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Research article
Fluorometric Detection of Low-Abundance EGFR Exon 19 Deletion Mutation Using
Tandem Gene Amplification
Department of Bioscience and Biotechnology, Konkuk University, Neundong-ro 120, Gwangjin-gu, Seoul 05029, Republic of Korea
J. Microbiol. Biotechnol. 2020; 30(5): 662-667
Published May 28, 2020 https://doi.org/10.4014/jmb.2004.04010
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
Lung cancer is one of the high mortality cancers, accounting for about 18.4% of the total cancer deaths, with around 18% of five-year survival rate [1]. Among various types of lung cancer, non-small cell lung cancer (NSCLC) patients comprise over 80% of all lung cancer cases [2]. Epidermal growth factor receptor (EGFR) mutations that are generally observed in the NSCLCs are thus genetic markers for diagnosis of NSCLC. Mutations in EGFR genes are also associated with response of tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, which have been standard provision for NSCLC patients [3]. Various EGFR mutations can alter the clinical response of EGFR-TKIs by changing the drug binding affinity to target region in EGFR [4, 5]. One of the most frequent EGFR kinase domain mutations is the in-frame deletion mutation in exon 19 (
Although tissue samples have been widely used to identify EGFR mutations, small proportion of mutant cancer cells is present at a low-abundance in clinically available tissue samples. Recently, instead of tissue biopsy sampling, circulating tumor DNA (ctDNA) in plasma has emerged as a reliable biomarker for detecting somatic mutations due to the advantages such as less-invasiveness and reflection of whole information of heterogenous tumor [7]. To date, several methods including Sanger DNA sequencing, next generation sequencing (NGS), and real-time PCR with TaqMan probe have been considered as a standard method for monitoring EGFR mutations [8, 9]. Alternatively, there are various PCR-based modifications using DNA-LNA blocker [10], universal oligo-quencher [11], allele-specific probe [12], and graphene oxide [13]. Despite several PCR-based modifications have been developed in an attempt to detect ctDNA, they still have limitations such as low sensitivity for detecting low frequency of mutant ctDNA in the plasma.
One of potential approaches to overcome shortcomings in PCR-based gen amplification methods is to improve sensitivity by combining two consecutive gene amplification methods such as PCR and isothermal gene amplification. Rolling circle amplification (RCA) is an effective isothermal amplification method that can be coupled with the other amplification methods [14-16]. The RCA reaction product, which is long stretch of single-stranded DNA (ssDNA) harboring tandem sequences generated by high fidelity of phi29 polymerase, can be simply detected by G-quadruplex (GQ) and thioflavin T (ThT)-based fluorometric system [17]. In this system, ThT can selectively intercalate into the four-stranded DNA structure of G-quadruplex and emit strong fluorescence with high signal to background (S/B) ratio [18]. Although there have been several attempts to combine RCA with PCR-amplified amplicon to improve sensitivity, their application is still limited due to the fact that single-stranded oligonucleotide is usually required for ligation of padlock DNA template for RCA initiation.
In this study, to overcome the uneasy preparation of ssDNA to initiate subsequent RCA, we developed a highly sensitive and selective detection method for low-abundance EGFR exon 19 deletion mutation (exon 19-del) in plasma using tandem gene amplification methods. The method consists of pre-amplification with PCR, thermal cycling of ligation by
Materials and Methods
Reagents, Oligonucleotides, and Lung Cancer Cells
DNA oligonucleotides including PCR primers and padlock probe DNA were chemically synthesized and purified by high performance liquid chromatography (Bionics, Korea). The padlock probe DNA that can hybridize to the sequence of EGFR exon 19-del site was modified with phosphate at the 5’-end to be ligated into a circular template for subsequent RCA. The sequences of the oligonucleotides are listed in Table S1. Ex
The A549 cell line was from the American Type Culture Collection (ATCC, CCL-185, USA) and PC9 cell line was from the European Collection of Authenticated Cell Cultures (ECACC, 90071810, UK). A549 cells possess the wild-type EGFR exon 19 gene, whereas PC9 cells contain EGFR exon 19 deletion mutation (E746-A750). All cells were cultured in DMEM high glucose (Hyclone, USA) supplemented with 10% fetal bocine serum (Hyclone) and 100 U/ml penicillin (WelGene, Korea) in a humidified incubator under 5% CO2 at 37°C.
