- Methodology article
- Open Access
High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay
- Osama A Alsmadi†1,
- Carole J Bornarth†1,
- Wanmin Song1,
- Michele Wisniewski1,
- Jing Du1,
- Joel P Brockman1,
- A Fawad Faruqi1,
- Seiyu Hosono1,
- Zhenyu Sun1,
- Yuefen Du1,
- Xiaohong Wu1,
- Michael Egholm1,
- Patricio Abarzúa1,
- Roger S Lasken1 and
- Mark D Driscoll1Email author
© Alsmadi et al; licensee BioMed Central Ltd. 2003
- Received: 4 February 2003
- Accepted: 30 May 2003
- Published: 30 May 2003
Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.
A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.
Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.
- Genotyping Assay
- Whole Genome Amplification
- Factor Versus Leiden
Sequencing of the human genome has led to the identification of mutations and single nucleotide polymorphisms (SNPs) that can be linked with specific phenotypes. A number of assays have been developed to genotype known SNPs and mutations. However, the majority of these methods have many steps and are difficult to automate. Currently, most genotyping methods require that the target region of the genomic DNA be amplified using PCR prior to the genotyping assay (reviewed in [1, 2]). Following amplification, the PCR product is either sequenced directly, or probed for the mutation of interest. Similarly, OCP ligation/ERCA can be used to accurately genotype PCR products in a biallelic format, where the probes for both alleles are present in a single reaction . The OCP ligation/ERCA method is based on ligation dependent circularization of allele specific DNA probes followed by exponential rolling circle amplification of the circularized probes [4, 5]. ERCA probes have also been variously described as 'padlock probes' [6, 7] or 'circularizable probes' . Although OCP ligation/ERCA can be used to genotype PCR products, it also provides a means for directly genotyping genomic DNA without requiring an initial amplification of the genomic DNA locus. For example, we recently described a high throughput ligation/ERCA protocol for genotyping genomic DNA samples in microtiter plates .
This report describes modifications to OCP ligation/ERCA genotyping that reduce reaction time and improve accuracy to above 99%. Methods for improving accuracy include simultaneous detection of two alleles per well, probes designed to contain hairpin sequences that regulate ligation discrimination and prevent nonspecific amplification, and introduction of a Q-PNA fluorescent detection system. Improvements in probe design, rolling circle reaction design, and assay methods were integrated into an improved high accuracy genotyping assay, and the utility of the method was demonstrated for four clinically important mutations. The Q-PNA fluorescent detection system produced a six-fold reduction in time to result.
In many instances only limited quantities of genomic DNA are available from clinical samples for genetic testing. DNA can also be limiting when large numbers of assays must be performed on a single sample. Recently, a whole genome amplification method (WGA) has been developed that is capable of accurately producing large quantities of genomic DNA from limited sample [10, 11]. In cases where genomic DNA was limiting, WGA genomic DNA was substituted for genomic DNA. Complete and faithful amplification of the entire genome resulted in WGA product that could be substituted for genomic DNA without affecting the accuracy of genotyping. WGA DNA amplification and OCP ligation/ERCA can be combined to create an automated high throughput mutation assay that includes DNA amplification from limiting samples and allele detection in a single tube.
Open Circle Probes
FV WT-no hairpin
5'-GCCTGTCC AGGGATCTGCTCTGTTATCGGCCGTCGCGCAGACACGATA AAGGAATACAACAAAATACCTGTATTCCTT-3'
FV Mut-no hairpin
H63 WT-no hairpin
H63 Mut-no hairpin
FV Normal P1
FV Mutant P1
FV 5' allele specific primer
FV 3' allele specific primer
FII Normal P1
FII Mutant P1
FII 5' allele specific primer
H63 Normal P1
H63D Mutant P1
H63 5' allele specific primer
H63 3' allele specific primer
C282 Normal P1
C282Y Mutant P1
5'-/5Cy3/ TCATTCGCAATCA CGGGCTGAGGTAGAA-3'
C282 5' allele specific primer
C282 3' allele specific primer
To increase the accuracy of genotyping, probes for both alleles were combined in a single tube format. For heterozygotes, the two probes are concurrently amplified in the ERCA reaction. Exponential amplification of circularized OCP requires primers P1 and P2 (see Figure 2). The concurrent amplification of mutant and normal probes is kept in balance by using the same P2 for each probe. P1 primers were routinely titrated to determine optimum concentration . This approach greatly reduces the difficulty of balancing the kinetics of biallelic ERCA amplifications. If the P2 primer is common to both OCPs, only P1 priming efficiencies need to be similar, greatly simplifying the process of obtaining balanced reaction kinetics in a biallelic assay.
