Invader Assay for Single Nucleotide Polymorphism Genotyping and Gene Copy Number Evaluation

Andrea Mast and Monika de Arruda

Summary

The Invader® assay (Third Wave Technologies) is a homogeneous, isothermal DNA probe-based method for sensitive detection of nucleic acid sequences. Invader reactions are performed directly on genomic DNA or total RNA targets; however, polymerase chain reaction- or reverse transcriptase polymerase chain reaction-amplified products can also be used. Detection is achieved through target-specific signal amplification instead of target amplification. The assay is a highly accurate and specific detection method for both qualitative and quantitative analysis of single-nucleotide changes, insertions or deletions, gene copy number, infectious agents, and gene expression.

Key Words: Genotyping; polymorphism; SNP; DNA; insertion; deletion; gene; FRET; probe; homogeneous; quantitative.

1. Introduction

The Invader (Invader® and Cleavase® are registered trademarks of Third Wave Technologies) assay is an isothermal DNA probe-based system for quantitative detection of specific nucleic acid sequences. Invader reactions can be performed directly on genomic DNA or total RNA targets or on polymerase chain reaction or reverse transcriptase polymerase chain reaction-amplified products. A target-specific signal is amplified, but not the target itself. The Invader assay is a highly accurate and specific detection method for both qualitative and quantitative analysis of single-nucleotide changes, insertions or deletions, gene copy number, infectious agents, and gene expression (1).

The Invader assay is based on the ability of a Cleavase enzyme to recognize and cleave a specific nucleic acid structure generated by an overlap of two oligonucleotides (oligos)—the invasive oligo and the primary probe—on the nucleic acid target (Fig. 1). Cleavase enzymes belong to a family of both natu-

From: Methods in Molecular Biology, vol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols Edited by: V. V. Didenko © Humana Press Inc., Totowa, NJ

Fig. 1. Schematic of the Invader DNA assay for single-nucleotide polymorphism detection. Invasive structure forms from singlebase overlap between the Invader oligo and the Probe when hybridized to complementary target DNA. The vertical arrow indicates the cleavage site. The cleaved 5' flaps from the primary reaction are used as Invader oligos in the secondary fluorescence resonance energy transfer (FRET) reaction. Specific cleavage of multiple FRET cassettes results in fluorescent signal generation. The primary and secondary Invader reactions occur simultaneously. (Figure provided by Third Wave Technologies Inc. Madison, WI.)

Fig. 1. Schematic of the Invader DNA assay for single-nucleotide polymorphism detection. Invasive structure forms from singlebase overlap between the Invader oligo and the Probe when hybridized to complementary target DNA. The vertical arrow indicates the cleavage site. The cleaved 5' flaps from the primary reaction are used as Invader oligos in the secondary fluorescence resonance energy transfer (FRET) reaction. Specific cleavage of multiple FRET cassettes results in fluorescent signal generation. The primary and secondary Invader reactions occur simultaneously. (Figure provided by Third Wave Technologies Inc. Madison, WI.)

c rally occurring and engineered thermophilic structure-specific 5' endonucleases (2). The DNA Cleavase enzymes are generated from members of the flap endonuclease-1 family, typically isolated from thermophilic archaebacteria, with no associated DNA polymerase activity (2). The RNA assays use Cleavase enzymes derived from the 5' exonuclease domain of DNA polymerase I found in thermophilic eubacteria (3). The optimal substrate for these nucleases is comprised of distinct upstream, downstream, and template strands, which mimics the replication fork formed during displacement synthesis. The enzymes cleave the 5'-end of downstream probe on the 3'-side of the invaded base, removing the single-stranded 5'-arm or flap (2,4).

