Abstract:
:Next-generation sequencing (NGS) of multigene panels performed for genetic clinical diagnostics requires 100% coverage of all targeted genes. In the genetic diagnostics laboratory, coverage gaps are typically filled with Sanger sequencing after NGS data are collected and analyzed. Libraries prepared using the hybridization-based custom capture HaloPlex method are covered at ~98% and include gaps in coverage because of the location of the restriction enzyme sites used for fragmentation and differences in the designed and actual library insert size. We describe a method for improving the coverage of HaloPlex libraries by generating a set of amplicons spanning known low-coverage regions that are pooled, indexed by sample, and sequenced together with the HaloPlex libraries. This approach reduces the number of post-NGS Sanger sequencing reactions required and complements any NGS library preparation method when complete gene coverage is necessary.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Coonrod EM,Durtschi JD,VanSant Webb C,Voelkerding KV,Kumánovics Adoi
10.2144/000114217subject
Has Abstractpub_date
2014-10-01 00:00:00pages
204-7issue
4eissn
0736-6205issn
1940-9818pii
000114217journal_volume
57pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract::We describe a multipurpose eukaryotic expression vector that incorporates the following features: restriction sites for in-frame insertion of cDNAs of interest between sequences encoding the glutathione-S-transferase (GST) and an oligohistidine element, allowing expression of the corresponding fusion proteins; a phosp...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1995-01-01 00:00:00
abstract::The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/01312pf01
更新日期:2001-08-01 00:00:00
abstract::DNA damage in the form of abasic sites, chemically altered nucleotides, and strand fragmentation is the foremost limitation in obtaining genetic information from many ancient samples. Upon cell death, DNA continues to endure various chemical attacks such as hydrolysis and oxidation, but repair pathways found in vivo n...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114307
更新日期:2015-07-01 00:00:00
abstract::The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for th...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/01313rr01
更新日期:2001-09-01 00:00:00
abstract::We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 microgr...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/03344pt03
更新日期:2003-04-01 00:00:00
abstract::We propose two methods for characterizing the spatio-temporal behavior of cell populations in culture. The first method, image auto-correlation microscopy (IACM), allows us to characterize the variation in the number of objects as a function of time, thus enabling the quantification of the clustering properties of cel...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112478
更新日期:2007-07-01 00:00:00
abstract::We report here a system for automation of the dideoxynucleotide DNA sequencing method. The system consists of a Beckman Biomek 1000 robotic workstation which has been modified by the addition of a thin heater/cooler block directly on the instrument table. The heater/cooler block, which is regulated by a user-specified...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1988-09-01 00:00:00
abstract::High stabilities of reporter proteins and their messenger RNAs (mRNAs) interfere with the detection of rapid transient changes in gene expression, such as transcriptional blocks posed by genotoxic DNA lesions. We have modified a green fluorescent protein (GFP) gene within the episomal pMARS vector by addition of a fra...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112479
更新日期:2007-08-01 00:00:00
abstract::A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguish...