Genomic DNA Extraction and PCR
The genomic DNA (gDNA) from A549 and PC9 was extracted with QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions and 50 ng of extracted gDNAs was used for PCR. The PCR mixture (20 μl) was composed of 0.5 μl of Ex
Circularization of Padlock DNA
Formation of closed circular padlock probe was accomplished by
Fluorogenic Rolling Circle Amplification
The fluorogenic RCA was performed in 25 μl of RCA mixtures containing 100 nM circular padlock probe, 100 nM RCA primer, 1× phi29 polymerase buffer, 15 μM ThT, 0.5 mM dNTPs, 50 mM KCl, and 173U phi29 polymerase. The mixture was incubated at 32°C for 60 min and heat inactivated at 65°C for 15 min. After adding 25 μl of DW to RCA product, the ThT fluorescence was scanned from 450 nm to 650 nm using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, USA) with excitation wavelength of 430 nm. To evaluate the sensitivity of PCR coupled ligation-mediated RCA assay for detecting EGFR exon 19 deletion mutation, the assay was performed with decreasing amounts of PC9 gDNA from 25 ng to 6 pg. The limit of detection (LOD) was determined by the following equation: LOD = 3.3 × standard deviation of the fluorescence intensity, SD/slope of the standard curve, S) based on the average signal of blank experiments and standard deviation obtained from triplicate measurements. To mimic the clinical sample, a fixed amount of gDNA mixture (2,000 ng) containing different mixing ratios (0-100% in mutant type gDNA) of A549 gDNA (wild-type) and PC9 gDNA (mutant type) was spiked into 100 μl of pooled normal human plasma. After gDNA was extracted using the QIAamp DNA mini kit and resolved in 100 μl, the 10 μl aliquot was used for the PCR coupled ligation-mediated RCA assay.
Quantitative Real-Time PCR
To compare the sensitivity for detection of EGFR exon 19 deletion mutation, conventional quantitative real-time PCR (qRT-PCR) was performed with the same PCR primers used for the PCR coupled ligation-mediated RCA assay. Decreasing amounts of PC9 gDNA from 25 ng to 6 pg were subjected to PCR in the 20 μl mixture containing 0.5 μl of Ex
Results and Discussion
Tandem Gene Amplification Strategy
To achieve highly sensitive and accurate detection of the EGFR exon 19-del mutant conferring different clinical response against EGFR-TKIs in NSCLC patient, we designed a fluorometric method consisting of three steps: i) PCR of gDNA as a pre-amplification, ii) circularization of padlock DNA by
-
Fig. 1.
Schematic illustration of tandem gene amplification for detection of the EGFR exon 19 deletion mutation. Segment a and c: adjacent sequences of the deletion site; segment b: deletion mutation site; segment d: Gquadruplex sequence; segment e: RCA primer.
To validate the eligibility of the tandem gene amplification assay, we first analyzed the PCR products amplified with A549 and PC9 gDNA containing wild-type and exon 19-del mutation (Fig. 2A). The amplicon with A549 gDNA was observed at a slightly higher position than amplicon with PC9 gDNA, which is consistent with the expected sizes of 109 bp for wild-type and 94 bp for mutant type. Next, we examined the circularization of padlock DNA complementary to sequence of the deletion site (Fig. 2B). Synthesized ssDNA and PCR-amplified dsDNA containing wild-type or mutant type sequence of EGFR were used as splint DNAs for cycling ligation with the padlock DNA. The circular padlock DNAs were only observed in the presence of mutant type DNAs (lane 1 and 3). Furthermore, the closed circular padlock DNA remained intact after treatment of exonuclease I & III, which can digest ssDNA and dsDNA with exposed ends, respectively (lane 5 and 7). In contrast, the padlock DNAs ligated with wild-type DNAs were fully degraded by exonuclease I & III (lane 6 and 8). This result demonstrates that the target mutant DNA sequence can be used as a proper splint DNA to conjugate both ends of padlock DNA. Next, we evaluated the G-quadruplex generating RCA and ThT fluorescence enhancement, in which the RCA product of ssDNA harboring multiple copies of GQ structure was monitored by ThT fluorescence. Increase in the ThT fluorescence of RCA product was obtained from both mutant-type sequence of ssDNA and dsDNA amplicon (Fig. 2C). The fold-increase in fluorescence intensity at 488 nm between RCA product with mutant and wild-type were calculated to be 92.8 (ssDNA) and 11.4 (dsDNA amplicon). The bright blue fluorescence with mutant type gene was visualized under UV light, consistent with fluorescence spectra. These results clearly show that circularization of padlock DNA and subsequent GQ-RCA with ThT occurs only in the presence of target mutant gDNA. To explore the optimal number of cycles and temperature for
-
Fig. 2.
