Synthesis using an appropriate primer and reverse transcriptase

↓First strand cDNA template ←

↓PCR Amplification using a thermostable DNA polymerase

and gene-specific primers↓

Double-stranded cDNA ←↓

Ligation into appropriate plasmid vector↓

Transformation of E. coli↓

Plating of transformants↓

Colonies representing individual cDNA clones↓

Verification of Cloning Fidelity. Does this clone contain the correct sequence?

↓Colony PCR of Test DNA cDNA PCR of Reference DNAGenerate PCR products Generate PCR productsderived from the cDNA insert derived from the original cDNAin the recombinant plasmid clone sequences used for cDNA cloning

Combine PCR Products↓

Heating and Cooling for Hybridization and Heteroduplex Formation↓

SURVEYOR Nuclease Treatment↓

Analysis by Agarose Gel Electrophoresis

Figure 3. Flowchart outlining the steps involved in cDNA clone generation and cloneverification using the SURVEYOR Check-It Kit. Note the cDNA obtained at the stages markedby an arrowhead (←), or a cDNA library, can be used as a source of reference cDNA foramplification.

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Results obtained while screening cDNA clones with PCR methods to check the insert sizes,and results obtained with the SURVEYOR Check-It Kit used to identify error-free clones areshown in Figure 4. Panel A shows the sizes of the inserts amplified by PCR methods, andPanel B shows SURVEYOR Nuclease digestion products obtained for these ampliconsannealed with amplified cDNA. When compared to the control in lanes labeled “c”, bona fideerror-free clones show no extra bands in their SURVEYOR Nuclease digests and are markedby asterisks. NADP+ clone #2, SUT1 clones #1 and #4, and Mat2 clones #4 and #5, are error-free.

Figure 4. Analysis of cDNA inserts by PCR (Panel A) and by digestion with SURVEYORNuclease (Panel B). Three different PCR primer pairs were used to generate and clone threedistinct cDNA inserts encoding a NADP+, SUT1 and Mat2 gene, respectively. Five coloniesfrom each group of transformants were picked for colony PCR. The PCR products for thesefifteen colonies were analyzed by agarose gel electrophoresis as shown in Panel A. In eachgroup the PCR fragments derived from individual colonies are shown in lanes 1 to 5, and thePCR fragment derived from the original cDNA is in lane c. Lane L shows the 1 Kb DNA ladder.Panel B shows the digestion products obtained by SURVEYOR Nuclease digestion of theamplified individual clone inserts (lanes 1-5) hybridized with the amplified original cDNA(lane c). Lanes for clones containing error-free inserts are marked with asterisks.

A

B

1 2 3 4 5 c L 1 2 3 4 5 c L 1 2 3 4 5 c L

NADP+ SUT1 Mat2

* * * * *

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Confirmation of Mutations Introduced by Site-Directed Mutagenesis

Generation of Clones Modified by Site-Directed MutagenesisTarget DNA template (cloned DNA in a plasmid cloning vector)

Also to be used as a source of reference DNA ↓

Site-Directed Mutagenesis↓

Transformation of E. coli↓

Plating of transformants↓

Colonies representing individual clones with modified sequence↓

Confirmation of Site-Directed Mutagenesis. Does this clone contain the desired modification in its sequence?

↓Colony PCR of Test DNA PCR of Reference DNA

Generate PCR products Generate PCR products derived from cloned mutant DNA derived from original cloned DNA

↓Combine PCR Products

↓Heating and Cooling for Hybridization and Heteroduplex Formation

↓SURVEYOR Nuclease Treatment

↓Analysis by Standard Gel Electrophoresis

Figure 5. Flowchart outlining the steps involved in site-directed mutagenesis and verificationof the presence of the desired mutation in the target sequence using the SURVEYOR Check-ItKit.

