omation mbers - Society for Laboratory Automation and Screening

WP001
Chris Barbagallo
Millipore Corporation
Research & Development
17 Cherry Hill Drive
Danvers, Massachusetts 01923
chris_barbagallo@millipore.com
Automating Large DNA Insert Preparation for Sequencing
192
Co-Author(s)
Masaharu Mabuchi, Janet Smith,
Peter Rapiejko, Joseph Hitti
Bacterial Artificial Chromosome (BAC) and fosmid libraries have proven to be powerful tools for both physical
mapping and the finishing process of genome sequencing. However, their unique properties (i.e., large DNA inserts)
have also presented several technical challenges when adapting their preparation to higher throughput. One major
obstacle to this process is the low copy number of the vectors harbored by the host cells. Typically, BAC and
fosmid inserts are maintained at only 1 – 2 copies per cell. As a result, yields obtained from a given volume of
culture are significantly lower than that for plasmid and cosmid DNA vectors. Modification of both bacterial growth
conditions and DNA purification methodologies have been essential to maximizing yields and obtaining high quality
template for downstream applications. We describe here methods that enable higher throughput processing for
end sequencing of BAC and fosmid templates. Isolation of these clones and subsequent sequencing cleanup
occur in a 96-well format, which is demonstrated on automation platforms to provide DNA of sufficient quantity
and purity for direct sequencing utilizing an ABI 3700 capillary sequencer.
WP002
Steven Eendhuizen
Spark Holland, Inc.
666 Plainsboro Road, Suite 1336
Plainsboro, New Jersey 08536
steven.eendhuizen@sparkholland.com
XLC-MS for Therapeutic Drug Monitoring
Co-Author(s)
Alex Berhitu, Emile Koster,
Peter Ringeling, Bert Ooms
Solid-phase extraction is integrated with the LC separation (“XLC”) to permit direct injection of “raw” biological
samples without prior filtration, centrifugation or protein precipitation. The XLC system was coupled to MS
(API2000) which is operated in positive multiple ion monitoring mode. Both thermally assisted electrospray
ionization (ESI) and atmospheric pressure chemical ionization (APCI) have been considered. In order to
demonstrate the XLC-MS concept for therapeutic drug monitoring (TDM) the important neuroleptic drug clozapine
(CLZ), and its metabolites desmethyl-clozapine (DMC) and clozapine-N-oxide (NOX) were determined in serum.
For LC-MS interfacing, detectability of CLZ and metabolites is better with the APCI interface, whereas selectivity
is better with the ESI interface. Recovery is essentially quantitative (100%), detection limits are in the sub ng/
mL range and the linear range is sufficiently large to cover the therapeutic range for clozapine in serum (10 to
1000 ng/mL). The within day precision is < 5% (at low therapeutic levels) and the accuracy within ± 10% of the
established concentrations. Optimization of SPE resulted in XLC-MS cycle times of only about 2 min. Because
SPE is performed within the LC run, throughput could only be increased further by changing LC conditions, e.g.,
column length, mobile phase flow rate, modifier percentage and pH. Summarizing, a sensitive, selective, robust
and fast method for the monitoring of “blood levels” of clozapine and metabolites is obtained. Moreover, seamless
integration of front-end sample prep and LC-MS (XLC-MS) seems to result in a universal and fully automated
platform for TDM.