Evaluation of tandem gene amplification with ThT fluorescence enhancement. (A ) Agarose gel (2%) electrophoresis showing the PCR products amplified with A549 gDNA (wild-type, W) or PC9 gDNA (mutant type, M). Marker lane is 100 bp DNA ladder. (B ) Target mutant gene-specific circularization of padlock DNA. Linear padlock DNA was hybridized and ligated byTaq ligase with synthesized ssDNA or PCR-amplified dsDNA amplicon containing wild-type or mutant type. After degradation by exonuclease I & III, digested products were resolved by denaturing 12% urea-PAGE and stained with SYBR Gold. (C) Fluorescence emission spectra of ThT (λex = 430 nm) obtained with synthesized ssDNA or PCRamplified dsDNA amplicon containing wild-type or mutant type. Inset image showed the RCA product visualized under UV light.
Sensitivity and Selectivity of Tandem Gene Amplification Assay
To examine the sensitivity of tandem gene amplification assay, decreasing amounts of PC9 gDNA (50 ng–6 pg) were used for the tandem gene amplification and scanned for fluorescence emission spectra of product. The ThT fluorescence intensity gradually decreased as the amount of PC9 gDNA decreased from 50 ng to 6 pg. The titration curve of fluorescence intensity at 488 nm obtained from the spectra showed a linear relationship between fluorescence intensity and the amount of PC9 gDNA with a calculated LOD value of 3.6 pg. Under UV light, the bright blue ThT fluorescence gradually increased as the amount of mutant gDNA increased, which is consistent with the titration curve.
Next, we compared sensitivity of our assay with conventional real-time PCR based on SYBR green intercalating dye method. As shown in Fig. 3A, the fluorescence intensity from tandem gene amplification assay was statistically significant at amount of PC9 gDNA as low as 6 pg. The samples with decreasing amount of PC9 gDNA were analyzed using quantitative real-time PCR with SYBR green (Fig. 3B). The cycle threshold (Ct) values that were gradually increased as the amount of PC9 gDNA decreases showed an LOD as low as 0.39 ng. These data indicate that our system had a 65-fold higher sensitivity than SYBR green-based quantitative real-time PCR for detection of the EGFR in-frame deletion mutation. In addition, the selectivity of tandem gene amplification assay was evaluated with gDNA mixture containing different fractions of PC9 mutant gDNA (0 to 100%) in a fixed amount of total gDNA (50 ng). The ThT fluorescence intensity at 488 nm provided a statistically significant LOD as low as 0.1%, and the blue colored ThT fluorescence gradually increased as the fractions of mutant gDNA increased under UV light (Fig. S3). Thus, tandem gene amplification followed by ThT fluorescence enhancement provides a superior sensitivity to conventional real-time PCR for detection of the EGFR in-frame deletion mutant present in genomic DNA sample.
-
Fig. 3.
Sensitivity of tandem gene amplification assay. (A ) Bar graphs represent fluorescence intensities obtained by tandem gene amplification for EGFR exon 19-del detection. The assay indicates a statistically significant positive detection at amount of mutant PC9 gDNA as low as 6 pg (***,p < 0.005 vs. No gDNA). (B ) Quantitative real-time PCR for EGFR exon 19-del detection. Bar graphs represent the threshold cycle (Ct) values obtained during real-time PCR with decreasing amount of PC9 gDNA. The real-time PCR shows statistically significant positive detection at an amount of mutant PC9 gDNA as low as 0.39 ng (*,p < 0.05 vs No gDNA). The data are presented as the mean ± standard deviation of three experiments.
