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'''Surveyor Nuclease Assay''' is an enzyme mismatch cleavage assay used to detect [[Single-nucleotide polymorphism|single base mismatches]] or small insertions or deletions ([[Indel|indels]]). |
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Surveyor nuclease is part of a family of mismatch-specific [[Endonuclease|endonucleases]] that were discovered in celery (CEL nucleases)<ref name=":2">{{Cite journal|title = Purification, cloning, and characterization of the CEL I nuclease|pmid = 10736152|journal = Biochemistry|date = 2000-04-04|issn = 0006-2960|pages = 3533–3541|volume = 39|issue = 13|first = B.|last = Yang|first2 = X.|last2 = Wen|first3 = N. S.|last3 = Kodali|first4 = C. A.|last4 = Oleykowski|first5 = C. G.|last5 = Miller|first6 = J.|last6 = Kulinski|first7 = D.|last7 = Besack|first8 = J. A.|last8 = Yeung|first9 = D.|last9 = Kowalski}}</ref>. The enzyme recognizes all base substitutions and insertions/deletions, and cleaves the 3′ side of mismatched sites in both DNA strands with high specificity<ref name=":0">{{Cite journal|title = Mutation detection using Surveyor nuclease|pmid = 15088388|journal = BioTechniques|date = 2004-04-01|issn = 0736-6205|pages = 702–707|volume = 36|issue = 4|first = Peter|last = Qiu|first2 = Harini|last2 = Shandilya|first3 = James M.|last3 = D'Alessio|first4 = Kevin|last4 = O'Connor|first5 = Jeffrey|last5 = Durocher|first6 = Gary F.|last6 = Gerard}}</ref> |
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This assay has been used to identify and analyze mutations in a variety of organisms and cell types, as well as to confirm genome modifications following genome editing (using [[CRISPR]]/[[TALENs]]/[[Zinc finger|Zinc-fingers]]). |
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[[File:Surveyor Assay Workflow.jpg|thumb|429x429px|Diagram of Surveyor nuclease assay workflow]] |
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== Background == |
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The ability to discover and detect known and unknown mutations is of great importance in biomedical research and genetic diagnosis (see <u>applications</u>). Therefore, multiple methods have been developed to enable research-based and clinical diagnostic detection of such mutations. |
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The most direct manner to identify sequence changes/differences is through reading the DNA sequence with traditional and high throughput DNA sequencing methods (see [[Sanger sequencing|Sanger Sequencing]] and [[DNA sequencing|DNA Sequencing]]). However, these methods provide large amounts of unnecessary data and are costly to use<ref name=":1">{{Cite journal|title = Enzymatic mutation detection technologies|pmid = 15948293|journal = BioTechniques|date = 2005-05-01|issn = 0736-6205|pages = 749–758|volume = 38|issue = 5|first = Anthony T.|last = Yeung|first2 = Deepali|last2 = Hattangadi|first3 = Lauryn|last3 = Blakesley|first4 = Emmanuelle|last4 = Nicolas}}</ref>. In addition, traditional sequencing can be useful for detection of germline mutations, but may be less successful in detecting somatic minor alleles at low frequencies ([[Mosaic (genetics)|mosaicism]]). Therefore, other non-sequencing based approaches to detect the mutation or polymorphisms are required. |
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Other widely used methods depend on physical properties of DNA, for example melting temperature-based systems such as [[Single-strand conformation polymorphism|Single-stranded conformational polymorphism]] analysis (SSCP) and [[Denaturing high performance liquid chromatography|Denaturing high-performance liquid chromatography]] (DHPLC). These techniques are generally limited to the analysis of short DNA fragments (< 1000 bp) and are only able to indicate the presence of polymorphism(s), but do not easily yield the location of a mutation within a DNA sequence. Therefore, this must be followed with additional techniques in order to pinpoint the mutation or map multiple mutations in the same fragment<ref name=":1" />. |
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Enzymatic mismatch cleavage assays utilize the properties of mismatch-specific endonucleases to detect and cleave mismatches. These methods are simple to run, require standard laboratory technique and equipment and can detect polymorphisms, single base pair mismatches, insertions and deletions at low frequencies.<ref>{{Cite journal|title = A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis|pmid = 22437793|journal = Electrophoresis|date = 2012-03-01|issn = 1522-2683|pages = 788–796|volume = 33|issue = 5|doi = 10.1002/elps.201100460|first = Mo Chao|last = Huang|first2 = Wai Chye|last2 = Cheong|first3 = Li Shi|last3 = Lim|first4 = Mo-Huang|last4 = Li}}</ref>. Several such enzymes have been discovered (including CEL I, T4 endonuclease VII, Endonuclease V, T7 endonuclease I)<ref name=":1" />. |
