Jae-Jin Choi

Peptide Nucleic Acid-Based Array for Detecting and Genotyping Human Papillomaviruses

ABSTRACT We describe a novel array for accurate and reliable genotyping of human papillomavirus (HPV) using peptide nucleic acid (PNA) probes. In order to exploit the superior hybridization properties of PNA with target HPV DNAs, we developed a novel PNA array (PANArray HPV). PANArray HPV enables the detection and genotyping of HPVs using 32 type-specific PNA capture probes for medically important HPVs. All tested HPV types showed highly unique hybridization patterns with type-specific PNA probes. PNA array results showed stable specificities and sensitivities after up to 13 months of storage at room temperature. Also, we demonstrated the superior specificity, sensitivity, and stability of PNA arrays for HPV genotyping. We compared the genotyping results of the PNA array to sequencing with MY09/11 PCR products derived from 72 clinical samples. The results showed excellent agreement between the PNA array and sequencing, except for samples reflecting multiple infections. The results from the PNA array were compared with those of type-specific PCR when discrepant results occurred owing to multiple infections. The results for the PNA array matched those of type-specific PCR in all cases. Newly developed PNA arrays show excellent specificity and sensitivity and long shelf life. Our results suggest that the PNA array represents a reliable alternative to conventional DNA arrays for HPV genotyping, as well as for diagnostics.

A PNA microarray platform for miRNA expression profiling using on-chip labeling technology

MicroRNAs (miRNAs) are short, non-coding RNAs that play a critical role in development, metabolism and other fundamental biological processes, and are also important in diseases such as cancer. To study miRNA expression levels in a systematic and parallel manner, we have developed a highly sensitive, specific and reproducible microarray for miRNA expression profiling using the Peptide Nucleic Acids (PNA) probes which have higher affinity and greater specificity for binding to RNA than do DNA probes. We developed a PNA microarray assay that optimizes PNA probe, hybridization conditions, and on-PNA chip labeling method. In this PANArray™ miRNA method, unlabeled RNA is hybridized to the PNA microarray and labeled by enzymatic ligation of pCp-Cy3 on the chip. This assay showed high reproducibility and low cross-hybridization for miRNAs belonging to the let-7 family and the miR-181 family, which differ by a single nucleotide. The PNA microarray (PANArray™ miRNA) is a rapid throughput technology for analyzing expression profiles with high fidelity, and could prove to be a powerful tool for cancer research, diagnosis and prognosic assessment.

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance

ABSTRACT The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 102 copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 104 copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms.

Abstract 3067: Improved detection of BRAF mutation in clinical samples using PNA-mediated real-time PCR clamping techniques

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The BRAF mutation is a somatic mutation with the highest pathogenic cancer-associated prevalence and a biomarker used for detecting cancer and monitoring prognosis of cancer patients. Usually, mutation in the Exon15 codons V600E mutation (1799 T>A) represent more than 90% of BRAF mutations. However, techniques using PCR and direct sequencing have limitations and are unable to detect low-level mutations in cancers to give false negative diagnoses. We have developed a highly sensitive and simple method for detecting BRAF mutations using PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM method is a simple, reliable and sensitive method with a detection limit of approximately 0.1% mutant alleles. The turnaround time for this method was only 2 hours. The method was utilized to detect BRAF mutations in fine needle aspiration biopsy (FNAB) specimens from papillary thyroid cancer (PTC) patients. V600E mutation was detected in 62% (52/84) of the FNAB specimens in the PTC samples, 10% higher rate compare with PCR and direct sequencing methods. In this report, we will discuss the results of clinical studies in detail including the comparison data. In summary, PNAClampTM method can serve a rapid, reliable, and economical alternative for BRAF mutation detection in clinical settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2011-3067

Abstract 3069: One-step sensitive PNA mediated real-time PCR clamping assay to detect PIK3CA mutations

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Phosphatidylinositol 3-kinases (PI3K) are important regulators of cellular growth, transformation, adhesion, apoptosis, survival and motility. Mutations in the PIK3CA oncogene encoding PI3K have been frequently found in various human cancers. The presence of PIK3CA mutations in a tumor has been suggested as a prognostic factor and may predict responses to select treatments. Therefore, detection of PIK3CA mutations may help predict drug responses and prognosis of cancer patients. We have developed a highly sensitive and simple method to detect 11 mutations in exons 9 and 20 of PIK3CA using one-step PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM relies on unique properties of PNA probes that PNA oligomers are not recognized by DNA serving as sequence-selective clamps during PCR amplification. This method can also detect 11 mutations through only three tube reactions each with 10 ng of the target DNA. PNAClampTM is simple and reliable with a detection limit of approximately 0.1% mutant alleles using 10 to 50 ng of normal DNA as a template. The turnaround time for performing this assay was only 2 hours. In clinical studies with breast cancer patients in Korea, PIK3CA mutations were detected utilizing PNAClampTM in 12 (35.3%) of clinical samples, and the results were compared with direct sequencing method. Among 34 patients, 1 (2.94%) had mutations in codon 542, 6 (17.65%) had mutations in codon 545 and 5 (14.71%) had mutations in codon 1047. The results will be discussed in detail in this report including the method development and optimization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2011-3069

Abstract 3068: Detection of K-RAS mutations in Korean patients using PNAClampTM KRAS