The use of several P1 and P2 primers, which initiate DNA synthesis from multiple positions on the probe and product, increases ERCA rate, sensitivity and specificity (, data not shown). Therefore, secondary amplification primers were designed for each probe. In genotyping reactions where two probes were present in a single reaction, the secondary primers were designed to anneal to target specific sequences shared by both OCPs, in the 5' and/or 3' arms (Table 1).
Table of patient sample numbers and genotyping accuracy.
Number of Individual Samples Mutant:Hetero:Normal
Single Assay Accuracy
Observed Assay Accuracy in Triplicate
Factor V Leiden
Factor II Prothrombin
OCP ligation/ERCA can be used to directly probe genomic DNA for SNPs or mutations, and the entire process can be performed in a single tube. The ERCA amplification reaction is rapid, generating signal in as little as 10 minutes, and is incubated at a single temperature. These characteristics make OCP ligation/ERCA easily adaptable to high throughput automated genotyping platforms. Accurate genotyping was obtained in screens designed to detect four clinically relevant mutations, Factor V Leiden, Factor II prothrombin, Hemochromatosis C282Y and Hemochromatosis H63D.
Several improvements to the ligation/ERCA method  have increased accuracy to levels acceptable for diagnostic applications and reduced reaction time. Other reports using ERCA based genotyping require PCR amplification of the locus of interest prior to genotyping [3, 16], which is prohibitively expensive, time consuming, and more difficult to automate. We have designed ERCA primers that are optimized for minimal misamplification and artifact formation. With any exponential signal amplification method, nonspecific amplification due to exponential artifacts presents a potential problem. In ERCA as in PCR, amplification of primer-primer artifacts can mimic specific signal if primers are not selected carefully. To avoid this problem, commercially available primer design software was used to design P1 and P2 primers that are optimal for amplification. This step does not necessarily have to be repeated for each target, however, which is an advantage over PCR based methodologies. The primary amplification primer sequences are present in the OCP backbone, not in the portion of the OCP that anneals to target, allowing optimized P1 and P2 pairs, to be used for many different targets. In principle, a small collection of optimized P1/P2 pairs should contain primers that can be used for any given target.
The Q-PNA detection system  was used as a fluorescent reporter during ERCA. During ERCA, Q-PNA is rapidly displaced from the ERCA product and is physically separated from the fluor in a bimolecular reaction, resulting in detectable signal in as little as 10 minutes. By comparison, Amplifluors typically required 60–120 minutes to generate signal. The substantial difference in time to generate signal may be due to the nature of Amplifluor design. In an Ampliflour, complementary DNA sequences, fluorescent reporter, and quencher are all covalently linked on the same DNA strand. The comparative stability of the unimolecular Amplifluor hairpin is likely to hinder displacement synthesis when compared to the bimolecular Q-PNA system. In addition, the high local concentration of fluor and quencher may serve to supress signal from the Amplifluor. The Q-PNA based reporter system consistently produces signal far more rapidly than the Amplifluor based system, allowing time to result of less than an hour for the entire assay.
Secondary allele specific, nonfluorescent, primers can also be used to increase the speed and specificity of ERCA. These primers significantly advanced the rate of ERCA amplification, cutting the reaction time in half for the Factor V Leiden probe set (data not shown), resulting in a 20-minute assay. Although maximum fluorescent signal decreased slightly as each additional nonfluorescent primer was added, the addition of one or two more primers to the reaction did not adversely influence signal strength to the extent that genotyping was compromised.
Each of the four assays developed for this report contained two probes, one for each allele. Using both probes in a single reaction has been demonstrated to reduce the levels of nonspecific amplification due to primer dimers and misamplification of unligated probe . At least one of the probes will always be amplified, suppressing low levels of nonspecific signal.
The introduction of a hairpin into the design of the open circle probes, similar to approaches taken in molecular beacon design , provides a means to regulate the degree of ligation discrimination. A 3' and/or 5' stem-loop structure may be designed to increase specific binding to difficult sequences. The ratio of the hairpin stability to target annealing stability directly affects the overall target annealing specificity. By modulating the stability of the hairpin any OCP can be fine tuned for ligation specificity.