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One of the commonly used enzymes is Surveyor nuclease (CEL II), which cleaves the 3′ side of both DNA strands with high specificity at sites of base substitution or insertion/deletion. This enzyme is capable of cleaving at multiple mutations in large DNA fragments, and produces detectable cleavage products from mismatch DNA representing only a small proportion of the DNA in a population, thus making it suitable for use in enzyme mismatch cleavage assays<ref name=":0" />. |
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== History == |
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== Surveyor Nuclease Assay Workflow == |
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=== DNA Extraction === |
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Initially, the DNA of interest (nuclear or mitochondrial DNA) is extracted from tissues or cell culture. This can be done by standard extraction methods such as Proteinase K digestion followed by ethanol precipitation or by other, commercially available methods. |
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If the DNA is predicted to be heterogeneous (e.g. from a pool of differentially modified cells or from heterozygous mutation carriers) there is no need for addition of control DNA. |
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=== [[Polymerase chain reaction|Polymerase Chain Reaction]] (PCR) === |
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The region of interest in both mutant and wild-type reference DNA is amplified by PCR. The PCR reaction should be carried out using a high-fidelity, proofreading polymerase to avoid introducing PCR errors that will be interpreted by the nuclease. The PCR reaction should be optimized to create a single, strong PCR band, as non-specific bands will increase the background of the assay. |
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If the allele of interest is anticipated to be present in low frequency, such as in the case of somatic mosaicism or heteroplasmic mitochondrial DNA, a modified Polymerase Chain Reaction protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA might be considered (e.g. [[COLD-PCR]]). |
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=== Formation of Hybrid DNA Duplex === |
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The DNA of interest is denatured and annealed in order to form heteroduplexes containing a mismatch at the point of the mutation, which can then be identified by the Surveyor nuclease. If the DNA is predicted to be homogenous (e.g. homoplasmic mitochondrial DNA or identical alleles on both chromosomes of genomic DNA) then DNA from a control sample is needed in order to form a heteroduplex which is then recognizable by the nuclease. If the DNA sample is heterogeneous, no additional control DNA is needed; however, the PCR products should still be denatured and annealed in order to create heteroduplexes. |
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=== Digestion === |
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The annealed DNA is treated with Surveyor nuclease to cleave the heteroduplexes. All types of mismatches are identifiable by Surveyor nuclease, although the mismatch cutting preferences fall into four groups from most to least preferred: CT, AC, and CC are preferred equally over TT, followed by AA and GG, and finally followed by the least preferred, AG and GT. Sequence context also influences the Surveyor nuclease digestion rate<ref name=":0" />. |
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=== Analysis === |
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Digested DNA products can be analyzed using conventional gel electrophoresis or high-resolution capillary electrophoresis. The detection of cleaved products indicates the presence of a heteroduplex formed by a mismatch. The location of the mutation/polymorphism can be inferred by observing the fragment length after cleavage. If fluorescently labelled primers are used to mark the 5’ and 3’ end of the PCR products, different colored bands will be observed in the analysis. The size of each band independently confirms the position of the mutation/polymorphism. Multiple mutations can be detected by the presence of several fragments<ref name=":2" /><ref>{{Cite journal|url = http://www.ncbi.nlm.nih.gov/pmc/articles/PMC147896/pdf/264597.pdf|pmc = 147896|title = Mutation detection using a novel plant endonuclease|last = Oleykowski|first = Catherine A.|date = 1998|journal = Nucleic Acids Research|doi = |pmid = 9753726|access-date = |last2 = Mullins|first2 = Colleen RB|issue = 26 (20)|pages = 4597–4602|last3 = Godwin|first3 = Andrew K|last4 = Yeung|first4 = Anthony T}}</ref>. |
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== Advantages and Limitations == |
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=== Advantages === |
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'''Advantages of mismatch nuclease assays:''' |
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* One of the main advantages of detecting mutations and polymorphisms using mismatch nuclease methods is that no previous knowledge is required regarding the nature of the mutation, as opposed to other methods, such as [[Restriction fragment length polymorphism|Restriction Fragment Length Polymorphisms]] (RFLP) that has been used for SNP analysis in the past. |