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Mutation of the K-RAS gene located in codons 12 and 13 may play an important role in prognosis of related diseases and responses to certain treatments targeting the pathway. Recent studies suggest that in colon cancer patients, K-RAS mutation status is a strong predictor of success in therapeutics of several tyrosine kinase inhibitors. Therefore, it is important to detect K-RAS mutations fast and accurately. To detect a minority of mutant K-RAS among abundant wild-type alleles, we have developed a highly sensitive and simple method for the detection of K-RAS mutations using PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM relies on the following two unique properties of PNA probes: 1) PNA-DNA duplexes generally have greater thermal stability than the corresponding DNA-DNA duplex; 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as a sequence-selective clamp during PCR amplification. This method is fast and accurate with a detection limit of approximately 0.1% mutant alleles using 10 ng to 25 ng of normal DNA as a template. The turnaround time for performance of this assay is only 2 hrs. The PNAClampTM method facilitates the detection of mutations in more than 1000-fold excess of wild-type DNA samples. K-RAS mutations were detected in 28.3% (36/127) of patients. In detail, 29 cases had mutations in codon 12 and 7 cases had mutations in codon 13. To confirm those results, the same clinical samples were analyzed with a direct sequencing assay. Concordant results were obtained from 124 (97.6%) of the 127 clinical samples in the two assays. The discrepancy was observed in three cases. For comparison, the three clinical samples were also analyzed using DxS K-RAS Mutation Kit (DxS Ltd., UK) to give the same outcome. In this study, we will discuss development of PNAClampTM KRAS and its optimization for higher sensitivity. The PNAClampTM KRAS may serve a rapid and reliable alternative for K-RAS mutation detection in the clinical field. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3068. doi:10.1158/1538-7445.AM2011-3068

Abstract 5059: Detection of K-RAS mutation in clinical samples by one-step PNA-mediated real-time PCR clamping techniques

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Mutations in the K-RAS oncogene are frequently found in human cancers. K-RAS mutations may indicate prognosis and drug response, and many new cancer therapies are being targeted to the K-RAS pathway. Many researchers suggest that in colon cancer patients the K-RAS mutation status is strong predictor of resistance to therapy with tyrosine kinase inhibitors such as Erbitux® (cetuximab/ Imclone system Inc) or Vectibix® (panitumumab/ Amgen Inc). Therefore, it is important that detection of K-RAS mutations is fast and simple with high accuracy and high sensitivity for predict drug response and prognosis. To detect a minority of mutant K-RAS among abundant wild-type alleles, we developed a high sensitivity and simple method for detection of K-RAS mutations using PNA-mediated real-time PCR clamping. PNA mediated real-time PCR clamping relies on the following two unique properties of PNA probes. 1) PNA-DNA duplexes generally have greater thermal stability than the corresponding DNA-DNA duplex and 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as sequence selective clamp during PCR amplification. One-step PNA-mediated real-time PCR clamping method was optimized for detection of K-RAS mutations in exon 12 and 13 with high sensitivity. This method was simple and correct with detection limit approximately 0.1% mutant alleles using 10ng to 50ng of normal DNA as the template. The turnaround time for performing this assay was only 2.5hrs. K-RAS mutations were detected in 24.4% (22/90) of clinical samples, of which 18 had mutations in codon 12 and 4 had these in codon 13. To confirm these results, same clinical samples were analyzed with sequencing assay. The concordant results were obtained from 88 (98%) of the 90 clinical samples by the two assays. The PNA-mediated real-time PCR clamping is a rapid and reliable tool for K-RAS mutation detection and allowed a minority of mutant in clinical field. Keywords: K-RAS, Mutation, PNA, cancer, PCR clamping This work was supported be the Small and Medium Business Administration funded by the Korean Government. (S1060400) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5059.

Abstract 3008: PNA based microarray for cancer & stem cell related miRNAs profiling from low total RNA with on-chip labeling

MicroRNAs(miRNAs) are short non-coding RNAs that play a critical role in many important biological processes. MiRNA expression profiling is important to understand the biological mechanisms, develop drugs, and diagnose. However, analyzed method of expression profiling is limited for short base of miRNA. Microarray which is hybridization based technique is the best for detection tool on miRNA characteristic. So, we have developed a PNA-based microarray that is highly sensitive, specific and reproducible to analyze miRNA expression profiles with direct miRNA labeling on chip after hybridization. PNA probes are well known to have stronger binding affinity, specificity to its complementary DNA or RNA strand and stability to biological enzymes. Unlabeled total RNA is hybridized to the PNA-based microarray for 4 hours and then only hybridized miRNA labeling is performed by using enzymatic ligation with pCp-Cy3. Labeling method of only hybridized miRNAs is an important technique for reproducibility and reliable results of microarray. Our data showed very low cross hybridization for miRNAs differing by single nucleotide such as human let7 family and miR-181 family. We used this microarray to determine the profile of microRNAs expressed in the developing cancer cell lines and Tissue. Array results have been validated by subsequent confirmation of miRNA expression using TaqMan qRT-PCR analysis. The results from the TaqMan qRT-PCR and PNA based microarray are shown that the range of correlation were 0.904-0.989. PNA-based microarray platform can detect small amounts of individual miRNAs from TM miRNA can be a powerful tool to analyze miRNA expression profiling in cancer diagnosis and prognosis for high throughput screening with high accuracy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3008.