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Figure 6 shows the results obtained while using the SURVEYOR Check-It Kit to check 11colony clones that had been modified by amplification-based site-directed mutagenesis. PanelA shows the amplicons for the 11 clones. Note that the amplicon for clone #3 is slightly largerthan the others indicating that this clone contains a cloning artifact. The other clones appear tobe identical. Panel B shows the cleavage products obtained when these amplicons werehybridized with the original reference DNA amplicon (lane 12) and then digested withSURVEYOR Nuclease. Clones #3 and #4 show patterns that differ from the others indicatingthat these contained undesired mutations. The fact that several extra bands appear in clone #4indicates that this colony DNA contains an undesired mutation in this region. All other clonescontain the desired mutation and no cloning artifacts or PCR errors.

Figure 6. Analysis of DNA derived from potential mutant colonies obtained by site-directedmutagenesis. Amplification-based site-directed mutagenesis was used to introduce a 3-baseinsertion into plasmid pQIS155. PCR products and SURVEYOR Nuclease digestion productswere analyzed by agarose gel electrophoresis.

Panel A: PCR products of individual colonies bearing a potential mutant plasmid wereanalyzed by agarose gel electrophoresis. Lanes 1 to 11 show PCR products from colony #1through #11. Lane 12 contains PCR products from the wild-type plasmid pQIS155.

Panel B: PCR products shown in panel A were annealed with amplified wild-type DNA(Reference DNA), digested with SURVEYOR Nuclease, and analyzed by electrophoresis (lanes1-11). Lane 12 contains PCR products from wild-type homoduplex reference DNA that wastreated with SURVEYOR Nuclease as well. Note that all clones contained the 3-base insertionintroduced by site-directed mutagenesis. However, clones #3 and #4 have additional bandingpatterns that differ from the other clones indicating that these two clones also contain cloningartifacts or PCR errors. Lane L shows a 100-bp ladder as a marker. Lanes for clonescontaining error-free inserts are marked with asterisks.

1 2 3 4 5 6 7 8 9 10 11 12 M

A

B

* * * * * * * * *

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Appendix A: Agarose Gel ElectrophoresisAppropriate preparation, electrophoresis, and photography of agarose gels are critical steps toachieving success with SURVEYOR Nuclease — particularly for achieving maximumdetection sensitivity.

Preparing Gels

• Select the percentage and type of agarose appropriate for the DNA fragment sizes to beanalyzed. For DNA fragments <1.5 kb, use a high resolution agarose such asTransOneK™ Agarose (Transgenomic Catalog No. 556001). TransOneK Agarose isspecifically formulated to resolve smaller DNA fragments at a lower percentage gel (seeTable 1), and produces minimal fluorescent background during transillumination andphotography. For DNA fragments >1.5 kb, use a standard agarose such as TransFiveK™Agarose (Transgenomic Catalog No. 556005). TransFiveK Agarose can be used to resolveDNA fragments in the 300 to 5,000 kb range at the appropriate percentage (see Table 1),and also produces minimal fluorescent background during gel analysis.

• Select the appropriate buffer to cast and run the gel. 1X TAE buffer [40 mM Tris-acetate(pH 8.3), 1 mM EDTA] is generally used for resolution of fragments >0.5 kb. For smallerfragments, particularly when analyzing digestion products in the 100 to 300 bp range, 1XTBE [89 mM Tris-borate (pH 8.3), 1 mM EDTA] should be used. 1X TBE helps to reducediffusion of small DNA fragments during electrophoresis, giving a sharper, more focusedband.

• To visualize DNA any time during an electrophoresis run, cast the gel in a UV-transparentgel tray and incorporate a DNA intercalating dye into the gel-casting buffer. Inclusion ofdye in the gel running buffer is not necessary. We recommend the use of ethidium bromide(EtBr) at 0.1 – 0.5 µg/mL in the gel. Within this concentration range, higherconcentrations of EtBr tend to give more intense staining of DNA bands at the cost ofhigher fluorescence background in the gel lanes.

• Before bringing the agarose into solution by boiling in a microwave oven, hydration of thesolid agarose by soaking in buffer for 20-30 min will reduce melting time and foaming.Weigh the container before and after bringing the agarose into solution and restore anyliquid lost during heating with water. Let the agarose cool to 55 to 65 ºC before pouringthe gel.

• Use a comb <1 mm thick to help sharpen bands. Make the gel only as thick as necessaryto accommodate in the wells the maximum volume of sample loaded.