Detection of the EGFR In-Frame Deletion Mutant gDNA in Blood Plasma
We further tested the detection capacity of our assay by using pooled normal human blood plasma samples (100 μl) spiked with gDNA mixture containing different proportions of mutant PC9 gDNA (0 to 100% in PC9 content) in a fixed amount of total gDNA (2.0 mg). After extraction of gDNA from blood plasma, 10 μl of gDNA was analyzed by the tandem gene amplification and the emission spectra of ThT fluorescence was scanned (Fig. 4A). The ThT fluorescence signal at 488 nm showed a linear correlation with the amount of PC9 gDNA and the LOD was found to be 1% (Fig. 4B). This result suggests that small numbers of mutant gDNA harboring the EGFR exon 19-del with abundant background of wild-type gDNA in plasma was able to be readily detected with our tandem gene amplification method.
-
Fig. 4.
Quantitative detection of EGFR 19-del mutant genes spiked in the pooled human plasma. (A ) Fluorescence emission spectra of ThT (λex = 430 nm) obtained at different fractions of mutant gDNA in a fixed amount of gDNA mixture (2.0 μg), which were spiked in plasma (100 μl). (B ) Bar graphs represent fluorescence intensities obtained at different fractions of mutant gDNA. The assay indicates a statistically significant positive detection at amount of mutant PC9 gDNA as low as 1% fraction (*,p < 0.05 vs. 0%). The data are presented as the mean ± standard deviation of three experiments. Inset image showed the RCA product visualized under UV light.
In conclusion, we have developed a sensitive fluorometric assay for detection of EGFR exon 19-del mutant present in genomic DNA samples using the tandem gene amplification method. Stringency of
Supplemental Material
Acknowledgments
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation funded by the Korean government (2016M3A9B6918892). This paper was supported by Konkuk University Researcher Fund in 2019.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
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et al . 2020. Differential significance of molecular subtypes which were classified into EGFR exon 19 deletion on the first line afatinib monotherapy.BMC Cancer 20 : 103. - Mitsudomi T, Yatabe Y. 2010. Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer.
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Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2020; 30(5): 662-667
Published online May 28, 2020 https://doi.org/10.4014/jmb.2004.04010
Copyright © The Korean Society for Microbiology and Biotechnology.
Fluorometric Detection of Low-Abundance EGFR Exon 19 Deletion Mutation Using
Tandem Gene Amplification
Dong-Min Kim†, Shichen Zhang†, Minhee Kim, and Dong-Eun Kim*
Department of Bioscience and Biotechnology, Konkuk University, Neundong-ro 120, Gwangjin-gu, Seoul 05029, Republic of Korea
Abstract
Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of lowabundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCRamplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.
Keywords: 5Epidermal growth factor receptor, G-quadruplex, In-frame deletion mutation, rolling circle amplification, thioflavin T
Introduction
Lung cancer is one of the high mortality cancers, accounting for about 18.4% of the total cancer deaths, with around 18% of five-year survival rate [1]. Among various types of lung cancer, non-small cell lung cancer (NSCLC) patients comprise over 80% of all lung cancer cases [2]. Epidermal growth factor receptor (EGFR) mutations that are generally observed in the NSCLCs are thus genetic markers for diagnosis of NSCLC. Mutations in EGFR genes are also associated with response of tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, which have been standard provision for NSCLC patients [3]. Various EGFR mutations can alter the clinical response of EGFR-TKIs by changing the drug binding affinity to target region in EGFR [4, 5]. One of the most frequent EGFR kinase domain mutations is the in-frame deletion mutation in exon 19 (
Although tissue samples have been widely used to identify EGFR mutations, small proportion of mutant cancer cells is present at a low-abundance in clinically available tissue samples. Recently, instead of tissue biopsy sampling, circulating tumor DNA (ctDNA) in plasma has emerged as a reliable biomarker for detecting somatic mutations due to the advantages such as less-invasiveness and reflection of whole information of heterogenous tumor [7]. To date, several methods including Sanger DNA sequencing, next generation sequencing (NGS), and real-time PCR with TaqMan probe have been considered as a standard method for monitoring EGFR mutations [8, 9]. Alternatively, there are various PCR-based modifications using DNA-LNA blocker [10], universal oligo-quencher [11], allele-specific probe [12], and graphene oxide [13]. Despite several PCR-based modifications have been developed in an attempt to detect ctDNA, they still have limitations such as low sensitivity for detecting low frequency of mutant ctDNA in the plasma.