The generation of the proper enzyme substrate is dependent on base pairing at an exact position between the primary probe and the target nucleic acid, which provides the ability to discriminate single base changes. The primary probe consists of two functionally distinct regions: a 5'-flap sequence and a 3' target-specific region (TSR). The sequence of the 5'-flap varies in length (typically 10-15 nucleotides), and because it is independent of the target, can consist of any sequence. The 3'-base of the invasive oligo overlaps with the TSR of the primary probe at a base referred to as "position 1," creating a substrate for the Cleavase enzyme. The specificity of the Cleavase enzymes requires that position 1 of the primary probe be complementary to the target for cleavage to occur. A noncomplementary base in the target at position 1 results in the formation of a nicked structure, rather than an invasive structure. Position 1 of the primary probe then becomes part of the flap that does not associate with the target. The nicked structure is a very poor substrate for the Cleavase enzyme, and, thus, the primary probe is not cleaved. Discrimination of specific targets relies upon enzymatic recognition of the properly assembled structure in addition to the sequence specificity of oligonucleotides to the target sequence.

In contrast to the invasive oligo, which forms a stable complex with the target, the duplex of primary probe and target is designed to be unstable at the reaction temperature to allow for the exchange of cleaved and uncleaved probes onto the target. When the specific sequence is present, the invasive oligo and TSR of the primary probe form an invasive structure on the target. The Cleavase enzyme removes the noncomplementary 5'-flap plus position 1 of the target specific region. Following cleavage, the primary probe dissociates from the target and is replaced with another uncleaved primary probe. Thus, numerous 5'-flaps are generated for each target molecule present, resulting in a linear amplification of signal without target amplification.

For detection, the 5'-flap forms another invasive structure with a generic sequence fluorescence resonance energy transfer (FRET) oligo, which contains a donor fluorophore on the overlapping base (position 1) and a quencher dye on the other side of the cleavage site. The Cleavase enzyme removes the position 1

nucleotide, thus, separating the donor fluorophore from the quencher dye and generating a fluorescence signal. Using two different 5'-flap sequences and their complementary FRET oligos with spectrally distinct fluorophores allows for two sequences to be detected in a single well. In the DNA assay, the 5'-flap forms an unstable, invasive duplex with a FRET oligo. After the fluorophore has been released, the 5'-flap dissociates and may form an invasive structure with a new, uncleaved FRET oligo. Both reactions, cleavage of the primary probes and of the FRET oligos, occur simultaneously at a single temperature near the melting temperature of the primary probe (Fig. 1).

The methods described here include the design, setup, and analysis of Invader assays for the A313G polymorphism on Glutathione-S-Transferase P gene (GSTP1_A313G) and for the copy number quantitation of the Glutathione-S-Transferase M gene (GSTM1_null).

2. Materials

1. Sample preparation: genomic DNA samples extracted by different methods and kits have been used in conjunction with Invader DNA assays (5-7). We have successfully used extracted DNA purchased from Coriell Cell Repositories or DNA extracted from whole blood, buffy coat, or cultured cells using the following DNA extraction kits: the QIAamp™ Blood kit (Qiagen, cat. no. 51104), the Puregene® kit (Gentra Systems, cat. no. D-5500), the MasterPure™ kit (Epicentre, cat. no. MG71100). These kits yield 20-60 |g/mL blood or 20-30 |g DNA/107 cells. The purified DNA should be dissolved in a volume sufficient to yield a DNA concentration of at least 15-20 ng/|L. Purified DNA can be stored at -20 to 4°C.

2. DNA quantitation: if using a DNA isolation/preparation method other than those recommended (or if modifying one of the recommended methods), verify that the yield of DNA is sufficient (at least 15-20 ng/|L), e.g., by using the PicoGreen® assay (Molecular Probes, Eugene, OR; cat. no. P7589).

3. Invader DNA Assay Reagents:

a. Assay specific probe mix: a number of Invader DNA assays for detection of gene-specific single-nucleotide polymorphism (SNP), insertion/deletions or gene copy number determination are available from Third Wave Technologies as Research Use Only kits. The assay-specific probe mix contains the primary probe oligonucleotide(s) and the invasive oligonucleotide(s). The probe mix typically contains 2.5 ||M each probe, 0.25 ||M invasive oligo in 10 mM 4-morpholinepropane sulphonic acid (MOPS) pH 7.5, and 0.1 mM EDTA.