journal_title:BioTechniques
pub_type:
doi:10.2144/03341dd09
更新日期:2003-01-01 00:00:00
abstract::Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02332rr04
更新日期:2002-08-01 00:00:00
abstract::Inverse PCR has been used for the recovery of genome regions flanking a known sequence, although its application to metagenome walking is limited due to inefficient amplification from low copy number fragments. Here we present an improved inverse PCR scheme that enables walking of rare fragments in environmental metag...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112210
更新日期:2006-08-01 00:00:00
abstract::Ribonucleases H (RNases H) are enzymes that specifically degrade the RNA of RNA-DNA hybrids. These enzymes are involved in DNA replication, reverse transcription (RT) and antisense oligodeoxyribonucleotide-mediated arrest of translation. One of the most valuable tools for assaying RNase H activity is the renaturation ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97235rr01
更新日期:1997-11-01 00:00:00
abstract::A labeled peptide nucleic acid (PNA) antisense probe was used to study the spatial distribution of triplet repeats (CTG) in human myotonic dystrophy (DM) cells by high-resolution fluorescence in situ hybridization (FISH). It was found that transcripts containing triplet repeats were present as a number of discrete foc...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98243rr02
更新日期:1998-03-01 00:00:00
abstract::We have utilized 5-bromo-2'deoxyuridine (BrdU) substituted DNA as a probe for a number of applications including, principally, for chromosome painting by fluorescence in situ hybridization (FISH) but also for DNA end-labeling to detect apoptotic cell death and for filter hybridization. Br-dUTP was used as a substitute...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1996-07-01 00:00:00
abstract::In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112476
更新日期:2007-06-01 00:00:00
abstract::Much effort has been directed toward the isolation and characterization of homeobox cDNAs from numerous cell types because they encode transcription factors important to many cellular processes, including pattern formation in the embryo, cell growth and cell differentiation. Many novel homeobox cDNAs have been isolate...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1994-05-01 00:00:00
abstract::Several methods and computer programs have been developed for estimating the size of DNA fragments from gel electrophoresis. However, methods are lacking that may facilitate in optimization of gel conditions. In this article, a computer program called DNAOPT is described, which was developed to assist researchers in t...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1995-02-01 00:00:00
abstract::A dot immunoblotting technique has been developed to estimate the relative expression levels of tagged recombinant human proteins in mammalian cell culture media. Variations in sample denaturation, blocking agents and membrane composition and treatment were used to optimize the signal-to-noise ratio of the defined pro...
journal_title:BioTechniques
pub_type:
doi:10.2144/00285st07
更新日期:2000-05-01 00:00:00
abstract::Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not co...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04371DD02
更新日期:2004-07-01 00:00:00
abstract::The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids th...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02335st03
更新日期:2002-11-01 00:00:00
abstract::The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converte...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112757
更新日期:2008-02-01 00:00:00
abstract::Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04363RR01
更新日期:2004-03-01 00:00:00
abstract::Expanding applications of cDNA microarrays such as fine needle aspiration biopsy and laser capture microdissection necessitate the ability to perform arrays with minute starting amounts of RNA. While methods for amplifying RNA have been advocated, the fidelity of array results using amplified material has not been ful...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02334mt04
更新日期:2002-10-01 00:00:00
abstract::Naturstoff reagent A (diphenylboric acid 2-aminoethyl ester [DPBA]) has been used historically in plant science to observe polyphenolic pigments, such as flavonoids, whose fluorescence requires enhancement to be visible by microscopy. Flavonoids are common dietary constituents and are the focus of considerable attenti...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2018-0084
更新日期:2019-02-01 00:00:00
abstract::Genome sequencing projects are either based on whole genome shotgun (WGS) or on a BAC-by-BAC strategy. Although WGS is in most cases the preferred choice, sometimes the BAC-by-BAC approach may be better because it requires a much simpler assembly process. Furthermore, when the study is limited to specific regions of t...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112686
更新日期:2008-01-01 00:00:00
abstract::Thymidine kinase 1 (TK1) is an enzyme involved in DNA precursor synthesis that has been used as a biomarker for prognosis and monitoring of different malignancies. In this study, we compared two immunoassays for measuring TK1 protein concentrations: the TK 210 ELISA (AroCell AB) and TK1 ELISA from Abcam. Overall, the ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2019-0148
更新日期:2020-06-01 00:00:00
abstract::Based on the requirement of a glutathione-deficient mutant strain of Saccharomyces cerevisiae to take up external glutathione for growth on synthetic media, a simple agar diffusion test for quantitative detection of total glutathione from various sources was established. Glutathione concentrations can be reliably dete...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/96215st07
更新日期:1996-11-01 00:00:00
abstract::We have developed a novel method for the rapid preparation of large quantities of 3' end-labeled single-stranded (ss) DNA (ssDNA) probes for transcript mapping. A recombinant phagemid vector containing the probe sequence was used to raise large quantities of ssDNA. Based on the DNA sequence of the probe, an oligonucle...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-05-01 00:00:00
abstract::MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114574
更新日期:2017-08-01 00:00:00
abstract::A fully automated nucleic acid analysis system is described, which offers positive sample identification, improved sensitivity and reduced user interaction compared to conventional techniques. The system relies on the sequence-specific capture of DNA onto solid-phase particles, confirming product identity without the ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/99271pf01
更新日期:1999-07-01 00:00:00