Short DNA Probes Developed for Sample Tracking and Quality Assurance in Gene Panel Testing.

Both multiplexing and target-enrichment technologies are key to reducing the cost of genetic testing using next-generation sequencing (NGS). Many diagnostic laboratories routinely handle thousands of targeted resequencing samples, leading to an increased risk of accidental sample mix-ups and cross contamination. Herein, we present a short DNA fragment that can be spiked into the original genomic DNA (gDNA) or whole blood sample and tracked through to the final targeted resequencing data. This DNA fragment comprises a 15-bp unique index sequence assembled with a 120-bp fixed sequence designed for recovery in a hybridization capture reaction. In a pilot study, the yield of the recovered probe was examined in a step-by-step genetic testing procedure, involving gDNA isolation from whole blood, library preparation for NGS, and capture hybridization. On the basis of the results, 10 fmol (6 × 109 molecules) and 10 amol (6 × 106 molecules) of the spike-in probe were estimated to be suitable for DNA and RNA probe-based library preparation and target enrichment from 200 ng (6.5 × 104 copies) gDNA, respectively. In fact, the number of NGS reads corresponding to the spike-in probe was almost equal to that corresponding to the genomic target regions and was sufficient for evaluating sample identification and cross-contamination events. Hence, this method may be useful for enhancing quality assurance in clinical genetic testing.

Closed-Tube PCR with Nested Serial Invasion Probe Visualization Using Gold Nanoparticles.

BACKGROUND: Detecting DNA biomarkers related to personalized medicine could improve the outcome of drug therapy. However, personalized medicine in a resource-restrained hospital is very difficult because DNA biomarker detection should be performed by well-trained staff and requires expensive laboratory facilities. METHODS: We developed a gold nanoparticle-based "Tube-Lab" to enable DNA analysis in a closed tube. Gold nanoparticle-modified probes (GNPs) were used to construct an inexpensive and simple DNA sensor for signal readout. The method consists of 3 steps (template amplification, sequence identification, and GNP-based signal readout), bridged by an invasive reaction. With temperature control at each step, the 3 reactions proceed sequentially and automatically in a closed tube without any liquid transfer. We used Tube-Lab to detect different biomarkers in blood, tissue, and plasma, including US Food and Drug Administration-approved pharmacogenomic biomarkers (single nucleotide polymorphisms, somatic mutations). RESULTS: The combination of PCR-based template replication and invader-based signal amplification allowed detection of approximately 6 copies of input DNA and the selective pick up 0.1% mutants from large amounts of background DNA. This method highly discriminated polymorphisms and somatic mutations from clinical samples and allowed a "liquid biopsy" assay with the naked eye. CONCLUSIONS: Tube-Lab provides a promising and cost-effective approach for DNA biomarker analysis, including polymorphisms and somatic mutations from blood DNA, tissue DNA, or circulating tumor DNA in plasma, which are critical for personalized medicine.

Establishment of a novel two-probe real-time PCR for simultaneously quantification of hepatitis B virus DNA and distinguishing genotype B from non-B genotypes.

OBJECTIVE: Establishment of a simple, rapid and economical method for quantification and genotyping of hepatitis B virus (HBV) is of great importance for clinical diagnosis and treatment of chronic hepatitis B patients. We hereby aim to develop a novel two-probe real-time PCR for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes. METHODS: Conserved primers and TaqMan probes for genotype B and non-B genotypes were designed. The linear range, detection sensitivity, specificity and repeatability of the method were assessed. 539 serum samples from HBV-infected patients were assayed, and the results were compared with commercial HBV quantification and HBV genotyping kits. RESULTS: The detection sensitivity of the two-probe real-time PCR was 500IU/ml; the linear range was 10(3)-10(9)IU/ml, and the intra-assay CVs and inter-assay CVs were between 0.84% and 2.80%. No cross-reaction was observed between genotypes B and non-B. Of the 539 detected samples, 509 samples were HBV DNA positive. The results showed that 54.0% (275/509) of the samples were genotype B, 39.5% (201/509) were genotype non-B and 6.5% (33/509) were mixed genotype. The coincidence rate between the method and a commercial HBV DNA genotyping kit was 95.9% (488/509, kappa=0.923, P<0.01). In addition, high correlation (R(2)=0.94, P<0.05) and good consistency between our assay and a commercial HBV DNA qPCR kit were achieved. CONCLUSIONS: A novel two-probe real-time PCR method for simultaneous quantification of HBV viral concentration and distinguishing genotype B from non-B genotypes was established. The assay was sensitive, specific and reproducible which can be applied to areas prevalent with HBV genotypes B and C, especially in China.