b. Generic reagents: Invader Reagent Core Kit (Third Wave Technologies, cat. no. 91-219), a generic reagent kit optimized for Invader DNA Assays, can be used for most Invader DNA assays for detection of gene-specific SNP, insertion/deletions, or gene copy number determination. This kit contains the following reagents:

i. Cleavase XI/MgCl2 Enzyme mix: 80 ng/mL Cleavase XI, 240 mM MgCl2.

ii. Cleavase XI FRET mix: 43 mM MOPS, pH 7.5, 10.7% polyethylenglycol 8000, and 1.37 mM each FRET oligo.

iii. No Target Blank: brewer yeast transfer RNA 10 mg/mL in 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0.

4. Microtiter plates and fluorescence microtiter plate reader: the assay described here can be performed in any type of 96-well microtiter plate and results can be read on any fluorescence microtiter plate reader equipped with filters or mono-chromator capable of detecting at least 109 molecules of fluorescein and Redmond Red™ Dye (Epoch Biosciences, Redmond, WA). We recommend using skirted or low profile polypropylene Microtiter Plates 96 (MJ Research, cat. no. MSP-9601 or MLL-9601, respectively) or equivalent plates and a GENios (Tecan Group, Maennedorf, Switzerland) or Cytofluor 4000 fluorescence (Applied Biosystems, Foster City, CA) microtiter plate readers. The Invader reactions performed in these microtiter plates can be directly quantitated by a Cytofluor 4000 or GENios reader, which eliminates an additional step of transferring the reactions into special read-out plates.

5. Incubation: any thermal cycler or air incubator, equipped to handle 96-well microtiter plates and capable of maintaining 63°C with precision of ±1°C can be used for the Invader assay described here (see Note 1).

3. Methods

3.1. Design of the Primary Probes, Invasive Oligos, and FRET Cassettes for SNP Genotyping and Gene Copy Number Determination

1. To design an Invader assay for SNP genotyping, the sequence of 50-60 nucle-otides flanking each side of the polymorphic site of interest on the target must be known. Although either the sense or antisense DNA strand can be used as target, certain features of the probes, such as four or more Gs in a row or sequences that might cause the TSR of the primary (signal) probe to form a secondary structure with its 5'-flap region, indicate that the opposite target strand should be chosen instead.

2. Primary probes used in the Invader assay have a 5'-flap and a TSR. For SNP or insertion/deletion detection, the base at the polymorphic site on the target DNA determines the base at the 5'-end of the TSR. In addition, the length of the TSR is chosen so that the melting temperature (Tm) of the probe-target duplex is approx 63°C. The Tm can be calculated with the Hyther program developed by Peyret and SantaLucia at Wayne State University (http://ozome2.chem.wayne.edu/ Hyther/hythermenu.html) or by any similar program using nearest-neighbor parameters for DNA (8,9) and including the concentrations of the probe 0.5 |M. Because the TSR of each primary probe will detect only one polymorphic nucleotide at the SNP site, two unique TSRs must be designed for a typical bi-allelic polymorphic locus (compare Fig. 1A,B). To complete the primary probe design, the TSR is extended at the 5'-end with one of the universal 5'-flap sequences.

These universal 5'-flap sequences are independent of the target sequence. As a result, practically any DNA assay can use primary probes designed with different TSRs but the same two 5'-flap sequences (e.g., one for each of two alleles being detected). Following these rules, we designed primary probes for the GSTP1_A313G polymorphism, which causes a substitution of an isoleucine at codon 105 for a valine (GSTP1_I105V): the A-specific (5'-ACG-GAC-GCG-GAG-ATC-TCC-CTC-ATC-TAC-ACC-hex-3') and G-specific (5'-CGC-GCC-GAG-GGT-CTC-CCT-CAT-CTA-CAC-C-hex-3') primary probes. The nucleotides complementary to the polymorphic site are shown in bold case and the universal 5'-flap sequences are in italic case. Typically, the primary probes are synthesized using controlled pore glass columns containing a C6 spacer (hex) or an amine group to minimize background signal generation.