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* In comparison to melting temperature-based methods, mismatch-specific endonuclease methods are not only a faster procedure, but are also superior to DHPLC and SSCP as a tool for gene mutation identification, as these methods can detect multiple mutations in large DNA fragments. |
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* The method is feasible for use in high-throughput systems due to an automated injection. This method is thus well suited for screening and may also reduce costs<ref name=":3">{{Cite journal|title = Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations|pmid = 18694524|journal = BMC biotechnology|date = 2008-01-01|issn = 1472-6750|pmc = 2525636|pages = 62|volume = 8|doi = 10.1186/1472-6750-8-62|first = Chia-Cheng|last = Hung|first2 = Yi-Ning|last2 = Su|first3 = Chia-Yun|last3 = Lin|first4 = Yin-Fei|last4 = Chang|first5 = Chien-Hui|last5 = Chang|first6 = Wen-Fang|last6 = Cheng|first7 = Chi-An|last7 = Chen|first8 = Chien-Nan|last8 = Lee|first9 = Win-Li|last9 = Lin}}</ref>. |
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* Surveyor nuclease is a reasonably sensitive enzyme. It can detect 1:32 heteroduplex to homoduplex for smaller PCR products (~0.6 bp), and 1:16 heteroduplex to homoduplex for longer PCR products (~2.3 kb), thus producing detectable cleavage products from sequences representing only a small proportion of DNA in the population. This property can be utilized to enhance clinical diagnosis by pooling samples (as this will increase the heteroduplex formation and the sensitivity)<ref name=":1" />. This is also useful for detection of minor variants in a heterogeneous population (such as heterogeneous tumor). In the case of genome editing by CRISPR or other methods, this property can enhance detection of rare editing events in a population of cells prior to creation and testing of individual edited clones. |
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* Surveyor nuclease cleaves all types of mismatches, even if some are more preferred then others: CT, AC, and CC are preferred equally over TT, followed by AA and GG, and finally followed by the least preferred, AG and GT. It also detects Indels up to at least 12 bp<ref name=":0" />. |
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* In addition, Surveyor nuclease assay can detect multiple mutations in the same fragment. However, this requires several additional processing steps that may also increase the background of the assay (see limitations). |
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=== Limitations === |
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'''PCR Amplification product:''' |
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* One of the main limitations of this assay is that it relies upon PCR amplification, and is therefore influenced by the quality of the amplified product. PCR artifacts (e.g. primer-dimers or truncated products) can increase the background and obscure the signal. Primer-dimers can also inhibit the activity of Surveyor nuclease thus reducing the signal even more. |
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* As the PCR method has the potential to erroneously introduce mutations during the amplification, these errors can also significantly increase Surveyor background. Therefore, it is recommended to use a high-fidelity polymerase to minimize the errors introduced that will result in erroneously-formed heteroduplexes in the annealing and detection stage. |
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* Mitochondrial DNA analysis might be susceptible to contamination by nuclear mitochondrial DNA sequences co-amplified during the PCR reaction, thus confounding the analysis of homoplasmic versus heteroplasmic mitochondrial DNA<ref>{{Cite journal|title = Interference of Co-amplified nuclear mitochondrial DNA sequences on the determination of human mtDNA heteroplasmy by Using the SURVEYOR nuclease and the WAVE HS system|pmid = 24664244|journal = PloS One|date = 2014-01-01|issn = 1932-6203|pmc = 3963942|pages = e92817|volume = 9|issue = 3|doi = 10.1371/journal.pone.0092817|first = Hsiu-Chuan|last = Yen|first2 = Shiue-Li|last2 = Li|first3 = Wei-Chien|last3 = Hsu|first4 = Petrus|last4 = Tang}}</ref>. |
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* In order to detect multiple mutations in the same fragment, post-PCR clean-up is required prior to Surveyor nuclease digestion. Detection of multiple mismatchs can also be improved by increasing time and amount of Surveyor nuclease in the reaction. However, these conditions also increase the background due to non-specific cleavage. |
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'''Limitations of the Surveyor Nuclease enzyme:''' |
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* Surveyor Nuclease also has a 5′ exonuclease activity that attacks the ends of double‐stranded DNA increasing background signal during extended incubation<ref name=":0" />. This can be reduced by shortening the digestion time and/or by addition of DNA polymerase. |
