High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification
© Faruqi et al; licensee BioMed Central Ltd. 2001
Received: 30 May 2001
Accepted: 1 August 2001
Published: 1 August 2001
Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability.
We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation.
SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe.
Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.
Sequencing studies of human transcriptomes and genomes have identified hundreds of thousands of single nucleotide polymorphisms (SNPs), the most common type of human genetic variation . Human SNPs are being assembled into extremely high-density genetic maps that are anticipated to considerably expand the remit of genetic analyses . Broad availability of SNPs for candidate genes will enhance pharmacogenomic and complex trait association studies. Furthermore, extremely dense SNP maps have the potential to make possible genome-wide association studies for complex traits that bypass shortcomings of current genetic linkage analyses [3, 4].
The emerging era of multiplexed SNP-based genetic analysis has underscored a need for simple and accurate genotyping methods that can accommodate thousands of loci with economy of cost and consumption of sample DNA. In general, current methods require pre-amplification of genomic DNA, typically by Polymerase Chain Reaction (PCR), followed by SNP genotyping with an allele discrimination method, such as DNA cleavage, ligation, single base extension or hybridization . Current methods are limited either by expense, inaccuracy, consumption of sample DNA, or lack of scalability.
Recently a new method for SNP detection from genomic DNA based on DNA ligase-mediated single nucleotide discrimination and signal amplification by Rolling Circle Amplification (RCA) has been described [6–10]. An oligonucleotide Open Circle Probe (OCP) anneals to the target SNP such that the 5' and 3' ends of the OCP can be ligated together forming a circle topologically linked to the target. A base-pair match between the 3' end of the OCP and the SNP allows DNA ligase to circularize the OCP. A mismatch between the OCP and the SNP prevents ligation and circularization. In this manner, single base selectivity is achieved not only by the specific hybridization of the OCP ends to target sequences adjacent to the SNP, but also by the highly discriminative nick closure activity of the thermostable DNA ligase toward a perfectly matched substrate. Upon OCP circularization, an isothermal exponential RCA (ERCA) reaction involving an exonuclease (-) DNA polymerase with strand-displacement activity and two primers rapidly amplifies the signal by as much as 1012-fold, allowing for direct SNP genotyping from small quantities of DNA target [6, 11, 12].
We describe here a simple, scalable assay for SNP genotyping directly from human genomic DNA that uses a 96-well plate format and fluorescent primers called Amplifluors™ [13–15, 12, 16]. Ten different SNPs have been characterized, each for DNA samples from 192 different individuals, using this reporter assay system. We also report that the ERCA allows genotyping with as few as ∼ 300 copies of the target sequence.
Upon ligation, the DNA circle creates an effective template for an exponential, or hyperbranching, RCA reaction [6, 11]. The region of the OCP between the target-specific ends, which we have designated the OCP backbone, provides a binding site for a complementary primer (P1). In addition to P1, a reverse primer (P2) is also present to achieve amplification with exponential kinetics (Fig. 1). P1 is an "Amplifluor™"-primer, with a 5' hairpin and loop structure with a fluorophore and quencher at the base of the hairpin arms [13, 12, 16]. The two allele-specific OCPs have different backbone sequences complementary either to a FAM- or TET- labeled P1 allowing discrimination of the polymorphic base. Both P1s have an internal DABCYL quencher. Amplifluor primers are designed such that, at the ambient temperature of the assay, the fluorophore and the quencher are in close proximity, preventing fluorescence. Incorporation of Amplifluor into a double-stranded ERCA product opens the hairpin separating the fluorophore and quencher, resulting in increased fluorescence. Two different allele-specific P2s are also used in the assay. They are partly target- and partly backbone-specific, corresponding to the 3' target-specific region of the OCP, and 7–12 nucleotides of the adjacent backbone sequence. The backbone-specific 5' region of the allele-specific P2s not only increases the Tm of the primer to that desired for the assay, but also confers OCP specificity. P2s have two abasic residues at the 5' end to reduce primer-dimer formation (unpublished data).
Analysis of allele discrimination during the OCP ligation step of the assay
Analysis of the ERCA signal amplification step of the assay
Sequence of oligonucleotides used in the study.
Oligonucleotides used in the studies of ligation
kinetics, affect of OCP 3' arm length, and ligation
x = T in wild type and A in mutant
Y = A in wild type and T in mutant. The number 12,
15, 20 or 23 refers to the number of base pairs the
3' end forms with the target.