3. Probes used in copy number assays are designed with the same criteria as those used in SNP assays, but the TSR of one probe detects the gene of interest and the other probe detects a reference gene (such as §-actin) that is not known to be polymorphic for either duplication or deletion. The site selection for both the reference gene and the gene sequence to be quantitated is assessed for homology with other genes across the genome. Sequence alignments between related genes can identify nonhomologous regions, which are the best candidates for cleavage site positioning. Sequence homologies can be identified using the UCSC Genome Bioinformatics site, http://genome.ucsc.edu/cgi-bin/hgBlat, or the NCBI BLAST site, http://www.ncbi.nlm.nih.gov/BLAST/. Following these recommendations, we designed the GSTM1 -specific (5'-ACG-GAC-GCG-GAG-GCA-CAA-GAT-GGC-GTT-hex-3') and the reference-specific (5'-CGC-GCC-GAG-GCA-GGT-AGT-CGG-TGA-GAT-C-hex-3') primary probes for the GSTM1 _null assay.

4. The design of the invasive oligo starts with its 3' terminal nucleotide. This nucleotide overlaps with the first base of the primary probe's TSR and should be noncomplementary to the polymorphic nucleotides at the interrogated site, following the preference order T = C > A > G. This design feature allows the use of the same invasive oligo with both primary probes in typical SNP assays. In copy number assays, there are two invasive oligos: one for the target gene and one for the reference gene. Except for its 3' terminal nucleotide, the invasive oligo is complementary to the target. The length of the invasive oligo is chosen so that the Tm of the probe-target duplex is approx of 73-78°C or 10-15°C higher than that of the primary probe. Following these rules, the sequence of the invasive oligo for the GSTP1 _A313G assay is 5'-GCG-TGG-AGG-ACC-TCC-GCT-GCA-AAT-ACT-3'. Analogously, the sequence of the invasive oligos for the GSTM1_null assay are 5'-CCA-CAC-AGG-TTG-TGC-TTG-CGG-GCA-ATG-TAT-3' and 5'-AGG-AGT-AGC-CAC-GCT-CGG-TGA-GGA-TCT-TCA-TT-3' for the GSTM1 and f>-actin TSR, respectively.

5. Synthetic target controls: the inclusion of synthetic target controls is optional. However, they are often useful, particularly in the case of SNP assays. Such targets should typically be designed to encompass the area targeted by the Invader and probe oligos plus and additional two to three bases on each end.

6. The two FRET cassettes complementing the 5' flaps of the primary probes complete the design of the Invader assay. Like the 5' flaps, the two FRET cassettes are designed to be universal; the identical FRET cassettes can be used successfully in practically any Invader reaction. The sequences of the FRET cassettes containing FAM dye (Glen Research, Sterling, VA) and Redmond Red™ dye (Epoch Biosciences, Redmond, WA) utilized for detection of the GSTP1 _A313G and GSTM1 _null assays are 5'-FAM-TCT-E-AGC-CGG-TTT-TCC-GGC-TGA-GAC-CTC-GGC-GCG-hex-3' and 5'-Redmond Red-TCT-E-AGC-CGG-TTT-TCC-GGC-TGA-GAC-TCC-GCG-TCC-GT-hex-3', respectively. Both probes use the Eclipse™ dye (Epoch Biosciences) as a quencher, but dabcyl-dT (Glen Research) can be used instead of the Eclipse dye.

3.2. Synthesis and Purification of the Oligonucleotides

1. Synthesis of oligonucleotides for the Invader assay may be carried out using standard phosphoramidite chemistry. The primary probes are typically purified by gel isolation or anion exchange high-performance liquid chromatography to reduce nonspecific background signal generation (10). The invasive oligo can be used without any further purification.

2. Dissolve the invasive and purified primary probes in 10 mM Tris-HCl, pH 7.8, 0.1 mM EDTA and determine their concentrations by measuring their absorption at 260 nm and using the extinction coefficients 15,400, 7400, 11,500, and 8700 A260 M-1 for A, C, G, and T, respectively.