One of potential approaches to overcome shortcomings in PCR-based gen amplification methods is to improve sensitivity by combining two consecutive gene amplification methods such as PCR and isothermal gene amplification. Rolling circle amplification (RCA) is an effective isothermal amplification method that can be coupled with the other amplification methods [14-16]. The RCA reaction product, which is long stretch of single-stranded DNA (ssDNA) harboring tandem sequences generated by high fidelity of phi29 polymerase, can be simply detected by G-quadruplex (GQ) and thioflavin T (ThT)-based fluorometric system [17]. In this system, ThT can selectively intercalate into the four-stranded DNA structure of G-quadruplex and emit strong fluorescence with high signal to background (S/B) ratio [18]. Although there have been several attempts to combine RCA with PCR-amplified amplicon to improve sensitivity, their application is still limited due to the fact that single-stranded oligonucleotide is usually required for ligation of padlock DNA template for RCA initiation.
In this study, to overcome the uneasy preparation of ssDNA to initiate subsequent RCA, we developed a highly sensitive and selective detection method for low-abundance EGFR exon 19 deletion mutation (exon 19-del) in plasma using tandem gene amplification methods. The method consists of pre-amplification with PCR, thermal cycling of ligation by
Materials and Methods
Reagents, Oligonucleotides, and Lung Cancer Cells
DNA oligonucleotides including PCR primers and padlock probe DNA were chemically synthesized and purified by high performance liquid chromatography (Bionics, Korea). The padlock probe DNA that can hybridize to the sequence of EGFR exon 19-del site was modified with phosphate at the 5’-end to be ligated into a circular template for subsequent RCA. The sequences of the oligonucleotides are listed in Table S1. Ex
The A549 cell line was from the American Type Culture Collection (ATCC, CCL-185, USA) and PC9 cell line was from the European Collection of Authenticated Cell Cultures (ECACC, 90071810, UK). A549 cells possess the wild-type EGFR exon 19 gene, whereas PC9 cells contain EGFR exon 19 deletion mutation (E746-A750). All cells were cultured in DMEM high glucose (Hyclone, USA) supplemented with 10% fetal bocine serum (Hyclone) and 100 U/ml penicillin (WelGene, Korea) in a humidified incubator under 5% CO2 at 37°C.
Genomic DNA Extraction and PCR
The genomic DNA (gDNA) from A549 and PC9 was extracted with QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions and 50 ng of extracted gDNAs was used for PCR. The PCR mixture (20 μl) was composed of 0.5 μl of Ex
Circularization of Padlock DNA
Formation of closed circular padlock probe was accomplished by
Fluorogenic Rolling Circle Amplification
The fluorogenic RCA was performed in 25 μl of RCA mixtures containing 100 nM circular padlock probe, 100 nM RCA primer, 1× phi29 polymerase buffer, 15 μM ThT, 0.5 mM dNTPs, 50 mM KCl, and 173U phi29 polymerase. The mixture was incubated at 32°C for 60 min and heat inactivated at 65°C for 15 min. After adding 25 μl of DW to RCA product, the ThT fluorescence was scanned from 450 nm to 650 nm using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, USA) with excitation wavelength of 430 nm. To evaluate the sensitivity of PCR coupled ligation-mediated RCA assay for detecting EGFR exon 19 deletion mutation, the assay was performed with decreasing amounts of PC9 gDNA from 25 ng to 6 pg. The limit of detection (LOD) was determined by the following equation: LOD = 3.3 × standard deviation of the fluorescence intensity, SD/slope of the standard curve, S) based on the average signal of blank experiments and standard deviation obtained from triplicate measurements. To mimic the clinical sample, a fixed amount of gDNA mixture (2,000 ng) containing different mixing ratios (0-100% in mutant type gDNA) of A549 gDNA (wild-type) and PC9 gDNA (mutant type) was spiked into 100 μl of pooled normal human plasma. After gDNA was extracted using the QIAamp DNA mini kit and resolved in 100 μl, the 10 μl aliquot was used for the PCR coupled ligation-mediated RCA assay.
