Guide-it SNP Screening Kit FAQs
The Guide-it SNP Screening Kit provides a sensitive and accurate method for high-throughput detection of single-nucleotide substitutions in clonal populations using a simple enzymatic assay.
The principle of the assay is described in the following video:
What is the kit workflow?
The assay employed by the Guide-it SNP Screening Kit consists of the following steps:
As indicated, this workflow enables analysis of 96 samples in less than 4 hours.
What oligos do I need to order to perform the assay?
Assaying a given nucleotide substitution with the Guide-it SNP Screening Kit requires the following oligos:
- Forward and reverse primers (orange) for PCR amplification of the genomic region containing the target site
- Displacement oligo (green) and flap-probe oligo (purple) for detecting the edited nucleotide in the PCR product, and SNP and wild-type (WT) control oligos (blue) for verifying the performance of the designed displacement and flap-probe oligos
Oligos used with the Guide-it SNP Screening Kit do not require special modifications such as fluorescent labels or quenchers, but the kit protocol does require the inclusion of a hexanediol (/3C6/) blocking modification on the 3' end of the flap-probe oligo. Inclusion of this modification is a standard option provided with oligo synthesis services, and no special purification step is needed in this case, only standard desalting. To simplify the oligo design process, we have developed an online software tool that outputs assay-specific oligo sequences in a ready-to-order format. Additional information regarding the design of the assay-specific oligos is provided in the Guide-it SNP Screening Kit User Manual.
How does Guide-it Flapase detect the edited nucleotide?
Guide-it Flapase is a recombinant, structure-specific nuclease from Takara Bio; it recognizes and cleaves double-flap structures, but is unable to cleave gapped structures. The Guide-it SNP Screening Kit employs this feature to assay for single-nucleotide substitutions: the main difference between the double-flap and the gapped structures is whether or not hybridization occurs between the flap-probe oligo and the PCR product at the position of the interrogated base.
Tripartite structures formed by hybridization of the displacement oligo, flap-probe oligo, and PCR product during the SNP screening assay. Scheme showing the different tripartite structures generated in an assay designed to detect a C→T substitution. Annealing of the displacement oligo (green) and flap-probe oligo (purple, encoding for the edited nucleotide, T) to the PCR product (blue) forms either of two structures depending on whether the interrogated base in the PCR product is edited or wild-type. Double-flap structure (left). In a scenario where the interrogated base is edited, the flap-probe oligo containing the complementary nucleotide forms a complete base pairing at the target site. Gapped structure (right). If the editing event is unsuccessful and the interrogated base is wild-type, there isn't a complete base pairing at the target site, and a gap is formed.
What is included in the kit?
The Guide-it SNP Screening Kit includes all necessary reagents for extraction and amplification of genomic DNA from mammalian cell samples, and for performing the Guide-it Flapase enzymatic assay:
| Guide-it SNP Screening Kit | 632652 (100 rxns) | 632653 (400 rxns) |
|---|---|---|
| MightyPrep Reagent for DNA (Store at 4°C) | 9182A | 9182 |
| MightyPrep Reagent for DNA | 5 ml | 20 ml |
| Guide-it SNP Control Set (Store at –20°C) | 632654 | 632654 |
| Guide-it SNP Positive Control Mix | 300 µl | 300 µl |
| Guide-it SNP Negative Control Mix | 300 µl | 300 µl |
| Guide-it Flap Reagents (Store at –20°C) | 632655 (100 rxns) | 632656 (400 rxns) |
| Terra PCR Direct Polymerase Mix | 50 µl | 200 µl |
| 2X Terra PCR Direct Buffer (with Mg2+, dNTP) | 750 µl x 2 | 1 ml x 5 |
| Dilution Buffer | 8 ml | 32 ml |
| RNase-free Water | 10 ml | 40 ml |
| Annealing Buffer | 350 µl | 1.4 ml |
| Flapase Buffer | 350 µl | 1.4 ml |
| Guide-it Flapase | 100 µl | 400 µl |
| Guide-it Flap Detector (40X) | 50 µl | 200 µl |
In addition to user-provided oligos specific for each assay, performing the assay requires a thermal cycler and fluorescence plate reader.
What do I need for the detection of the assay signal?
Detection of the assay signal requires only a standard fluorescence plate reader that enables excitation at 485 nm and capture at 535 nm. If the plate reader to be used has different possible filter sets, the Guide-it SNP Control Set provided in the kit can be used to determine the optimal combination of filters.
