Solid-phase reversible immobilization for the isolation of PCR products

DeAngelis, Margaret M.; Wang, David G.; Hawkins, Trevor

doi:10.1093/nar/23.22.4742

Cited by 320 publications

(270 citation statements)

References 3 publications

Supporting

Mentioning

270

Contrasting

Order By: Relevance

“…We use in-house in vitro transcription as a cost-effective alternative to synthetic sgRNAs, whereas using commercial synthetic sgRNAs could further shorten the time needed to conduct the experiments. We routinely use column-based methods for sgRNA purification, but solid-phase reversible immobilization (SPRI) magnetic beads can be used to the same effect and are best suited for large-scale preparation in a multiwell format (24). The final electroporation step is done in a 96-well format so that a large number of cell lines can be processed in parallel.…”

Section: Resultsmentioning

confidence: 99%

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Leonetti

Sekine

Kamiyama

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

222

201

View full text Add to dashboard Cite

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context. CRISPR/Cas9 | GFP library | genome engineering M ore than a decade after the completion of the Human Genome Project (1), over 30% of human genes still lack clear functional annotation (2, 3). Functional tagging is a powerful strategy to characterize the cellular role of proteins. In particular, tags allow access to two key features of protein function: localization (using fluorescent tags) and interaction partners (using epitope tags and immunoprecipitation). Hence, by tagging proteins in a systematic manner, a comprehensive functional description of an organism's proteome can be achieved. The power of systematic tagging approaches is best illustrated by studies conducted in the budding yeast Saccharomyces cerevisiae (4). In particular, a genome-wide collection of GFP-tagged yeast strains enabled the systematic study of protein localization in live cells (5), whereas libraries of strains expressing TAP epitope-fusion proteins paved the way for the large-scale isolation and proteomic analysis of protein complexes (6, 7). One of the great advantages of yeast genetics (especially in S. cerevisiae) is the efficiency and relative simplicity of PCR-based homologous recombination (8). As a result, functional tags can be easily inserted in a gene locus of interest, preserving endogenous expression levels and minimizing genomic disruption. Together, these genome-wide tagged libraries helped provide a comprehensive snapshot of the yeast protein landscape under near-native conditions (4, 5, 9-11).The development of clustered regularly interspersed short palindromic repeat associated protein 9 (CRISPR/Cas9)-based methods has profoundly transformed our ability to dir...

show abstract

Section: Resultsmentioning

confidence: 99%

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Leonetti

Sekine

Kamiyama

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

222

201

View full text Add to dashboard Cite

show abstract

“…PCR products were prepared for sequencing using solidphase reversible immobilization (SPRI) using Bangs Estapor SuperParamagnetic Microspheres (Bangs Laboratories, IN, USA, and Seradyn Uniform Microparticles, MN, USA) as described. 35 Sequencing was performed using BigDye Terminator Chemistry (PerkinElmer) on a capillary ABI 3700. SNP detection was as described previously.…”

Section: Snp Identificationmentioning

confidence: 99%

Family-based association study of 76 candidate genes in bipolar disorder: BDNF is a potential risk locus

et al. 2002

View full text Add to dashboard Cite

Identification of the genetic bases for bipolar disorder remains a challenge for the understanding of this disease. Association between 76 candidate genes and bipolar disorder was tested by genotyping 90 single-nucleotide polymorphisms (SNPs) in these genes in 136 parent-proband trios. In this preliminary analysis, SNPs in two genes, brain-derived neurotrophic factor (BDNF) and the alpha subunit of the voltage-dependent calcium channel were associated with bipolar disorder at the PϽ0.05 level. In view of the large number of hypotheses tested, the two nominally positive associations were then tested in independent populations of bipolar patients and only BDNF remains a potential risk gene. In the replication samples, excess transmission of the valine allele of amino acid 66 of BDNF was observed in the direction of the original result in an additional sample of 334 parent-proband trios (T/U=108/87, P=0.066). Resequencing of 29 kb surrounding the BDNF gene identified 44 additional SNPs. Genotyping eight common SNPs identified three additional markers transmitted to bipolar probands at the P Ͻ 0.05 level. Strong LD was observed across this region and all adjacent pairwise haplotypes showed excess transmission to the bipolar proband. Analysis of these haplotypes using TRANSMIT revealed a global P value of 0.03. A single haplotype was identified that is shared by both the original dataset and the replication sample that is uniquely marked by both the rare A allele of the original SNP and a novel allele 11.5 kb 3Ј. Therefore, this study of 76 candidate genes has identified BDNF as a potential risk allele that will require additional study to confirm.

show abstract

“…comm. ), to automate all of the reaction steps and conditions required for successful completion of the Solid-Phase Reversible Immobilization (SPRI) process (Hawkins et al 1994;DeAngelis et al 1995;Hawkins et al 1997). The system uses a CRS A465 six degrees of freedom articulated robot, POLARA software, and modules for liquid handling, shaking, washing, dispensing, bead settling, lidding and de-lidding, barcode identification, plate feeding, and storage.…”

Section: Dna Template Preparation Systemsmentioning

confidence: 99%

Automation for Genomics, Part One: Preparation for Sequencing

Meldrum

2000

Genome Res.

View full text Add to dashboard Cite

In the past four years, automation for genomics has enabled a 43-fold increase in the total finished human genomic sequence in the world. This two-part noncomprehensive review will provide an overview of different types of automation equipment used in genome sequencing. Part One focuses on equipment involved in DNA preparation, DNA sequencing reactions, and other automated procedures for preparing DNA for running on sequencers or subsequent analysis; it also includes information on the development of these machines at various genome centers. Part Two, to be published in the next issue, will cover sequencing machinery and array technology, and conclude with a look at the future technologies that will revolutionize molecular biology. "Alternate" sequencing technologies (including mass spectrometry, biochips, and single-molecule analysis) will also be examined.

show abstract

Solid-phase reversible immobilization for the isolation of PCR products

Cited by 320 publications

References 3 publications

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

Family-based association study of 76 candidate genes in bipolar disorder: BDNF is a potential risk locus

Automation for Genomics, Part One: Preparation for Sequencing

Contact Info

Product

Resources

About