Quantitative Real-Time PCR
To compare the sensitivity for detection of EGFR exon 19 deletion mutation, conventional quantitative real-time PCR (qRT-PCR) was performed with the same PCR primers used for the PCR coupled ligation-mediated RCA assay. Decreasing amounts of PC9 gDNA from 25 ng to 6 pg were subjected to PCR in the 20 μl mixture containing 0.5 μl of Ex
Results and Discussion
Tandem Gene Amplification Strategy
To achieve highly sensitive and accurate detection of the EGFR exon 19-del mutant conferring different clinical response against EGFR-TKIs in NSCLC patient, we designed a fluorometric method consisting of three steps: i) PCR of gDNA as a pre-amplification, ii) circularization of padlock DNA by
-
Figure 1.
Schematic illustration of tandem gene amplification for detection of the EGFR exon 19 deletion mutation. Segment a and c: adjacent sequences of the deletion site; segment b: deletion mutation site; segment d: Gquadruplex sequence; segment e: RCA primer.
To validate the eligibility of the tandem gene amplification assay, we first analyzed the PCR products amplified with A549 and PC9 gDNA containing wild-type and exon 19-del mutation (Fig. 2A). The amplicon with A549 gDNA was observed at a slightly higher position than amplicon with PC9 gDNA, which is consistent with the expected sizes of 109 bp for wild-type and 94 bp for mutant type. Next, we examined the circularization of padlock DNA complementary to sequence of the deletion site (Fig. 2B). Synthesized ssDNA and PCR-amplified dsDNA containing wild-type or mutant type sequence of EGFR were used as splint DNAs for cycling ligation with the padlock DNA. The circular padlock DNAs were only observed in the presence of mutant type DNAs (lane 1 and 3). Furthermore, the closed circular padlock DNA remained intact after treatment of exonuclease I & III, which can digest ssDNA and dsDNA with exposed ends, respectively (lane 5 and 7). In contrast, the padlock DNAs ligated with wild-type DNAs were fully degraded by exonuclease I & III (lane 6 and 8). This result demonstrates that the target mutant DNA sequence can be used as a proper splint DNA to conjugate both ends of padlock DNA. Next, we evaluated the G-quadruplex generating RCA and ThT fluorescence enhancement, in which the RCA product of ssDNA harboring multiple copies of GQ structure was monitored by ThT fluorescence. Increase in the ThT fluorescence of RCA product was obtained from both mutant-type sequence of ssDNA and dsDNA amplicon (Fig. 2C). The fold-increase in fluorescence intensity at 488 nm between RCA product with mutant and wild-type were calculated to be 92.8 (ssDNA) and 11.4 (dsDNA amplicon). The bright blue fluorescence with mutant type gene was visualized under UV light, consistent with fluorescence spectra. These results clearly show that circularization of padlock DNA and subsequent GQ-RCA with ThT occurs only in the presence of target mutant gDNA. To explore the optimal number of cycles and temperature for
-
Figure 2.
Evaluation of tandem gene amplification with ThT fluorescence enhancement. (A ) Agarose gel (2%) electrophoresis showing the PCR products amplified with A549 gDNA (wild-type, W) or PC9 gDNA (mutant type, M). Marker lane is 100 bp DNA ladder. (B ) Target mutant gene-specific circularization of padlock DNA. Linear padlock DNA was hybridized and ligated byTaq ligase with synthesized ssDNA or PCR-amplified dsDNA amplicon containing wild-type or mutant type. After degradation by exonuclease I & III, digested products were resolved by denaturing 12% urea-PAGE and stained with SYBR Gold. (C) Fluorescence emission spectra of ThT (λex = 430 nm) obtained with synthesized ssDNA or PCRamplified dsDNA amplicon containing wild-type or mutant type. Inset image showed the RCA product visualized under UV light.