The OCP 3' hairpin also decreases the level of nonspecific DNA synthesis caused by misamplification of OCP sequences. Analysis of misamplified products has shown that OCP sequences are usually found in the misamplified DNA products. This means that uncircularized OCP is able to provide a template for primer-based misamplification. Removing the unligated OCP helps to reduce background amplification , but digestion of the unused OCP or purification of the ligated circles introduces time consuming and expensive steps. Designing a 3' hairpin into the OCP creates a self-priming sequence in unligated OCP. During ERCA, the polymerase will extend unligated OCP starting at the 3' end in a suicide pathway that renders the OCP double stranded and inert. This approach eliminates nonspecific amplification and improves genotyping accuracy without the introduction of separate isolation or purification steps. As demonstrated above, removal of the hairpin sequence either by deletion or point mutation of the base paired region resulted in greatly decreased genotyping accuracy for both Factor V Leiden and Hemochromatosis H63D genotyping assays.
Additional methods to improve ERCA specificity were integrated into the genotyping protocol. Careful design allowed the same P2 sequence to be used in the ERCA reaction for both alleles. For each set of OCPs, the P2 sequence was generic. The generic P2 was not part of the target specific portion of the probe, which allowed it to be used in both the normal and mutant OCP. Because both probes in each assay contained the same P2, the reaction kinetics could be more easily balanced, promoting uniform amplification for both alleles. Uniform amplification is especially important when the genotype is heterozygous so that both alleles are accurately represented.
Genotyping accuracy depends on using sufficient quantities of genomic DNA target. As expected, OCP ligation/ERCA results were influenced by the amount of genomic DNA used in the assay. Typically, genotyping was possible with as little as 50 ng target DNA. However, specific signal strength increased with more DNA target, up to 1000 ng, after which no further benefit was seen (data not shown). WGA DNA can be produced in milligram quantities from nanograms  of original sample allowing use of high DNA concentration for optimal signal. As a result, 1000 ng WGA DNA was used to obtain optimal genotyping reaction conditions. The results obtained for the WGA DNA agreed in every instance with the known genotypes determined by RFLP. WGA product should also be compatible with other genotyping assays where better results can be obtained using more target DNA.
Many aspects of probe design are open to manipulation, and in practice probes can be successfully designed for most target sequences of interest. Pickering et al. demonstrated a 95% success rate in designing OCPs for 99 different targets on the first attempt . Probes can be designed against either strand of genomic DNA, and the backbone portion can also be varied. Optimization of Tm parameters, and sequence allows rapid design of OCPs with a high degree of success.
The degree to which a genotyping assay needs to be optimized depends in part on the purpose of the assay. A genotyping system intended for diagnostic purposes needs to meet the highest standards of accuracy, robustness, and throughput, as compared to a system that is intended solely for research purposes. In recognition of the expectation that 100% accuracy is a requirement, even under suboptimal conditions, the assays developed for the targets in this report have undergone extensive optimization to maximize accuracy even under less than optimal conditions. As a result, the concentrations of primers, probes and polymerase vary between assays. After determining the best conditions for each individual genotyping assay, the expectation is that these conditions would be the best possible for genotyping large numbers of samples with high accuracy. The critical nature of the information derived from the output of the assay justifies the extra optimization effort. To minimize the potential for error, the accuracy for a single assay was compared to the accuray for the same assay performed in triplicate. Depending on the assay requirements, the level of accuracy can be selected by increasing or decreasing the number of repeats. The demonstration that high levels of input genomic DNA improve robustness of genotyping results is paralleled by PCR results. WGA can be used to generate sufficient DNA for genotyping using large quantities of template. The cost of performing WGA is offset by the fact that sample prep is unnecessary; WGA product can be performed directly on crude blood sample and yields enough DNA for dozens or hundreds of genotyping reactions. Under different circumstances, it may be necessary to rapidly generate assays for a large number of SNPs. For this, the emphasis could be placed on optimization of assay design. Amounts of input DNA, primers and polymerase could be standardized, rapidly yielding accurate genotyping assays but without the highest accuracy under suboptimal conditions. The process of OCP ligation followed by ERCA has been designed such that genomic DNA can be genotyped directly in less than an hour. Hundreds of individual samples were genotyped with over 99% accuracy. Accurate genotyping was demonstrated for both genomic DNA and whole genome amplified DNA. Isothermal exponential amplification produces a fluorescent signal from ligated OCP using nanomolar amounts of probe. Because OCP ligation/ ERCA does not rely on thermal cycling for amplification, amplification kinetics are not limited by cycling rate. The exponential nature of the reaction produces millions of fold amplification in as little as 20 minutes. Output can be read on a fluorescent plate reader, real time PCR instrument, fluorescent imager, or other device equipped with the ability to measure fluorescent signals. OCP ligation/ ERCA probes can be multiplexed. OCP ligation and ERCA reactions for each SNP target are performed in a single tube, and are easily scalable from 96 to 384 well formats, making OCP ligation/ ERCA ideal for high throughput screening.