Running Gels

• Carry out electrophoresis at 5 – 10 V/cm where distance in cm is measured betweenelectrodes.

• Small SURVEYOR Nuclease digestion products (<100 bp) are more easily detected earlyduring a gel electrophoresis run, particularly when larger substrates are analyzed. Werecommend that the electrophoresis be stopped after the bromophenol blue (BPB) hasmigrated about 1/3 of the length of the gel for the purpose of photographing the gel earlyin the run. Then continue electrophoresis until the BPB has run 2/3 of the length of the geland photograph the gel a second time.

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Photographing GelsThe sensitivity of DNA fragment detection in agarose gels varies both with the wavelength ofthe UV transilluminator used and the sensitivity of the photography system. Most transillumi-nators allow selection for illumination of DNA at several wavelengths. Use illumination at theshortest wavelength possible to increase signal intensity. We have used several differentcommercial digital photography systems, and have found the AlphaImager™ 2200 ImagingSystem from Alpha Innotech Corp. (San Leandro, CA) to give the most sensitive detection ofEtBr stained DNA. Photograph the gel at several different exposure settings to achieve the bestratio of signal to background fluorescence.

Table 1 – Relationship of DNA Substrate Size, Agarose Type and Gel Concentration, and Running Buffer

aSpecifically formulated agarose for high resolution of small fragmentsbMulti-purpose agarose formulated for separation of larger fragments

Agarose Type DNA Fragment Size (kb)

Agarose (%) Buffer

TransOneK Agarosea

<1.5 2 TBE or TAE

TransFiveK Agaroseb

1.5 – 2.5 1.5 TAE or TBE

TransFiveK Agaroseb

2.5 - 5 1.2 TAE

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Appendix B: TroubleshootingEffective use of the SURVEYOR Mutation Detection Kit depends upon successful completionof a number of steps. One of the most critical is PCR amplification that must result in theproduction of specific, uniform-sized DNAs in sufficient quantity to be detected afterhybridization and cleavage. Also critical is matching the amount of DNA and SURVEYORNuclease used. If you are a first-time user, you should process the control DNAs providedthrough all the steps as described in Control Experiments – Using Control G and Control CPlasmid 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 theSURVEYOR Mutation Detection Kit and how to resolve them.

Problem 1 – Low PCR yield or no PCR product

POSSIBLE CAUSE SOLUTION

Inhibition of PCR by colony

Use less cell suspension or dilute the original cell suspension.

Not enough template and/or too few cycles

Increase the volume of cell suspension and/or add more PCR cycles.

Suboptimal PCR parameters

Do one or more of the following:

• Decrease the annealing temperature in increments of 2 ºC.

• Increase the extension time. For Taq DNA polymerase,use 1 min per 1 kb.

Suboptimal DNA polymerase for target

Use Taq DNA polymerase or a “hot-start” versionof Taq DNA polymerase. Increase the amount ofDNA polymerase.

Target fragment too large for Taq DNA polymerase

Use a Taq DNA polymerase blend (a mixturecontaining Taq DNA polymerase and a proofreading DNA polymerase) such as MaximasePolymerase (Transgenomic Catalog No. 703245).

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Problem 2 – Multiple PCR products

Problem 3 – No cleavage products observed upon analysis after SURVEYOR Nuclease treatment of known heteroduplex

POSSIBLE CAUSE SOLUTION

Poor primer design Redesign primers to improve specificity, Tm, and GC content.

Annealing temperature too low

Increase the annealing temperature in increments of 2 ºC.

Extension time too long Reduce the extension time. For Taq DNA polymerase, use 1 min per 1 kb.

Cycle number too high Reduce cycle number in increments of 2.

Suboptimal PCR conditions

Use “hot-start” PCR and/or touchdown PCR or perform nested PCR.

POSSIBLE CAUSE SOLUTION

Proportion of mismatch target too low

Mix equal amounts of test and reference DNA before annealing.

Cleavage site too close to PCR product end

Redesign the primer set to move the target siteaway from ends.

Inactive SURVEYOR Nuclease

Perform the Control reaction to verify enzyme performance.