3. Dilute the primary probes and the invasive oligos together to a final concentration of 2.5 ||M for each primary probe and 0.25 |M for each invasive oligo(s) in 10 mM MOPS (pH 7.5), 0.1 mM EDTA.

3.3. Sample Preparation

Recommended quantities of genomic DNA per assay are typically 100-150 ng DNA in a 15 |L reaction in the 96-well microtiter plate format described in this protocol, although some Invader assays for other applications require 4-10 times less DNA per reaction. A 7.5-|L aliquot of genomic DNA at 15-20 ng/|L, prepared with a standard procedure for DNA extraction (see Subheading 2.1.), is required for the reaction.

3.4. Invader Assay Setup

The following procedure applies to a manual set up of the Invader DNA assays.

1. Plan the microtiter plate assay layout.

2. Prepare the appropriate amount of Master Mix, by combining the Probe mix, FRET mix, and Cleavase XI/MgCl2 solution at the ratios listed on Table 1.

3. Dispense 7.5 ||L of DNA sample at 15-20 ng/|L (approx 100-150 ng of genomic DNA) to the appropriate wells. Use one well as a blank by adding 7.5 |L of the No Target Blank solution (see Note 2). Overlay each well with 15 |L of mineral

180 Mast and de Arruda Table 1

Sample Reaction Setup

Master Mix (A) Volume (B) Number (A) x (B) = Volume component per reaction of reactions required of reagent

Cleavase XI FRET mix 3.5 |L

Cleavase XI enzyme/ 1.0 ||L

MgCl2 solution

Total volume 7.5 |L

oil (Sigma, cat. no. M 3516) to prevent evaporation (see Note 3). Denature the DNA by incubating the microtiter plate at 95°C for 5 min in a thermal cycler.

4. Remove the plate from the thermal cycler.

5. Add 7.5 |L of Master Mix into the appropriate wells of the 96-well plate and seal the plate with an adhesive cover.

6. It is optional to spin the plate at 250g in a Beckman GS-15R centrifuge (or equivalent) for 10 s to force the probe and target into the bottom of the wells. Alternatively, mix the reagents in each well by pipetting the solution up and down several times avoiding bubble formation.

7. Incubate the plate(s) at 63°C for 4 h in a thermal cycler or other incubation device.

8. After 4 h incubation at 63°C, lower the temperature to 4-10°C for thermal cyclers or to room temperature for incubators.

9. Directly read the signal from the FAM and Redmond Red dyes using a GENios or CytoFluor fluorescence plate reader with the following excitation and emission wavelength and bandwidth that are listed on Table 2. If the plates cannot be read immediately after the 4-h incubation at 63°C, store the plate(s) at 4-25°C. Fluorescence signal from reactions kept at less than 25°C in the dark is stable for up to 24 h.

3.5. Data Analysis

1. Import the fluorescence data into Microsoft Excel® or other spreadsheet analysis program. Determine the Fold Over Zero (FOZ) values for the FAM signal (G Allele or P-actin gene) and the Redmond Red signal (A allele or GSTM1 gene) by dividing the raw counts form the sample well by the raw counts of the No Target Blank well.

2. Calculate the Allelic Ratio of each sample according to the equation below:

Allelic Ratio (GSTP1 A313G) = Net FOZ for GSTP1 "G" probe

Net FOZ for GSTP1 "A" probe

*„ v r, • Net FOZ for GSTM1 probe Allelic Ratio (GSTM1) = -¡--

Net FOZ for a-actin probe

Table 2

Recommended Excitation and Emission Filters

Dye specific settings CytoFluor GENios

F Dye excitation 485/20 nm 485/20 nm

F Dye emission 530/25 nm 535/25 nm

R Dye excitation 560/20 nm 560/20 nm

R Dye emission 620/40 nm 612/10 nm

See Note 8.

Where Net FOZ = FOZ - 1. For cases in which the Net FOZ value is <0.1, set the value to 0.1 for the calculation of the Allelic Ratio value. A minimum Net FOZ of 0.6 is typically recommended for at least one of the alleles in order to make a valid allelic ratio calculation (see Notes 4 and 5).

