Polymerase Chain Reaction Capillary Electrophoresis Research Articles

Integrated slidable and valveless polymerase chain reaction–capillary electrophoresis microdevice for pathogen detection

An integrated slidable and valveless polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for pathogen detection on a portable genetic analysis microsystem. The proposed microdevice does not need any microvalve or external pneumatic actuators, thereby simplifying the chip fabrication and the chip operation process. The slidable PCR–CE chip consists of three glass layers: a channel wafer that includes a sample injection and CE microchannel, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a slidable plate in which a PCR chamber was fabricated. First, the slidable PCR plate was placed in the sample injection zone to load a PCR cocktail, and then moved to the PCR zone for thermal cycling. After PCR, the slidable PCR chamber was switched to the CE region for separation, the resultant amplicons were then analyzed by a miniaturized laser-induced fluorescence detector. Non-specific DNA adsorption was prevented by treating the glass surface with hydrophobic decyltrichlorosilane, and the leakage and evaporation of the PCR cocktail during the movement and PCR could be minimized by surrounding the slidable chamber with mineral oil. The target protein A gene (101 bp) of Staphylococcus aureus (S. aureus) was detected with a limit of detection (LOD) of 6 copies, even with a background of 1000-fold excess of Escherichia coli K12 (E. Coli K12) templates. This demonstrates a highly sensitive and selective genetic analysis was achieved on our slidable and valveless PCR–CE microdevice.

Mutant NPM1 is a Reliable marker of Minimal Residual leukemia in patients with Acute Myeloid Leukemia.

Abstract 2484 Background:Mutations in NPM1 (Nucleophosmin-1) have been described in about 35% of adult patients with de novo AML and 45–60 % of AML patients with a diploid karyotype. NPM1 mutations predict for achieving higher complete remission (CR) rates and better outcomes in AML. Few studies have reported on the reliability of mutated NPM1 as a marker for minimal residual disease (MRD) detection in patients with AML. Methods:We conducted a retrospective analysis of patients (n=360) with newly diagnosed AML treated at our institution between 2008 and 2012. The study was approved by the Institutional Review Board. NPM1 mutation status was determined from DNA from unsorted bone marrow (BM) aspirate samples by a PCR-based method at baseline, remission, and relapse. Genomic DNA from bone marrow samples was isolated using the Autopure extractor (QIAGEN/Gentra, Valencia, CA). Mutations in exon 12 of NPM1 were assessed by a DNA-based semi-quantitative polymerase chain reaction capillary electrophoresis (PCR-CE) assay with analytical sensitivity of approximately 2.5%. Results:Data on remission and relapse samples from 360 newly diagnosed and previously untreated patients with AML, with available NPM1 analysis on their BM at the time of initial diagnosis, was collected (see flow chart below). Median age was 60 years (range 21 – 81 years). 262 patients (72%) had de novo AML, and 98 (27%) secondary AML. Cytogenetics was diploid in 137 (38%) patients, t(8;21) in 17 (5%), inversion 16 in 26 (7%), deletion 5 alone in 27 (7.5%), del 7 and 5 in 26 (7%), deletion 7 alone in 21 (5.8%), trisomy 8 in 17 (5%) and miscellaneous in 89 (24.7%) patients, respectively. Overall, 60 (16.6%) patients including 46 of the 137 (33.5%) diploid patients had NPM1 mutation at baseline. Secondary leukemia was more common in the NPM1 wild type (30%) than in the NPM1 mutated (13%) category.When analysed by age, in patients < 60 years (n=175), OS (overall survival), EFS (event free survival) and response rates were significantly superior in NPM1 mutated subgroup (p=0.001, 0.007, 0.02 respectively), while among patients ≥ 60 years (n=185) EFS and response rates were significantly higher in the NPM1 mutated subgroup (p=0.008, 0.03 respectively). Among the patients with diploid cytogenetics who were younger than 60 years (n=60) OS, EFS and CR duration was significantly better in the NPM1 mutated subset (p=0.007, 0.007 and 0.02 respectively), while in those ≥60 years (n=77) there was no statistically significant difference in the outcomes for the NPM1 mutated and wild-type subsets.Among the 60 NPM1 mutated patients 54 (90%) including 41/46 (89%) of those with diploid cytogenetics achieved complete response (CR) or CR without platelet recovery. Thirty nine patients (including 30 with diploid karyotype) had available NPM1 status at the time of CR and all (100%) were negative for NPM1 mutation.Among the patients with mutated NPM1 at baseline who have achieved a NPM1 negative status at CR, 10/39 overall (25%) and 7/30 diploid (23%) patients relapsed. NPM1 status was available for 6 patients overall including 4 with diploid karyotype at the time of relapse. Among them, 5/6 overall (83%) and 3/4 diploid (75%) patients had mutated NPM1, while 1/6 overall (16%) and 1/4 diploid (25%) patients remained NPM1 wild type. This patient relapsed with extramedullary disease (leukemia cutis) without any BM involvement.Among the 300 patients (including 91 with diploid karyotype) with wild type NPM1 at diagnosis, none acquired a mutated NPM1 clone, either at CR or at the time of relapse. Conclusions:These data suggest that mutated NPM1 is a reliable and stable marker for the detection of MRD at the time of CR. Hence, NPM1 mutations can be used to detect MRD and their recurrence may predict pending relapse. Disclosures:Cortes:Celgene: Research Funding. Ravandi:Johnson and Johnson: Honoraria; Celgene: Research Funding.

