We have developed a procedure for massively parallel resequencing of multiple human genes by combining a highly multiplexed and target-specific amplification process with a high-throughput parallel sequencing technology. The amplification process is based on oligonucleotide constructs, called selectors, that guide the circularization of specific DNA target regions. Subsequently, the circularized target sequences are amplified in multiplex and analyzed by using a highly parallel sequencing-by-synthesis technology. As a proof-of-concept study, we demonstrate parallel resequencing of 10 cancer genes covering 177 exons with average sequence coverage per sample of 93%. Seven cancer cell lines and one normal genomic DNA sample were studied with multiple mutations and polymorphisms identified among the 10 genes. Mutations and polymorphisms in the TP53 gene were confirmed by traditional sequencing.cancer analysis ͉ high-throughput sequencing ͉ multiplex amplification
2Transcript levels of several Escherichia coli genes involved in chromosome replication and cell division were measured in dnaC2(Ts) mutants synchronized for chromosome replication by temperature shifts. Levels of transcripts from four of the genes, dam, nrdA, mukB, and seqA, were reduced at a certain stage during chromosome replication. The magnitudes of the decreases were similar to those reported previously for ftsQ and ftsZ (P. Zhou and C. E. Helmstetter, J. Bacteriol. 176:6100-6106, 1994) but considerably less than those seen with dnaA, gidA, and mioC (P. W. Theisen, J. E. Grimwade, A. C. Leonard, J. A. Bogan, and C. E. Helmstetter, Mol. Microbiol. 10:575-584, 1993). The decreases in transcripts appeared to correlate with the estimated time at which the genes replicated. This same conclusion was reached in studies with synchronous cultures obtained with the baby machine in those instances in which periodicities in transcript levels were clearly evident. The transcriptional levels for two genes, minE and tus, did not fluctuate significantly, whereas the transcripts for one gene, iciA, appeared to increase transiently. The results support the idea that cell cycle timing in E. coli is not governed by timed bursts of gene expression, since the overall findings summarized in this report are generally consistent with cell cycle-dependent transient inhibitions of transcription rather than stimulations.
Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.
Evidence suggests that the practice of sharing clinicians’ notes with patients via online patient portals may increase patient engagement and improve patient–clinician relationships while requiring little change in providers’ workflow. Authors examined clinical social workers’ experiences and attitudes related to open psychotherapy notes using focus groups and telephone interviews. Twenty-four of 29 eligible therapists agreed to open their notes to patients, and nine participated in this study. Participants were generally positive about their experiences and reported few disruptions to their workload or practice. However, they were hesitant to bring up notes to patients during sessions, and they discussed the benefits of open therapy notes mostly hypothetically. The five therapists who did not share notes worried that open notes would be detrimental to therapeutic relationships, patient well-being, and workflow. However, the concern they discussed most often related to the electronic health record rather than to open notes, because therapy notes are visible to all authorized clinicians as part of the general medical record. Future research is needed to deepen our understanding of the risks and benefits of open psychotherapy notes and to inform development of training programs to support therapists in opening notes.
Childhood leukemia, which accounts for > 30% of newly diagnosed childhood malignancies, is one of the leading causes of death for children with cancer. Genome-wide studies using microarray chips to identify copy number changes in human cancer are becoming more common. In this pilot study, 45 pediatric leukemia samples were analyzed for gene copy aberrations using novel molecular inversion probe (MIP) technology. Acute leukemia subtypes included precursor B-cell acute lymphoblastic leukemia (ALL) (n = 23), precursor T-cell ALL (n = 6), and acute myeloid leukemia (n = 14). The MIP analysis identified 69 regions of recurring copy number changes, of which 41 have not been identified with other DNA microarray platforms. Copy number gains and losses were validated in 98% of clinical karyotypes and 100% of fluorescence in situ hybridization studies available. We report unique patterns of copy number loss in samples with 9p21.3 (CDKN2A) deletion in the precursor B-cell ALL patients, compared with the precursor T-cell ALL patients. MIPs represent an attractive technology for identifying novel copy number aberrations, validating previously reported copy number changes, and translating molecular findings into clinically relevant targets for further investigation.
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