Detection of DNA variations in the polymorphic hydroxymethylbilane synthase gene by high-resolution melting analysis.

Acute intermittent porphyria (AIP) represents the most frequent type of acute porphyria. The underlying cause is a defect in the hydroxymethylbilane synthase (HMBS) gene. Diagnosis of AIP is crucial for preventing life-threatening, acute attacks among both symptomatic and asymptomatic carriers. We established the diagnostic tool, high-resolution melting (HRM), for diagnosing AIP. Of 13 amplicons amplified by PCR in the presence of the LCGreen Plus dye, 4 showed polymorphic backgrounds. The ability of the HRM method to detect DNA variations in the HMBS gene was tested on a DNA sample with 10 known mutations by a curve shape scan using the LightScanner instrument. Furthermore, genomic DNA (gDNA) samples from 97 individuals with suspected hepatic porphyria were tested. All possible genotypes from each of four polymorphic amplicons were detected. Each of the 10 mutations tested had an altered melting profile compared with the melting profile of the controls. Screening the group of subjects with suspected hepatic porphyria revealed nine different DNA variations, four of which were novel. In conclusion, HRM is a fast, cost-effective prescreening method for detecting DNA variations in the HMBS gene. Therefore, the screening can be easily applied to a porphyria family if misdiagnosis or rare dual porphyria is suspected.

Levels of minimal residual disease detected by quantitative molecular monitoring herald relapse in patients with multiple myeloma.

BACKGROUND AND OBJECTIVES: Detection of minimal residual disease (MRD) has helped to improve the treatment of patients with leukemia. At present MRD testing in patients with multiple myeloma (MM) is not applied as a standard diagnostic or prognostic method. DESIGN AND METHODS: Immunoglobulin heavy chain (IgH) polymerase chain reaction (PCR) using patient-specific TaqMan probes together with LightCycler technology was performed to quantify minimal residual disease in MM. Relative levels of clonotypic cells were assessed as IgH/2beta-actin ratios with a sensitivity of 10(-4) to 10(-5). RESULTS: Following stem cell transplantation, a significant reduction of clonotypic cells was observed in bone marrow (BM) and peripheral blood (PB) samples of 11 patients, comparing pre-treatment values with those of best response (median: 13% to 0.09% and 0.03% to 0%, respectively). In 5 patients with ongoing clinical remission IgH/2beta-actin ratios remained stable at a low level, while in 6 patients an increase to 2% in BM and 0.4% in PB was associated with progression of the disease. In 4 of these 6 patients the increase of clonotypic cells in PB was detectable a median of 3 months (range: 0.5-6) before relapse. Furthermore, time-to-progression of patients with pre-transplantation IgH/2b-actin ratios > 0.03% in BM was significantly shorter than that of patients with lower MRD levels. INTERPRETATION AND CONCLUSIONS: MRD in patients with MM can be quantified reliably using TaqMan chemistry adapted to the LightCycler system. Residual tumor cell levels before transplantation as well as results of sequential molecular monitoring are predictive of relapse.

DNA hybridization to mismatched templates: a chip study.

High-density oligonucleotide arrays are among the most rapidly expanding technologies in biology today. In the GeneChip system, the reconstruction of the sample mRNA concentrations depends upon the differential signal generated by hybridizing the RNA to two nearly identical templates: a perfect match probe (PM) containing the exact biological sequence; and a single mismatch (MM) differing from the PM by a single base substitution. It has been observed that a large fraction of MMs repetitively bind targets better than the PMs, against the obvious expectation of sequence specificity. We examine this problem via statistical analysis of a large set of microarray experiments. We classify the probes according to their signal to noise (S/N) ratio, defined as the eccentricity of a (PM,MM) pair's "trajectory" across many experiments. Of those probes having large S/N (>3) only a fraction behave consistently with the commonly assumed hybridization model. Our results imply that the physics of DNA hybridization in microarrays is more complex than expected, and suggest estimators for the target RNA concentration.

Detection of serum hepatitis B virus using a nested polymerase chain reaction assay.

We described a nested polymerase chain reaction (PCR) assay to detect serum hepatitis B virus (HBV) DNA in patients. We compared the sensitivity of the PCR assay to that of slot-blot hybridization for detecting HBV DNA in serum and found that analysis by PCR provides a > 10(4) fold increase over the slot-blot assay. Most of the HBe Ag-positive patients were positive in PCR, and most of those being anti-HBe-positive were negative with this PCR assay.