Microwave Heating of Long-term Formalin-fixed Surgical Pathology Specimens Improves Quality of Extracted DNA

A large amount of DNA of high quality is essential for molecular analysis. The amount of DNA in routine paraffin sections is small. Surgical specimens retained in formalin for the long-term (several months) left over from the sampling required for wax embedding can be referred to as "long-term formalin-fixed" specimens, and clearly this material is a rich source of DNA; however, it is difficult to extract. In the current study, we designed a microwave-heating method for DNA extraction from these specimens. We found that the heating procedure achieved greater DNA yields than a common nonheating method used for comparison (DNA contents mean±SD, heating 2.16±0.95 μg/μL vs. common 1.75±0.90 μg/μL, P<0.05). Fluorescence multiplex polymerase chain reaction-capillary electrophoresis successfully detected microsatellite instability (MSI) in the DNA samples derived from this heating procedure (98.4%, 689 of 700 sample tests), at significantly higher levels than from the conventional method (82.3%, 247 of 300 sample tests, P<0.05). We identified 10 (14.3%) MSI with high frequency and 6 (8.6%) MSI with low frequency colorectal cancers. MSI with high frequency cancers showed distinct clinicopathologic features including higher incidence of right-sided location, high histologic grade, mucin-production, and prominent intraepithelial lymphocyte infiltration. We concluded that the microwave-heating method was efficient for DNA isolation from long-term formalin-fixed tissue samples. The successful fluorescence multiplex polymerase chain reaction-capillary electrophoresis analysis in these samples might facilitate MSI detection in clinical practice.

Inhibition of on-chip PCR using PDMS–glass hybrid microfluidic chips

In the course of developing a microfluidic analytical platform incorporating the polymerase chain reaction (PCR) and subsequent capillary electrophoresis (CE) analysis for a variety of bio-assays, we examined PCR inhibition through surface interactions with the chip materials. Our devices perform PCR in a three-layer chip, a glass–poly(dimethylsiloxane)–glass sandwich in which the poly(dimethylsiloxane) (PDMS, a silicone rubber) layer is used for pneumatic membrane pumping and valving of the PCR reagents. Initial on-chip PCR–CE tests of BK virus replicated in multiple uncoated chips showed variable results, usually yielding no detectable product at the target sample concentrations used. Subsequent “chip-flush” experiments, where water or reagents were flushed through a chip and subsequently incorporated in off-chip PCR, highlighted bovine serum albumin (BSA) amongst other pre-treatments, chip materials and PCR recipes as being effective in mitigating inhibition. When the BSA channel pre-coating was applied to on-chip PCR–CE experiments, a substantial improvement (10× to 40×) in signal-to-noise (S/N) of the CE product peak was conferred, and was shown with high confidence despite high S/N variability. This is the first study to quantitatively examine BSA’s ability to reduce inhibition of PCR performed on PDMS chips, and one of very few microfluidic PCR inhibition studies of any kind to use a large number of microfluidic chips (~400). The simplicity and effectiveness of our BSA coating suggest that passivating materials applied to microfluidic device channel networks may provide a viable pathway for development of bio-compatible devices with reduced complexity and cost.

Integrated allele-specific polymerase chain reaction–capillary electrophoresis microdevice for single nucleotide polymorphism genotyping

An integrated allele-specific (AS) polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for multiplex single nucleotide polymorphism (SNP) genotyping on a portable instrumentation, which was applied for on-site identification of HANWOO (Korean indigenous beef cattle). Twelve sets of primers were designed for targeting beef cattle's eleven SNP loci for HANWOO verification and one primer set for a positive PCR control, and the success rate for identification of HANWOO was demonstrated statistically. The AS PCR and CE separation for multiplex SNP typing was carried out on a glass-based microchip consisting of four layers: a microchannel plate for microfluidic control, a Pt-electrode plate for a resistance temperature detector (RTD), a poly(dimethylsiloxane) (PDMS) membrane and a manifold glass for microvalve function. The operation of the sample loading, AS PCR, microvalve, and CE on a chip was automated with a portable genetic analyzer, and the laser-induced fluorescence detection was performed on a miniaturized fluorescence detector. The blind samples were correctly identified as a HANWOO by showing one or two amplicon peaks in the electropherogram, while the imported beef cattle revealed more than five peaks. Our genetic analysis platform provides rapid, accurate, and on-site multiplex SNP typing.