Can a qPCR thermal cycler be used for fluorescence detection?
Yes, a qPCR thermal cycler can be used. The Guide-it SNP Screening Kit employs an end-point assay that doesn't require real-time monitoring of the flapase reaction, but a qPCR machine can be used during the enzymatic assay to periodically measure fluorescence (e.g., every 3 minutes) to follow the kinetics of the reaction and upon completion of the reaction.
Does the Guide-it SNP Screening Kit allow for detection of any nucleotide substitution regardless of the genomic target site?
Yes. The Guide-it SNP Screening Kit has been used successfully to analyze SNPs at 18 different loci within the human genome (corresponding to 14 different genes, see table); a subset of the results are shown in the graph below.
Detecting different nucleotide substitutions from genomic DNA. Genomic DNA samples (obtained from the Coriell Institute) which were either wild-type or homozygous for the indicated substitutions were analyzed using the Guide-it SNP Screening Kit. All substitutions were successfully detected, as demonstrated by the strong fluorescent signals obtained for samples that were homozygous (+/+, blue) for the indicated substitutions relative to signals obtained for wild-type (–/–, orange) and negative control (NT, gray) samples.
All the genomic loci and the nucleotide substitutions we have tested so far are listed in the table below:
| Gene | Nucleotide substitution | Position |
|---|---|---|
| F2 | G→A | g.20210 |
| A→G | ||
| HFE | C→G | c.187C→G (p.His63Asp) |
| G→C | c.187G→C | |
| ITPA | C→A | c.94C→A (p.Pro32Thr) |
| A→C | c.94A→C | |
| MAN2B1 | A→T | c.212A→T (p.His71Leu) |
| T→A | c.212T→A | |
| DBT(E2) | G→T | c.487G→T(p.Glu163Ter) |
| T→G | c.487T→G | |
| GALT | G→C | c.292G→C (p.Asp98His) |
| C→G | c.292C→G | |
| MTHFR | A→C | c.1289A→C (p.Glu429Ala) |
| C→A | c.1289 | |
| SMPD1 | T→C | c.905T→C (p.Leu302Pro) |
| C→T | c.905C→T | |
| NCP1 | C→T | c.2793C→T(p.Asn931Asn) |
| T→C | c.2793T→C | |
| A→G | c.2572A→G(p.Ile858Val) | |
| G→A | c.2572G→A | |
| CFTR | T→A | c.3434T→A (p. Met1101Lys) |
| A→T | c.3434A→T | |
| REQCL4 | C→T | g.1488C→T (p.Ser246Ser) |
| G→C | g.1551G→C (p.Glu267Asp) | |
| G→A | g.5321G→A (p.Arg1005Gln) | |
| DPM1 | C→G | c.274C→G (p.Arg92Gly) |
| TUBGCP6 | G→T | c.5458G→T (p.Gly1820Ter) |
| T→G | c.5458T→G | |
| FAH | G→A | c.786G→A (p.Trp262Ter) |
| G→A | c.1009G→A (p.Gly337Ser) |
Can I use the kit if I have no idea what the substitution is?
Each assay performed with the Guide-it SNP Screening Kit is designed to detect a specific single-nucleotide substitution at a particular genomic location. The sequence of the flap-probe oligo determines which nucleotide will be detected at a chosen target site. To be able to identify any nucleotide at a given site, one must perform four independent assays, with each assay using a different flap-probe oligo designed to specifically detect a G, C, A, or T at the target site (i.e., one specific flap-probe oligo per possible nucleotide).
Can the Guide-it SNP Screening Kit be used for genotyping?
Yes, you will need to design one specific flap-probe per possible nucleotide at the target site and perform independent assays.
Using the Guide-it SNP Screening Kit for genotyping. Samples from the Coriell Institute carrying SNPs at either of two genomic loci (NCP1 or CFTR genes) were analyzed using the Guide-it SNP Screening Kit (graphs, top) and by Sanger sequencing (chromatograms, bottom). Panel A. Analysis of NCP1. The analysis determined which samples were homozygous or heterozygous for an A→G substitution by employing two flap-probe oligos in independent assays designed to detect either A or G. Panel B. Analysis of CFTR. Analysis of samples that were either wild-type or homozygous for the indicated T→A substitution. All results for both panels were confirmed by Sanger sequencing (displayed below the graphs).
Can the kit be used to detect successful homologous recombination (HR) in edited populations?