The Allelic Ratio and the FOZ values are used to confirm the validity of each assay and determine the genotype or gene copy number of the samples (see Note 6). Based on these calculations, the results of the genotyping GSTP1_A313G assay can be classified for each sample as:

a. Homozygous for A Allele.

b. Homozygous for G Allele.

c. Heterozygous.

d. Equivocal.

e. Low signal.

Results for the copy number assay can be classified into these categories:

c. Two copies.

d. Equivocal.

e. Low signal.

3. Generic interpretations of the allelic ratio values for the GSTP1_A313G Invader DNA assay and SNP Invader DNA assays are listed in Table 3. However, each laboratory is encouraged to establish its own Allelic Ratio ranges for interpreting results of individual Invader assays (see Note 7). Cluster analysis of the GSTP1_A313G Invader DNA assay data obtained for 40 genomic DNA samples following the procedures described in Subheadings 3.4. and 3.5. is shown in Fig. 2. The homozygous A/A samples are depicted as squares, the homozygous G/G samples are depicted as circles and the heterozygous samples are depicted as diamonds.

4. Interpretation for the allelic ratio of the GSTM1 _null Invader DNA assay and copy number Invader DNA assays in general. For Invader copy number assays, sample results can be analyzed in any of three ways:

a. Graphing results using a scatter plot and visually distinguish individual genotypes by clusters.

Table 3

Interpretation for the Allelic Ratio of the GSTP1_A313G Invader DNA Assay and SNP Invader DNA Assays in General

Allelic ratio

Genotype

Equivocal (see Note 7)

Fig. 2. Example of graphical analysis of the results obtained for the analysis of 40 samples of human genomic DNA using the GSTP1 _A313G Invader DNA Assay. (A) Plot of Net FAM FOZ and Net RED FOZ values. (B) Scatter plot of the allelic ratio values. The cluster of diamonds consists of samples homozygous for the A allele (A/ A). The cluster of circles represents the homozygous for the G-allele (G/G). The cluster of squares consists of heterozygous samples (A/G).

Fig. 2. Example of graphical analysis of the results obtained for the analysis of 40 samples of human genomic DNA using the GSTP1 _A313G Invader DNA Assay. (A) Plot of Net FAM FOZ and Net RED FOZ values. (B) Scatter plot of the allelic ratio values. The cluster of diamonds consists of samples homozygous for the A allele (A/ A). The cluster of circles represents the homozygous for the G-allele (G/G). The cluster of squares consists of heterozygous samples (A/G).

b. Using the allelic ratio values as demonstrated in Subheading 3.5.2. and interpreting it as listed on Table 4.

c. Using a normalized allelic ratio strategy as described by Neville et al. (11). Briefly, to minimize run-to-run variation and intrinsic differences in probes performance, the allelic ratio can be normalized using a reference sample that has been verified to contain two copies of the gene of interest. Subsequently, determine the allelic ratio of the sample of interest and divide it by the allelic ratio of the reference sample. This value is multiplied by two to obtain the final normalized allelic ratio.

Table 4

Interpretation for the Allelic Ratio of the GS7M7_Null Invader DNA Assay and Gene Copy Number Invader DNA Assays in General

Allelic ratio Normalized allelic ratio Gene copy number

0.2, 0.6 or 0.9, 1.2 0.25, 0.65 or 1.35, 1.65 EQ (see Note 7)

Normalized Allelic Ratio -

Allelic ratio of two copies reference sample

Generic interpretations of the allelic ratio values for the GSTM1 _null Invader Assay and copy number Invader DNA assays meant to distinguish between null, one and two gene copies per genome are listed in Table 4. As in the case of the SNP assays, each laboratory is encouraged to establish its own Allelic Ratio ranges for interpreting results of individual Invader assays.

Figure 3 indicates a graphical representation of the different types of analysis for the GSTM1 _null Invader DNA assay. Samples marked as circles have no copies of GSTM1 (null), samples marked as squares have one copy of GSTM1 gene, and samples marked as diamonds have two copies of GSTM1 gene.

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