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== Main Applications == |
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=== Confirming genome modifications using CRISPR and other methods === |
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A number of genome editing technologies have emerged in recent years, including [[Zinc finger nuclease|zinc-finger nucleases]] (ZFNs), transcription activator–like effector nucleases ([[TALENs]]) and the RNA-guided [[CRISPR|CRISPR/Cas9]] nuclease system. All three methods promote genome editing by introduction of a double strand DNA break, followed by repair through the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. While HDR is expected to introduce a consistent modification to the genome, NHEJ can introduce a heterogeneous mix of mutations (usually small indels). As both point mutations and indels can be detected by surveyor nuclease assay, the assay can be used to detect genome editing in a pool of cells without the need for clonal expansion prior to analysis, as well as estimate the targeting efficiency achieved<ref>{{Cite journal|title = A rapid and general assay for monitoring endogenous gene modification|pmid = 20680839|journal = Methods in Molecular Biology (Clifton, N.J.)|date = 2010-01-01|issn = 1940-6029|pages = 247–256|volume = 649|doi = 10.1007/978-1-60761-753-2_15|first = Dmitry Y.|last = Guschin|first2 = Adam J.|last2 = Waite|first3 = George E.|last3 = Katibah|first4 = Jeffrey C.|last4 = Miller|first5 = Michael C.|last5 = Holmes|first6 = Edward J.|last6 = Rebar}}</ref><ref>{{Cite journal|title = Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity|pmid = 23992846|journal = Cell|date = 2013-09-12|issn = 1097-4172|pmc = 3856256|pages = 1380–1389|volume = 154|issue = 6|doi = 10.1016/j.cell.2013.08.021|first = F. Ann|last = Ran|first2 = Patrick D.|last2 = Hsu|first3 = Chie-Yu|last3 = Lin|first4 = Jonathan S.|last4 = Gootenberg|first5 = Silvana|last5 = Konermann|first6 = Alexandro E.|last6 = Trevino|first7 = David A.|last7 = Scott|first8 = Azusa|last8 = Inoue|first9 = Shogo|last9 = Matoba}}</ref><ref>{{Cite journal|title = One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering|pmid = 23643243|journal = Cell|date = 2013-05-09|issn = 1097-4172|pmc = 3969854|pages = 910–918|volume = 153|issue = 4|doi = 10.1016/j.cell.2013.04.025|first = Haoyi|last = Wang|first2 = Hui|last2 = Yang|first3 = Chikdu S.|last3 = Shivalila|first4 = Meelad M.|last4 = Dawlaty|first5 = Albert W.|last5 = Cheng|first6 = Feng|last6 = Zhang|first7 = Rudolf|last7 = Jaenisch}}</ref>. |
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=== Detection of germline mutations in human genes === |
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Surveyor nuclease assay has been used for detection of germline mutations in human genes. For example, ATRX for X-linked mental retardation<ref>{{Cite journal|title = A new detection method for ATRX gene mutations using a mismatch-specific endonuclease|pmid = 16763962|journal = American Journal of Medical Genetics Part A|date = 2006-07-15|issn = 1552-4825|pages = 1519–1523|volume = 140|issue = 14|doi = 10.1002/ajmg.a.31310|first = Takahito|last = Wada|first2 = Yoshimitsu|last2 = Fukushima|first3 = Shinji|last3 = Saitoh}}</ref>, HBB gene linked to β-thalassemia<ref name=":3" />. The assay has also been used for detection of mitochondrial and nuclear DNA mutations associated with respiratory chain defects<ref>{{Cite journal|title = Surveyor Nuclease: a new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects|pmid = 15880407|journal = Human Mutation|date = 2005-06-01|issn = 1098-1004|pages = 575–582|volume = 25|issue = 6|doi = 10.1002/humu.20177|first = Sylvie|last = Bannwarth|first2 = Vincent|last2 = Procaccio|first3 = Veronique|last3 = Paquis-Flucklinger}}</ref>, and mutations associated with kidney disease<ref>{{Cite journal|title = Novel method for genomic analysis of PKD1 and PKD2 mutations in autosomal dominant polycystic kidney disease|pmid = 18837007|journal = Human Mutation|date = 2009-02-01|issn = 1098-1004|pages = 264–273|volume = 30|issue = 2|doi = 10.1002/humu.20842|first = Ying-Cai|last = Tan|first2 = Jon D.|last2 = Blumenfeld|first3 = Raluca|last3 = Anghel|first4 = Stephanie|last4 = Donahue|first5 = Rimma|last5 = Belenkaya|first6 = Marina|last6 = Balina|first7 = Thomas|last7 = Parker|first8 = Daniel|last8 = Levine|first9 = Debra G. B.|last9 = Leonard}}</ref><ref>{{Cite journal|title = Screening for mutations in kidney-related genes using SURVEYOR nuclease for cleavage at heteroduplex mismatches|pmid = 19525337|journal = The Journal of molecular diagnostics: JMD|date = 2009-07-01|issn = 1943-7811|pmc = 2710707|pages = 311–318|volume = 11|issue = 4|doi = 10.2353/jmoldx.2009.080144|first = Konstantinos|last = Voskarides|first2 = Constantinos|last2 = Deltas}}</ref> |
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==References== |
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{{reflist}}{{Reflist}} |
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Latest revision as of 02:23, 12 June 2017
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