Although the Guide-it SNP Screening Kit was specifically intended for detection of SNPs in clonal cell lines, it can be used to detect successful HR in an edited population with a detection limit around 5%*. Download our poster to see data relating to this application. Visit our technical support page if you need more information about how to use the kit for the detection of HR.
*For this particular application, we recommend that genomic DNA should be extracted from the edited population using NucleoSpin Tissue Columns (Cat. #740952.250S).
Detection of a CRISPR/Cas9-generated nucleotide substitution in a pool of edited cells using RFLP analysis and the Guide-it SNP Screening Kit. Creation of an isogenic hiPSC line carrying a SNP related to tyrosinemia (c.1009G→A) in the FAH gene using CRISPR/Cas9 technology. Panel A. Design of sgRNAs and HR donor template. Different sgRNAs (indicated by #1, #2, and #3) located around the target site (in bold) were tested. A synthetic short oligo encoding for the SNP (in red) was used as a donor template. The edited sequence encodes for a new PvuII restriction site. Panel B. Results of RFLP analysis. Analysis of the cells electroporated with Cas9-sgRNA complexes (RNPs) without (1) or with (2) the donor template. The results suggest that only sgRNA #3 triggered the desired G→A substitution due to the appearance of the extra band in Lane 2 (indicated by blue arrow). Panel C. Results of analysis using the Guide-it SNP Screening Kit. After extraction of genomic DNA using NucleoSpin Tissue Columns, The Guide-it SNP Screening Kit was used to determine which experimental conditions were successful in introducing the SNP. Consistent with the RFLP results, presence of the G→A substitution was only detected in cells edited using the RNP with sgRNA #3 in combination with the HR donor template. By analysis of more than 100 clones it was determined that the percentage of HR was approximately 24%. Panel D. PCR products obtained from the edited cell population using sgRNA #3 were serially diluted with wild-type PCR products, such that the G→A substitution was represented at the frequencies indicated in the graph. The samples were then analyzed using the Guide-it SNP Screening Kit in order to determine the detection limit (gray line).
Do I need to use column-purified genomic DNA?
No. The cells are lysed with MightyPrep Reagent for DNA, and the supernatant from the lysate is used directly as template for the PCR to amplify the genomic target sequence without the need for extra purification steps.
Which cell types have been analyzed using the kit? Can the kit be used for suspension cells?
The Guide-it SNP Screening Kit has been used successfully to analyze a variety of cell types, including cells grown in suspension (Jurkat cells) and adherent cells (fibroblasts, hiPSCs).
Analysis of Jurkat cells using the Guide-it SNP Screening Kit. Jurkat cells were plated at different cell densities in a 96-well plate. After 48 hours, genomic DNA was extracted using MightyPrep Reagent for DNA and the Guide-it SNP Screening Kit was successfully applied to detect the indicated A→G substitution.
What cell densities are suitable for analysis with the kit?
Clonal cell lines in a 96-well plate can have different growth rates, and therefore the protocol has been optimized for a wide range of cell densities (from 2 x 104 to 2 x 105 cells per well).
However, if this assay is being performed for the detection of homologous recombination we recommend that the assay be performed with genomic DNA that has been purified using NucleoSpin Tissue Columns (Cat. # 740952.250S).
Assessing the performance of the Guide-it SNP Screening Kit across a range of cell densities. Panel A. The Guide-it SNP Screening Kit was used to assay for a C→G substitution in the DPM1 gene. Two cell lines (one wild-type, the other carrying the SNP) were plated at different densities in a 96-well plate. After 48 hours, they were analyzed using the Guide-it SNP Screening Kit. Fluorescent signals (arbitrary units) and signal-to-noise ratios were largely comparable over a range of 1.8 x 103–1.2 x 105 cells. Panel B. Microscopy images of the primary fibroblasts that were seeded at different cell densities from 2 x 105 (left) to 2 x 104 (right) cells per well.
Can the protocol for the Guide-it SNP Screening Kit be adapted for use with 384-well plates?
We have not tested the kit protocol using 384-well plates; however, theorizing such an adaptation, the only modification we can foresee at this time is a reduction in reaction volumes. Please note that some troubleshooting may be required to ensure successful setup of the PCR reaction and enzymatic assay and to confirm that the resulting fluorescent signal is strong enough to be detected using your chosen method. If you need more assistance with this, please contact our Technical Support team.
Can the kit assay be used to distinguish homozygotes from heterozygotes?