Hairpin OCP Design
All oligomers were ordered from Integrated DNA Technologies, Inc.(Coralville, IA). Sequences of oligomers are shown in Table 1. All OCPs were 5' phosphorylated and gel purified. OCPs were designed such that the 3' terminal nucleotide annealed opposite the mutation of interest (Figure 1). The Tm for the 3' arm of the probe annealed to target was designed to be between 60–65°C. The 5' arm of all OCPs annealed to target upstream of the mutation and was designed to have a Tm of approximately 70°C. Oligo 6 (Molecular Biology Insights, Inc., Cascade, CO) was used to calculate Tm values.
To reduce or eliminate the artifactual amplification of unligated OCPs, a hairpin was designed into the 3' end of the OCP, with approximately 10 bases in the stem and 10 in the loop. The hairpin was calculated to be stable at 60°C in the context of the entire OCP using the DNA folding server at Michael Zucker's website http://bioinfo.math.rpi.edu/~zukerm/ OCP sequence was chosen such that the hairpin was the only stable structure in the OCP, with a ΔG of -0.5 to -1.5 kcal/mol.
Generic P1 and P2 design
ERCA primers P1 and P2 were chosen using Oligo 6. These were generic primer sequences, and could be used to amplify any circle sequence. First, 10–20 kb of randomly generated DNA sequence was scanned for primers using settings limiting 3' dimer ΔG to -1.0 kcal/mol, 3' dimer length to 3, 3' terminal stability range -5.5 to -8.0 kcal/mol, GC clamp stability to -10 kcal/mol, no acceptable loop, Tm range 52.1 to 56.2°C. Compatible primers were selected using the multiplex function of Oligo 6. A subset of these compatible primers was utilized as generic P1 and P2 primers (see Table 1). ERCA P1 primers were labeled with FAM (if paired with the normal OCP) or 5-Cy3 (if paired with the mutant OCP) and were RP-HPLC purified. P2 primers were purified by desalting.
Allele Specific ERCA primers
The sequence of these primers was chosen from the target specific 5' and 3'-arms of the OCP, either complementary to the OCP (5'-arm) or the exact sequence of the OCP (3'-arm). These primers annealed to both the normal and mutant OCPs. The sequences of these allele specific primers are shown in Table 1. Allele specific primers were desalted after synthesis.
Exponential amplification of circularized OCP (see Figure 2) was accomplished using generic primers P1 and P2, along with allele specific primer(s). Each P1 primer contained a universal 13 base 5'-tail (see Table 1) with a fluorescent dye at the 5'-end. Fluorescence was quenched in unincorporated primers by the annealing of a 13-residue PNA molecule, Q-PNA-13 (Applied Biosystems/Boston Probes, Bedford, MA). The sequence of Q-PNA-13 is Ac-X-OO-TGA-TTG-CGA-ATG-A-Lys (Dabcyl). The C-terminal dabcyl was positioned so that it was proximal to the 5'-fluorescent moiety on the DNA reporter, quenching fluorescence when the reporter and Q-PNA-13 were annealed, as shown in Figure 2. After P1 primer is incorporated into ERCA product, Q-PNA is displaced, producing a fluorescent signal.
Genomic DNA samples were purchased from Coriell Cell Repositories, Camden, NJ. Human Variation Panel, Caucasian panel of 100, Reference # HD100CAU, was used as non-mutant controls and for general screening purposes. Human samples containing specific mutations were also purchased from Coriell: Factor V Leiden mutation (heterozygous NA14642 and homozygous NA14899); Hemochromatosis C282Y (heterozygous NA14642, homozygous NA14620); and Hemochromatosis H63D (heterozygous NA14641 and homozygous NA13591); Factor II Prothrombin mutation (heterozygous NA16028 and homozygous NA16000). Other patient DNA samples were genotyped using RFLP, and an aliquot of the purified DNA was subsequently used either directly in genotyping reactions, or used after Whole Genome Amplification (WGA).
Whole Genome Amplification (WGA) of genomic DNA samples
For some rare genotypes, it was only possible to obtain a few nanograms of sample, insufficient for more than one genotyping reaction. As a result, the rare samples were subjected to whole genome amplification (WGA), which yielded enough DNA for 200 or more reactions. WGA was performed as described previously . Briefly, 10 ng of each individual genomic sample was amplified into 40 μg of product by adding 99 μl of amplification mix and incubating at 30°C for 6 hours. Quality control assays demonstrating that whole genome amplification was successful were performed for locus 979 and 1004 as described , and amplified DNA was used directly in genotyping reactions at the same concentration as the original genomic DNA, typically 200 ng/reaction.