An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis

A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 μl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.

Impact of DNMT3A mutations on Clinical Response to the Hypomethylating Agent Decitabine in Older Patients (pts) with Acute Myeloid Leukemia (AML)

Abstract 944Loss of function mutations of the DNA methyltransferase 3A (DNMT3A) gene have been associated with a negative impact on outcome in AML pts treated with conventional chemotherapy and/or stem cell transplantation. DNA methyltransferases (DNMTs) such as DNMT3A are responsible for catalyzing the addition of methyl groups to CpG dinucleotides. In AML, aberrant DNMT activity plays a role in epigenetic silencing of genes involved in hematopoiesis, and inhibition of DNMT activity by hypomethylating agents such as azacitidine and decitabine has been explored as a way to reverse hypermethylated gene silencing and induce clinical response. However, whether DNMT3A mutations influence response to hypomethylating agents is unknown. We studied the frequency and impact of DNMT3A mutations in a cohort of AML pts similarly treated with low dose decitabine. Genomic DNA from pretreatment bone marrow specimens from 46 pts (age 32–85) with untreated or relapsed AML who received decitabine 20mg/m2/d for 10 days every 28 days alone (n=39) or in combination with escalating doses of bortezomib (1.3mg/m2/d on days 5, 8, 12 & 15; n=7) were evaluated. DNMT3A was studied for mutations in the “hot spot” regions, and the mutational status of NPM1, CEBPA, IDH1/IDH2 and TET2 was assessed using polymerase chain reaction (PCR) and direct sequencing. Mutations within FLT3 were determined by quantitative fluorescence-based PCR capillary electrophoresis. Of the 46 pts, 17 had cytogenetically normal (CN) AML. Ten pts belonged to the Favorable genetic group as defined by the European LeukemiaNet (ELN) reporting system, nine pts fell into the ELN Intermediate-I, 11 were in the Intermediate-II, and 16 in the Adverse. Of the 46 pts, eight (17%) carried a DNMT3A mutation: six had codon R882 missense mutations, one nonsense (c.1729A>T; p.K576X) and one splice-site (c.2322+1G>A) mutation. DNMT3A mutations were distributed across all ELN genetic groups: three in the ELN Favorable, one in the ELN Intermediate-I, one in the ELN Intermediate-II, and three in the ELN Adverse group. Among the other molecular markers, only NPM1 mutations were significantly associated with DNMT3A mutations (P=.004). The complete remission (CR) rate for the entire cohort was 41% (19/46, 95% CI: 27% to 57%). Six of eight DNMT3A-mutated pts (75%) reached CR, compared to 13 of 38 with wild-type DNMT3A (34%; P=.05). All five pts with mutated DNMT3A and mutated NPM1 achieved CR, a significantly better response rate than that observed among the remaining pts (P=.008). The median overall survival of DNMT3A-mutated pts was longer than that of DNMT3A-wild-type pts (16.8 vs 11.0 months), but the difference did not reach statistical significance, likely due to the relatively small number of pts. Our group has shown that high levels of microRNA miR-29b, which targets and downregulates DNMT3A, are significantly associated with response to decitabine. (Blum W et al, PNAS 107:7473) Among the pts achieving CR, DNMT3A mutations were present in the pts with the lowest miR-29b expression levels (Wilcoxon’s P=.02 for the comparison of miR-29b expression between CR pts with and without DNMT3A mutations). Indeed, the miR-29b expression levels in these three pts was similar to that of pts who did not achieve CR, thereby supporting an impact of the mutation independent from that of the miR. These initial findings, while based on a small sample size, suggest a potential link between DNMT3A mutations and response to decitabine. If confirmed in a larger cohort, screening for DNMT3A mutation status could be used for risk-adapted stratification of AML pts to decitabine-based regimens.Supported by NIH/NCI grants NO1 CM62207; UO1 CA76576; K23CA120708 (WB); R01 CA102031 (GM), K12CA133250 (JCB, AW); AW is an ASH-AMFDP Scholar.Table 1:Pt Characteristics and Response to Decitabine TreatmentCR n=19No CR n=27PAge (years) median (range)72 (62 – 85)76 (32 – 84).09WBC (×103/μl) median (range)2.7 (1.1 – 119.5)2.2 (0.6 – 150).74Bone marrow blast count median (range)35 (20 – 87)39 (18 – 92).23ELN genetic category no..77favorable55intermediate-I45intermediate-II38adverse79DNMT3A mutated no.62.05NPM1 mutated no.63.13DNMT3A mutated &NPM1mutated no.50.008IDH1 or IDH2 mutated no.341.0TET2 mutated no.351.0FLT3-ITD no.21.56FLT3-TKD no.011.0CEBPA mutated no.14.39P values are from the Wilcoxon test for continuous data or Fisher's exact test for count data and not adjusted for multiple comparisons Disclosures:Off Label Use: Decitabine alone or in combination with bortezomib is not approved for the treatment of AML.