Too little enzyme Increase the amount of SURVEYOR Nuclease 2-fold and repeat digestion.

Too little substrate Concentrate the PCR products by ethanol precipitation before annealing.

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Problem 4 – High background after SURVEYOR Nuclease treatment

POSSIBLE CAUSE SOLUTION

Suboptimal hybridization step

Do the following:1 Make sure the DNA concentration is in the range of >25

ng/µL to <50 ng/µL.

2 Repeat the hybridization step, taking care to cool theannealing mixture slowly.

3 Add 1X PCR reaction buffer to precipitated productsbefore annealing.

4 Use sufficient sample volume (>20 µL) duringhybridization in a water bath.

5 Add 1/10 volume 0.5 M KCl to PCR product contained inlow salt 1X PCR reaction buffer

Errors introduced by PCR enzyme

Use a Taq DNA polymerase-based blend with higher fidelity.

Incubation time too long Reduce the SURVEYOR Nuclease digestion time by increments of 5 min.

Too much SURVEYOR Nuclease

Reduce the SURVEYOR Nuclease amount 2-fold and repeat digestion.

DNA amount too lowIncrease the DNA amount to at least 200 ng of substrate per 1 µL of SURVEYOR Nuclease used.

Nonspecific PCR products Optimize the PCR parameters to increase specificity.Always use an appropriate substrate as a control toidentify background.

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References1 Oleykowski, C.A., Bronson Mullins, C.R., Godwin, A.K. and Yeung, A.T. (1998)

Mutation detection using a novel plant endonuclease. Nucleic Acids Res. 26, 4597-4602.

2 Yang, B., Wen, X., Kodali, N.S., Oleykowski, C.A., Miller, C.G., Kulinski, J., Besack, D.,Yeung, J.A., Kowalski, D. and Yeung, A.T. (2000) Purification, cloning, andcharacterization of CEL I nuclease. Biochemistry 39, 3533-3541.

3 Sokurenko, E.V., Tchesnokova, V., Yeung, A.T., Oleykowski, C.A., Trintchina, E.,Hughes, K.T., Rashid, R.A., Brint, J.M., Moseley, S.L. and Lory, S. (2001) Detection ofsimple mutations and polymorphisms in large genomic regions. Nucleic Acids Res. 29,e111.

4 Qiu, P. Shandilya, H., D’Alessio, J.M., O’Connor, K., Durocher, J. and Gerard, G.F. (2004)Mutation detection using Surveyor Nuclease. BioTechniques, 36, 702-707.

5 Scaffino, M.F., Pilotto, A., Papadimitriou, S., Sbalzarini, M., Ansaldi, S., Diegoli, M.,Porcu, E., Grasso, M., Brega, A. and Arbustini, E. (2004) Heteroduplex detection with aplant DNA endonuclease for standard gel electrophoresis. Transgenics 4, 157-166.

6 Caldwell, D.G., McCallum, N., Shaw, P., Muehlbauer, G.J., Marshall, D.F. and Waugh, R.(2004) A structured mutant population for forward and reverse genetics in Barley(Hordeum vulgare L.). The Plant Journal doi: 10.1111/j.1365-313X.2004.02190.x.

7 Qiu, P., Shandilya, H. and Gerard, G.F. (2005) A method for clone confirmation using amismatch-specific DNA endonuclease. Mol. Biotechnol. 29, 11-18.

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www.transgenomic.com

This product is manufactured under exclusive license to US Patents �6,391,557; 5,869,245; and other patents pending. Use of SURVEYOR �Nuclease requires a license from Transgenomic. Academic, not-for-profit �and for-profit organizations have limited rights to use SURVEYOR Nuclease �for research purposes with purchase of this product. Resale or other uses are strictly prohibited. Please contact Transgenomic for more information.

“TRANSGENOMIC”, “transforming the world” and the globe logo are �registered trademarks, and “SURVEYOR”, TransOneK”, “TransFiveK” and �“Maximase” are trademarks of Transgenomic, Inc. All other trademarks are trademarks of their respective holders.

© 2005 Transgenomic, Inc. All rights reserved. Printed in USA.

Document Part No. 482108-01