Sensitivity and Selectivity of Tandem Gene Amplification Assay
To examine the sensitivity of tandem gene amplification assay, decreasing amounts of PC9 gDNA (50 ng–6 pg) were used for the tandem gene amplification and scanned for fluorescence emission spectra of product. The ThT fluorescence intensity gradually decreased as the amount of PC9 gDNA decreased from 50 ng to 6 pg. The titration curve of fluorescence intensity at 488 nm obtained from the spectra showed a linear relationship between fluorescence intensity and the amount of PC9 gDNA with a calculated LOD value of 3.6 pg. Under UV light, the bright blue ThT fluorescence gradually increased as the amount of mutant gDNA increased, which is consistent with the titration curve.
Next, we compared sensitivity of our assay with conventional real-time PCR based on SYBR green intercalating dye method. As shown in Fig. 3A, the fluorescence intensity from tandem gene amplification assay was statistically significant at amount of PC9 gDNA as low as 6 pg. The samples with decreasing amount of PC9 gDNA were analyzed using quantitative real-time PCR with SYBR green (Fig. 3B). The cycle threshold (Ct) values that were gradually increased as the amount of PC9 gDNA decreases showed an LOD as low as 0.39 ng. These data indicate that our system had a 65-fold higher sensitivity than SYBR green-based quantitative real-time PCR for detection of the EGFR in-frame deletion mutation. In addition, the selectivity of tandem gene amplification assay was evaluated with gDNA mixture containing different fractions of PC9 mutant gDNA (0 to 100%) in a fixed amount of total gDNA (50 ng). The ThT fluorescence intensity at 488 nm provided a statistically significant LOD as low as 0.1%, and the blue colored ThT fluorescence gradually increased as the fractions of mutant gDNA increased under UV light (Fig. S3). Thus, tandem gene amplification followed by ThT fluorescence enhancement provides a superior sensitivity to conventional real-time PCR for detection of the EGFR in-frame deletion mutant present in genomic DNA sample.
-
Figure 3.
Sensitivity of tandem gene amplification assay. (A ) Bar graphs represent fluorescence intensities obtained by tandem gene amplification for EGFR exon 19-del detection. The assay indicates a statistically significant positive detection at amount of mutant PC9 gDNA as low as 6 pg (***,p < 0.005 vs. No gDNA). (B ) Quantitative real-time PCR for EGFR exon 19-del detection. Bar graphs represent the threshold cycle (Ct) values obtained during real-time PCR with decreasing amount of PC9 gDNA. The real-time PCR shows statistically significant positive detection at an amount of mutant PC9 gDNA as low as 0.39 ng (*,p < 0.05 vs No gDNA). The data are presented as the mean ± standard deviation of three experiments.
Detection of the EGFR In-Frame Deletion Mutant gDNA in Blood Plasma
We further tested the detection capacity of our assay by using pooled normal human blood plasma samples (100 μl) spiked with gDNA mixture containing different proportions of mutant PC9 gDNA (0 to 100% in PC9 content) in a fixed amount of total gDNA (2.0 mg). After extraction of gDNA from blood plasma, 10 μl of gDNA was analyzed by the tandem gene amplification and the emission spectra of ThT fluorescence was scanned (Fig. 4A). The ThT fluorescence signal at 488 nm showed a linear correlation with the amount of PC9 gDNA and the LOD was found to be 1% (Fig. 4B). This result suggests that small numbers of mutant gDNA harboring the EGFR exon 19-del with abundant background of wild-type gDNA in plasma was able to be readily detected with our tandem gene amplification method.
-
Figure 4.
Quantitative detection of EGFR 19-del mutant genes spiked in the pooled human plasma. (A ) Fluorescence emission spectra of ThT (λex = 430 nm) obtained at different fractions of mutant gDNA in a fixed amount of gDNA mixture (2.0 μg), which were spiked in plasma (100 μl). (B ) Bar graphs represent fluorescence intensities obtained at different fractions of mutant gDNA. The assay indicates a statistically significant positive detection at amount of mutant PC9 gDNA as low as 1% fraction (*,p < 0.05 vs. 0%). The data are presented as the mean ± standard deviation of three experiments. Inset image showed the RCA product visualized under UV light.
In conclusion, we have developed a sensitive fluorometric assay for detection of EGFR exon 19-del mutant present in genomic DNA samples using the tandem gene amplification method. Stringency of
Supplemental Material
Acknowledgments
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation funded by the Korean government (2016M3A9B6918892). This paper was supported by Konkuk University Researcher Fund in 2019.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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