NFKB1Insertion/Deletion Promoter Polymorphism Increases the Risk of Advanced Ovarian Cancer in a Chinese Population

Ovarian cancer is the leading cause of death among all gynecological cancers. This is mainly attributed to its frequent presentation at an advanced stage (International Federation of Gynecology and Obstetrics stage III-IV). Nuclear factor-kappaB (NF-κB) is critically involved in the carcinogenesis and development of ovarian cancer. A functional insertion/deletion polymorphism (-94 ins/del ATTG) in the promoter region of the NFKB1 gene, which encodes the p50 subunit of the NF-κB protein, has been recently identified and shown to increase the susceptibility to many diseases. The purpose of this study was to explore the association between this polymorphism and the risk of advanced ovarian cancer in a Chinese population. A total of 179 advanced ovarian cancer patients and 223 healthy controls were recruited into this study. Genotypes were determined using polymerase chain reaction-capillary electrophoresis method. The insertion increased the risk of advanced ovarian cancer (odds ratio = 2.111, 95% confidence intervals = 1.125-3.961, p = 0.019 for heterozygote insertion, and odds ratio = 2.656, 95% confidence intervals = 1.397-5.051, p = 0.002 for homozygote insertion) compared with homozygote deletion. Similar results were seen in age-adjusted analyses (p < 0.05). Our preliminary results suggest that NFKB1-94 ins/del ATTG promoter polymorphism may be associated with increased susceptibility to advanced ovarian cancer.

Multichannel PCR-CE Microdevice for Genetic Analysis

We have developed a fully integrated multichannel polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice with nanoliter reactor volumes for highly parallel genetic analyses. Resistance temperature detectors and heaters made out of Ti/Pt are integrated on the microchip using a scalable radial design to provide precise temperature control of the four parallel PCR-CE reactor systems. Heating rates of >15 degrees C s(-1) and cooling rates of >10 degrees C s(-1) allow cycle times of 50 s and 30 complete PCR cycles in <27 min. PDMS membrane valves control and localize PCR reagents in the 380-nL reactors. By directly integrating PCR reactors with the CE separation system, efficient coupling of amplification with separation is achieved. The microdevice demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380-nL reactor (approximately 43 aM) with signal-to-noise ratio greater than 10. Parallel PCR-CE multiplex amplification and genetic analyses of four different samples with (1) both M13mp18 control template and E. coli K12 cells, (2) only M13mp18 template, (3) only E. coli K12 cells, and (4) negative control are completed in less than 30 min in a single run.

Quantification of BCR-ABL transcripts in CML patients in cytogenetic remission after interferon-α-based therapy

We measured using a competitive quantitative polymerase chain reaction-capillary electrophoresis (PCR-CE)-based assay, the levels of bcr-abl transcripts in 44 patients with chronic myeloid leukemia (CML) after interferon-alpha (IFN-alpha) therapy, who achieved a major (10 patients, MCR group) or complete (34 patients, CCR group) cytogenetic response. All 34 CCR patients had molecular evidence of residual disease detected in bone marrow samples at the time of best karyotypic response. The median number of bcr-abl transcripts of 34 evaluable patients in the CCR group at the time of complete cytogenetic remission was 4/microg RNA (range 3-4600), while the median number of bcr-abl transcripts of 10 patients in the MCR group at the time of best cytogenetic response was 4490/microg RNA (range 600-23 900) (P = 0.000024). In nine CCR and five MCR patients we were able to quantify the amount of bcr-abl transcript both at diagnosis and after interferon therapy: no statistical difference (P = 0.18) was found between the two groups at diagnosis (median bcr-abl transcripts/microg RNA was 30 000 vs. 39 650, respectively). During IFN-alpha therapy, the two groups were evaluable at the time of major karyotypic conversion: at this point, there was a statistical difference of expression of bcr-abl transcript between the CCR group (17 patients) (median 2700; range 76-40 000) and the MCR group (10 patients) (median 4490; range 600-23 900), respectively (P = 0.046). No differences of bcr-abl amount of transcript were found in patients with CCR obtained either by IFN-alpha therapy alone (20 patients) vs. IFN-alpha plus ABMT (13 patients) (P = 0.47). We firstly demonstrated that although the CCR and MCR groups were clinically, cytogenetically and molecularly indistinguishable at diagnosis, the two groups could be recognized successfully during interferon therapy based on the level of bcr-abl transcript.