“Molecular Biology Core Facilities”的意思、由来-开放百科全书

This is an example of a molecular biology core developed in an academic institution over the past 35 years. These molecular biology cores are now commonplace and necessary in that they provide NIH funded academic labs access to expensive instrumentation in a shared use setting.

The Molecular Biology Core Facilities (MBCF) was created to allow investigators at the Dana-Farber Cancer Institute (DFCI) access to cutting edge molecular biology tools which would be tested and developed in a shared setting. Collaborations can be set up with anyone in the world. Although these services are primarily focused on Cancer and AIDS research, there is a broad spectrum of research that uses these resources.^[1]

History

The MBCF at DFCI was first started in 1984 to supply small oligonucleotides to researchers. Marvin H. Caruthers at the University of Colorado determined how to synthesize small oligonucleotides by solid-phase synthesis using the phosphoramidite method.^[2]^[3] Caruthers and Leroy Hood at the California Institute of Technology constructed the first automated DNA Synthesizer which the MBCF successfully tested after discussions with Don Taylor.

Because of the growing demand for oligonucleotide primers to initiate DNA replication and for probes, a plan was put into place to develop a core facility to produce reagents for molecular biologists as well as instrumentation for the analysis of DNA and protein samples. This plan stated that a charge-back method would be put in place to fairly spread the resources as a shared facility.

A Peptide Synthesizer using Bruce Merrifield's solid phase peptide synthesis^[4] was brought online in 1988 when Fmoc chemistry became commercially available. A Protein Sequencer using Edman degradation^[5]^[6] was installed in 1989 quickly followed by several DNA Sequencers which were the first to use fluorescent dye terminator chemistry.^[7]^[8] Mass Spectrometers were acquired to provide analysis of synthesized peptides but soon grew into a stand-alone service in high demand. Biacore^[9] instrumentation added for ligand kinetics in 1996 (and updated by Shared Instrumentation Grant to a Biacore 3000 in 2004). In 2007 a large expansion of high throughput proteomics using mass spectrometry has been funded by private donation. Blais Proteomics Center. (BPC)

The Molecular Biology Core Facilities has had a continuous web presence since 1992 and began receiving primer sequence for high throughput DNA synthesis by web forms in 1995 when new high volume DNA synthesizers were brought on-line. Since 2004 all interactions between researchers and the core services have been moved to a web-based LIMS system.

The MBCF is constantly evaluating new instrumentation which could eventually become a shared resource. The future massively parallel DNA sequencers which can sequence entire genomes in just a few days, (454, Solexa+Illumina, Helicos, SOLiD, Visigen, PacBio, ION Torrent, Nanopore), are being made available to researchers by various methods. In 2009 the Heliscope from Helicos BioSciences began being evaluated in the genomics facility.^[10] After successfully completing 2,365 Chip-Seq samples the Helicos service was shut down in 2011. An Illumina MiSeq was evaluated on multiple projects and now a series of three MiSeqs have been streamlined for fast analysis of Next Gen samples. Library prep has been automated on an IntegenX Apollo 324 and Beckman robots and several instruments have been incorporated into quality control of samples including the Pippin Prep by Sage Biosciences, an Agilent Tapestation Bioanalyzer and an Echo QPCR system from Illumina. The Mass Spectrometry proteomics facility has also undergone a large expansion to undertake more complex high-throughput projects as part of the Blais Proteomics Center.

Recent expansion of genomics includes three, now four Illumina NextSeq 500s as well as an Illumina Neoprep Library prep robot (which was returned as a rebate on another NextSeq500). The current platform for genomics in 2017 changes faster than a wiki page. MBCF can send you sample into a pipeline that is small and proof of concept or large production runs. We can run with Illumina, Pacbio, or Oxford Nanopore instruments. But that was yesterday. The big deal is library prep and amplification. We are a small production NGS lab that has done a thousand different types of NGS. We know how to prep a sample. If you want whole genome of human or chicken, go to BGI in China. They are much cheaper. That is where we send our full genome samples.

Research tools available

Funding

The MBCF is for the most part funded as a straight fee for service chargeback core facility. At times other sources of funding for research and development or expansion are incorporated into the overall budget.

References

1. ^{{cite web |url = http://cbmi.catalyst.harvard.edu/cores/cat/core.html?core_id=67&category_id=8&navMode=cat|title = The Molecular Biology Core Facilities at Dana-Farber Cancer Institute/ Harvard Cores|accessdate = 18 April 2009 |publisher = Harvard Catalyst}}
2. ^Beaucage SL, Caruthers MH. (1981) Tetrahedron Lett.22, 1859-62.
3. ^McBride LJ, Caruthers MH. (1983) Tetrahedron Lett. 24, 245-8.
4. ^{{cite journal | author = R. B. Merrifield | title = Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide | year = 1963 | journal = J. Am. Chem. Soc. | volume = 85 | issue = 14 | pages = 2149–2154 | doi = 10.1021/ja00897a025 | authorlink = Robert Bruce Merrifield}}
5. ^Edman, P. Acta Chem. Scand. 1950, 4, 283.
6. ^{{cite book |author=Niall HD |title=Automated Edman degradation: the protein sequenator |journal=Meth. Enzymol.|volume=27 |issue= |pages=942–1010 |year=1973 |pmid=4773306 |doi= 10.1016/S0076-6879(73)27039-8|url=|series=Methods in Enzymology |isbn=9780121818906 }}
7. ^Nature. 1986 Jun 12-18;321(6071):674-9. Fluorescence detection in automated DNA sequence analysis. We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.
8. ^Nucleic Acids Res. 1985 Apr 11;13(7):2399-412. The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. Note that Oxford University Press, the publishers of the journal Nucleic Acids Research, make the full contents of this journal available online for free - you can download a copy of this paper for yourself !!
9. ^H. M. Hiep et al. "A localized surface plasmon resonance based immunosensor for the detection of casein in milk" Sci. Technol. Adv. Mater. 8 (2007) 331 [https://dx.doi.org/10.1016/j.stam.2006.12.010 free download]
10. ^GenomeWeb InSequence; Oct 13, 2009 | Interview of Paul T Morrison discussing Heliscope in a Core Lab.http://www.genomeweb.com/sequencing/dana-farbers-paul-morrison-running-helicos-sequencer-core-facility