SNP genotyping on human genomic DNA
The G1822A SNP on chromosome 13q32 was used for genotyping with OCPs using the SNP assay (B. Grimwade, unpublished). Human genomic DNA was digested with the restriction endonuclease Alu I and assayed in two different tubes, each containing one of the two allele specific OCP. Reactions contained 0.5 pM OCP, and 100 ng of the genomic DNA, corresponding to a gene copy number of approximately 30,000. OCP ligation was performed with Ampligase for 20 min at 60°C, approximately 15°C above the Tm for the OCP 3' arm, in order to maximize specificity of ligation. Since the 5' arm of the OCP has a Tm above that of the ligation temperature, the 5' arm hybridizes to its target in a stable manner, and SNP specificity is achieved via the 3' arm.
Validation of the assay for ten SNPs for 192 individuals
Summary of 10 SNPs genotyped on genomic DNA.
96 NIMH DNA samples
96 Coriell DNA samples (HD100CAU)
Single-tube assay for SNP genotyping using a low copy number of targets
The above experiments involved a two-tube assay with 100 ng of genomic DNA and one OCP/P2 combination per tube. Therefore, a total of 200 ng genomic DNA was used per DNA sample to be investigated. One of the objectives for high-throughput screening of SNPs is the ability to accurately detect a SNP from a small amount of genomic DNA. Previously, it was shown that 20 ng of genomic DNA, equivalent to ∼ 6000 copies of the gene, was sufficient to detect a C/T polymorphism using the serial invasive signal amplification reaction (SISAR) . In order to investigate the sensitivity of ERCA for detecting SNPs, 1 ng of genomic DNA, equivalent to ∼ 300 copies of the gene, was used as target for circle formation with OCPs for the SNP G1822A.
We have demonstrated a solution-based, efficient, homogeneous and robust assay for genotyping SNPs directly from human genomic DNA utilizing ligation of open circle probes and rolling circle amplification. The design of the assay is fairly straightforward and the assay can be carried out in three hours (30 min for the denaturation / annealing / ligation reaction and 2 1/2 h for the amplification reaction) in a 96-well format. Specificity of the OCP to its target sequence is achieved by the complementarity of the two ends of the OCP to the target and the requirement of these ends to be adjacent for ligation. Single nucleotide discrimination is achieved at the ligation step by the use of the thermostable DNA ligase, Ampligase, which has a high affinity for a perfectly matched substrate at the 3' end of a DNA molecule . We were able to enhance allele discrimination at the ligation step by designing the OCP such that the 5' complementary region is firmly hybridized to the target sequence whereas the 3' region is in equilibrium with its target at the ligation temperature . This would result in increased specificity since the correctly matched OCP will have a greater chance to act as substrate for the ligase. It has been previously reported that 3'-T/G and 3' G/T mismatches are not good substrates for single nucleotide discrimination . However, we found that the G1822A SNP, which would result in a 3'-T/G mismatch, was efficient for allele discrimination. The ability of this assay to genotype any SNP regardless of the base pair involved is an important advantage over assays based on primer extension such as PCR.
Allele discrimination achieved at the ligation step results in small circular DNA molecules topologically linked to the target DNA strand. These DNA circles are amplified in the powerful homogeneous ERCA reaction capable of 1012-fold signal amplification , similar to that achieved with PCR technology, the current gold standard in genetic analysis and quantitation. However, PCR involves exponential target amplification, thereby increasing the risk of amplicon cross-contamination. Even though this shortcoming can be overcome, it increases the cost and complexity of the assay, making it less attractive for high-throughput analysis. Since ERCA is a signal (circle) amplification method, it does not have the problems associated with PCR. In addition, ERCA is an isothermal reaction and the reaction endpoint can be used as the assay readout. Even though the present study was conducted on a real-time ABI 7700 Sequence Detector instrument the strategy can be easily adapted for a simple fluorescence plate reader coupled to an adjustable heating block. These properties make it an ideal system for high-throughput analysis.
The assay was tested for 10 SNPS on two sets of 96 different DNA samples (Table II). Results were compared to the known genotypes that we determined by RFLP or single nucleotide sequencing reactions. The assay had an average genotyping accuracy of 93% when samples were screened initially. When the mis-scored samples were repeated in triplicate, the genotyping accuracy jumped to over 99%. The majority of the mis-scoring involved a homozygous sample being called heterozygous, i.e., an amplification signal was observed with both sets of OCP/primer combinations. A DNA sample homozygous for one allele was never genotyped as homozygous for the other allele. This implied that there is a low frequency of DNA synthesis artifacts resulting in a fluorescence signal.
Indeed when these reactions were analyzed on an agarose gel, the size of the characteristic ERCA DNA ladder was different from that obtained with amplification of the OCP (data not shown). We have used abasic residues at the 5' ends of P2 primers in order to reduce these artifacts. Other nucleotide modifications that have been reported to reduce primer-dimer formation will be tested to improve the accuracy of SNP genotyping. In addition, reagents that have been shown to reduce primer-dimer formation in PCR will be tested in the ERCA reaction.