As demonstrated by the data shown below, each assay performed with the kit provides a qualitative output rather than a quantitative measure of a substitution's frequency. Therefore, a single Guide-it SNP screening assay using the current single-color chemistry cannot be used to distinguish homozygous clones from heterozygous clones (see FAQ above, "Could the Guide-it SNP Screening Kit be used for genotyping?" for information about how the kit can be used to assess zygosity). We are developing a new version of the kit that will enable simultaneous detection of either of two bases at the same position (e.g., SNP and wild type) using two Guide-it flap detectors in tandem (one triggering a red fluorescent signal and the other one producing a green signal).
Comparison of assay results obtained for homozygous, heterozygous, and wild-type cell samples. The Guide-it SNP Screening Kit was used to assay for each indicated substitution in samples that were homozygous (+/+, blue), heterozygous (+/–, purple), or wild-type (–/–, orange). For each case, fluorescent signals obtained for homozygous and heterozygous samples (blue bars and purple bars, respectively) were of comparable value.
How clean does the PCR of the genomic target sequence need to be?
The assay performs well regardless of whether extra PCR products are generated during amplification of the genomic target sequence. See the image below for example PCR amplification results that have been successfully used to detect nucleotide substitutions with this kit.
Examples of PCR-amplified target DNAs used as samples with the Guide-it SNP Screening Kit. PCR products visible in each gel lane were successfully analyzed for nucleotide substitutions involving the indicated genomic loci using the Guide-it SNP Screening Kit. As indicated by the gel, the size of the amplified target can range between 200 bp and 700 bp. For SNP detection assays involving the DBT(E2) and MTHFR gene sequences, amplicons of two different sizes were successfully used.
How sensitive is the Guide-it SNP Screening Kit to the presence of mutations close to the site of the nucleotide substitution being assayed?
The Guide-it SNP Screening Kit is highly sensitive to the presence of mutations in the genomic target sequence surrounding the site of the substitution being assayed. The displacement oligo and the flap-probe oligo anneal to the region immediately adjacent to the SNP, and the presence of unknown mutations in that region would affect the annealing of the oligos with the PCR product and the formation of the double-flap structure.
Sensitivity of the Guide-it SNP Screening Kit to mutations involving the genomic target sequence. The c.786G→A substitution in the FAH gene was engineered in hiPSCs by RNP electroporation together with either of two HR templates; one HR template (#1) encoded a sequence which included only the G→A substitution (in red), another template (#2) included an additional G→T substitution (in green) which generated a new SmlI restriction site (allowing detection of the substitution via RFLP). Two different displacement oligos were designed for both scenarios (i.e., G→A substitution vs. G→A and G→T substitutions in tandem), and used to assay samples obtained from the various edited cell populations. The oligo designed to assay the G→A substitution (displacement #1) only generated a strong signal for the sample electroporated with template #1. The complementary result was true for the displacement oligo designed to assay the two substitutions. These results show the sensitivity of the assay towards the sequence surrounding the edited base.
Are there any limitations or special considerations regarding what target sequences can be analyzed with the kit?
The only design limitation one must consider when designing the probes is that the displacement oligo must not encode the sequences 5'-GGAGn-3' or 5'-GGAGNn-3' at its 3' terminus (where N can be any base, and n is the extra noncomplementary base that depends on the substitution being assayed). Displacement oligos containing these sequences will generate a background signal in the enzymatic assay. In these scenarios it is advisable to redesign the assay to target the opposite strand of the PCR product, such that the displacement oligo doesn't encode the restricted sequence at its 3' terminus (see example in figure below).
Example of a limitation in the design of the displacement oligo. In this scenario, the displacement oligo designed to hybridize with the PCR product (in the 3'→5' orientation) encodes the restricted sequence at its 3' terminus (Panel A). A viable alternative (Panel B) targets the opposite strand of the PCR product (in the 5'→3' orientation), avoiding the inclusion of the restricted sequence at the 3' terminus of the displacement oligo.
Can the kit only be used to detect single-nucleotide substitutions? Would it be suitable for detecting longer insertions?
The current version of the Guide-it SNP Screening Kit can only be used to detect single-nucleotide substitutions, not longer insertions.
We are, however, in the process of developing a new version of the kit that will enable detection of longer insertions using two Guide-it Flap Detectors in tandem. One detector yields a red fluorescent signal while the other one generates a green signal, allowing simultaneous detection of seamless insertions at both the 5' and 3' ends of the recombinant sequence. We have applied this updated approach for detecting the fusion of a myc tag to an endogenous gene in hiPSCs (a poster including this data is available for download).
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