DNA Resource Core
The DF/HCC DNA Resource Core provides DF/HCC members, Harvard affiliates, and outside researchers with quality and inexpensive DNA sequencing services and sequenced verified plasmid constructs, paired with comprehensive quality control and excellent customer support. The Core meets the growing need for small- and large-scale DNA sequencing projects, including preparation of DNA from glycerol stocks in advance of sequencing, a plasmid repository and distribution service, fully automated low-temperature plasmid storage, and plasma annotation and informatics support to optimize utilization of the core.
Users of the Core employ sequencing in many types of experiments, including to confirm construction of new plasmid vectors, to perform serial analysis of gene expression (SAGE), to confirm the results of site-directed mutagenesis, to determine or confirm the identity of specific cDNA and genomic library clones, and to identify single nucleotide mutations associated with heritable human disease. The Core fills a niche as a moderate-throughput sequencing center that is equally able to manage relatively large and small sample numbers, rather than being restricted, say, to only genome-scale projects. The range of orders the Core has processed for an individual user is from one to thousands of samples. Additionally, the Core has processed samples that were submitted as glycerol stocks, as bacterial pellets, and as purified DNA, with the flexibility of using either standard or user-supplied sequencing primers.
- DNA Sequencing and Sample Preps
- Plasmid Distribution
- Plasmid Storage
Project Title: Preconfiguration of the antigen-binding site during affinity maturation of a broadly neutralizing influenza virus antibody
Principal Investigator: Stephen Harrison, PhD (BCH)
Description of the Project: Affinity maturation refines a naive B-cell response by selecting mutations in antibody variable domains that enhance antigen binding. The project describes a B-cell lineage expressing broadly neutralizing influenza virus antibodies derived from a subject immunized with the 2007 trivalent vaccine. The lineage comprises three mature antibodies, the unmutated common ancestor, and a common intermediate. Their heavy-chain complementarity-determining region inserts into the conserved receptor-binding pocket of influenza HA.
The research show by analysis of structures, binding kinetics and long time-scale molecular dynamics simulations that antibody evolution in this lineage has rigidified the initially flexible heavy-chain complementarity determining region by two nearly independent pathways and that this preconfiguration accounts for most of the affinity gain. The results advance our understanding of strategies for developing more broadly effective influenza vaccines.
Contribution of the Core: The DF/HCC DNA Resource Core was instrumental in sequencing all the Fab fragments and influenza constructs that were used in the biochemistry experiments (i.e. the kinetic analysis using SPR) and those that were eventually overexpressed and solved by x-ray crystallography.
Project Title: Gygi/Harper Interactome Project
Principal Investigators: J. Wade Harper, PhD (HMS); Steven Gygi, PhD
Description of the Project: The goal of this project is to identify interacting proteins for approximately half of the proteome using affinity purification-mass spectrometry and informatics approaches. Human protein coding genes derived from the CCSB Orfeome Library (provided by Marc Vidal and David Hill, DFCI) were used to create a library of lentiviral vectors which are, in turn, used to express proteins in tissue culture cells. Purified complexes are employed for proteomics.
Contribution of the Core: The DF/HCC DNA Resource Core is providing 2 services for this project. First, each expression clone is sequenced as a validation step prior to cell line generation. Second, all expression vectors are being deposited into the DNA Resource Core for distribution to the community upon publication.
Publication: in process.
If research supported by this core facility results in publication, please acknowledge this support by including the following in your publication(s):
"We thank Dana-Farber/Harvard Cancer Center in Boston, MA, for the use of the DNA Resource Core Facility, which provided __________ service. Dana-Farber/Harvard Cancer Center is supported in part by an NCI Cancer Center Support Grant # NIH 5 P30 CA06516."