Background signal is sometimes related to the amount of OCP used in the assay. For each SNP, the optimal OCP concentration needs to be determined before screening. Excess OCP concentrations result in an increase in non-specific fluorescence signal, therefore lowering the accuracy rate of genotyping. Potentially, un-ligated OCP can act as template or primer giving rise to a low level of non-specific DNA synthesis and subsequent fluorescent signal. We are currently developing a modified OCP design to overcome the need for concentration optimization. In this design, the 3' region of the OCP forms a stable hairpin-loop structure and opens up only to anneal to it target sequence (O. Alsmadi, unpublished). As with molecular beacons, this may also improve target specificity [19, 20]. Any unused OCP is self primed and extended to form an inert double stranded molecule, thus eliminating OCP as a source for non-specific amplification. Initial experiments with this design have been encouraging.
We have described a solution-based SNP genotyping assay that is simple, sensitive and robust and can be easily formatted to high-throughput analysis of single nucleotide polymorphisms and mutation detection directly from human genomic DNA. The SNP genotyping assay uses a simple, homogeneous, fluorescence readout and can be carried out on inexpensive instruments already available in many academic and industrial laboratories.
Amplifluor is a trademark of Intergen Company.
Ampligase is a trademark of Epicentre Technologies Corp.
Materials and methods
Table I shows the sequences of the oligonucleotides used in the study. Gel-purified and phosphorylated OCP oligonucleotides were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Abasic and Amplifluor primers were synthesized in-house and purified by HPLC. The two Amplifluors are labeled with a differently colored fluorophore: fluorescein (FAM) and tetrachloro-6-carboxyfluorescein (TET) and contain an internal nonfluorescent quencher DABCYL.
DNA denaturation, annealing and ligation reactions were carried out in an Eppendorf Master Cycler (Eppendorf Scientific, Germany). ERCA reactions were performed in the Real-Time ABI 7700 Sequence Detector (Perkin Elmer).
Genomic DNA samples were obtained from (a) National Institute of Mental Health (NIMH) Center for Genetic Studies, Rutgers University Cell and DNA Repository, Piscataway, NJ and (b) Coriell Cell Repository (HD 100 CAU). The DNA samples were digested with the restriction endonuclease Alu I before being used as template in the ligation reaction.
DNA annealing and ligation
The reactions were set up in 96-well MicroAmp Optical plates (Perkin Elmer) in a 10 μl reaction volume containing 1 U Ampligase (Epicentre Technologies), 20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, and 0.01% Triton® X-100. Standard reactions contained 0.5 pM OCP and 100 ng of Alu I digested genomic DNA. DNA was denatured by heating the reactions at 95°C for 3 min followed by annealing and ligation at 60°C for 20 min.
20 μl of ERCA mix was added to the 10 μl ligation reaction. The ERCA mix contained 5% tetramethyl ammonium oxalate , 400 μM dNTP mix, 1 uM each of the two primers, 8 u of Bst polymerase (New England Biolabs, MA), and 1X modified ThermoPol reaction buffer containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4 and 0.1% Triton X-100.
Kinetics of the ligation reaction
All ligation reactions were in 1x Ampligase Reaction Buffer supplied by Epicentre Technologies: 20 mM Tris-HCl (pH8.3 at 25°C), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD+, and 0.01% Triton X-100. The OCP were synthesized with a 5' phosphate and labeled with 32P by the T4 kinase exchange reaction (following Life Technologies suggested protocol for T4 PNK). 40 nM CFTR M1101K12 OCP (Table I) was annealed to 100 nM oligo target, at 95°C, 2 min, and slow cooled to room temperature. Reactions were started by adding Ampligase at 0.2 units/μl final concentration in a reaction volume of 0.1 ml. Reactions time points were taken at 15, 30, and 60 sec for matched OCP and 1, 2, 4, and 6 hours for mismatched OCP. 3.5 μl was resolved on an 8% PAGE sequencing gel.
OCP 3' arm length and ligation reaction temperature
OCP were designed with 12, 15, 20, or 23 nt 3' arms (Table I). All components except Ampligase were pre-warmed to the reaction temperature (40, 47, 55, or 63°C) for 20 min to allow OCP annealing to come to equilibrium. Reactions were started by adding Ampligase at 0.2 units/μl final concentration and a reaction volume of 0.1 ml. Reactions conditions were the same as for kinetics of the ligation reaction above except for the reaction temperatures indicated. Reactions were stopped at 1 min for matched OCP and 4 hours for mismatched OCP by digestion with 0.75 units/μl Exonuclease I and 1.25 units/μl T7 gene 6 exonuclease (Amersham Pharmacia Biotech, Inc, NJ) at 37°C for 1 h, except the zero time points. The digestion removes all but circular DNA. 3.5 μl was resolved on an 8% PAGE sequencing gel.
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