50 ng to 1 μg of either genomic DNA or WGA genomic sample was mixed with OCP (typically 0.5 nM final concentration) and 0.5 unit of Ampligase (Epicentre Technologies, Madison, WI) in 1x Ampligase buffer (Epicentre), for a total volume of 10 μl. The reaction was heated to 95°C for 10 seconds, and cooled to 63–68°C for 5–20 minutes, during which time OCP annealed to genomic target and was circularized by ligase. The reaction was subsequently heated to 95°C for 10 minutes to release ligated circles from genomic DNA. The reaction was cooled to 4°C, and 20 μl ERCA reaction mix was added (typically 16 units BST polymerase (New England Biolabs, Beverly, MA), 6 mM dNTPs, 0.5 μM P1, 0.5 μM P2, 4 μM Q-PNA, 7.5 μM TMAO in 1x ThermoPol Buffer II, all concentrations final). Reactions were incubated at 60°C for 1 hour in an I-Cycler (BioRad, Hercules, CA) reading both FAM and Cy3 channels. Signals typically appeared after 10–20 minutes.
Specific reaction component concentrations
FV ligation reaction conditions
0.5 nM wt OCP and 0.5 nM mut OCP (final concentration); 0.5 U ampligase.
FV ERCA reaction conditions
0.5 μM WT P1; 0.5 μM mutant P1; 0.75 μM P2; 0.75 μM 5'-allele specific primer; 0.75 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 24–32 U DNA polymerase.
FII ligation reaction conditions
0.5 nM wt OCP and 0.5 nM mut OCP (final concentration); 0.5 U ampligase.
FII ERCA reaction conditions
0.5 μM WT P1; 0.7 μM mutant P1; 0.9 μM P2; 0.4 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 16 U DNA polymerase.
Hemochromatosis H63 ligation reaction conditions
0.1 nM wt OCP and 1.2 nM mut OCP (final concentration); 0.5 U ampligase.
Hemochromatosis H63 ERCA reaction conditions
0.4 μM WT P1; 0.5 μM mutant P1; 0.75 μM P2; 0.5 μM 5'-allele specific primer; 0.5 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 16 U DNA polymerase.
Hemochromatosis C282 ligation reaction conditions
0.1 nM wt OCP and 1.2 nM mut OCP (final concentration); 0.5 U ampligase.
Hemochromatosis C282 ERCA reaction conditions
0.4 μM WT P1; 0.5 μM mutant P1; 0.75 μM P2; 0.5 μM 5'-allele specific primer; 0.5 μM 3'-allele specific primer; 4 μM PNA (final concentrations); 16 U DNA polymerase.
Determination of genotype
In this retrospective study, the genotype of each sample was known. Genotyping by RFLP analysis (data not shown) was performed prior to OCP ligation/ ERCA genotyping, and the results of OCP ligation/ ERCA analysis were compared to the RFLP analysis results.
Real Time OCP ligation/ ERCA
The genotype for each sample was determined by amplitude of amplification. The average amplification threshold time for all amplified reactions was determined using the I-Cycler software. Fluorescence traces were normalized using early cycles as a baseline, and a threshold value was determined, typically at 10-fold above the average standard deviation of the baseline values. Threshold cycle for each trace was measured at the point where the trace crossed the threshold value. Threshold cycle values fell into three distinct clusters, one each for homozygous normal, heterozygous, and homozygous mutant. Reactions with times more than 2 standard deviations beyond the mean cluster value were considered failures and repeated. In general, amplification reactions with an increase of less than 300 units were scored as negative. Any reaction with amplification of 300 fluorescence units above baseline or greater was scored as positive. Reactions with fluorescent units between 101–299 above baseline were repeated. Any reaction where signal was baseline for both alleles was repeated.
End Point OCP ligation/ ERCA
ERCA reactions were allowed to incubate at 60°C until the reaction was expected to be complete (30 to 40 minutes, depending on the assay). After the reaction was complete, the results could be read at any time. Some reactions were allowed to remain at room temperature overnight, some were stored at 4°C, some were frozen at -20°C. In each case, the reaction was protected from light to prevent photobleaching of the fluorescent reporters. The genotype of each sample was determined automatically using a modified fuzzy c-means clustering algorithm , which groups the data into three genotypes plus a negative control, and assigns a confidence level to each genotyping call from 0 (not in cluster) to 1 (100% certainty that point belongs to cluster).
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