SCIENTIFIC REPORT 2004 - Sylvester Comprehensive Cancer Center
SCIENTIFIC REPORT 2004 - Sylvester Comprehensive Cancer Center
SCIENTIFIC REPORT 2004 - Sylvester Comprehensive Cancer Center
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S H A R E D R E S O U R C E S<br />
M O L E C U L A R A N A L Y S I S C O R E<br />
MANAGER<br />
Roland Jurecic, Ph.D.<br />
Assistant Professor of Microbiology and<br />
Immunology<br />
PURPOSE<br />
The Molecular Analysis Core provides UM/<strong>Sylvester</strong><br />
members with: 1) capillary-based DNA<br />
sequencing of plasmids and polymerase chain<br />
reaction (PCR) fragments, 2) shotgun sequencing<br />
of large cDNA and genomic DNA inserts<br />
(transgenic and knockout/targeting vectors), 3)<br />
basic DNA fragment analysis, and 4) real time<br />
quantitative PCR and reverse transcriptase-PCR<br />
(RT-PCR) using LightCycler technology.<br />
3) Basic DNA fragment analysis. Detection of<br />
isoforms, alternative transcripts, and DNA rearrangements,<br />
etc.<br />
4) Automated heterozygote detection.<br />
Quantitative Real Time PCR Techniques Using<br />
the Roche LightCycler<br />
1) Real time quantitative PCR and RT-PCR.<br />
2) Full melt curve analysis of amplified products.<br />
3) Design and testing of fluorogenic probes for<br />
rapid and sensitive detection of gene targets.<br />
4) Genotyping of transgenic and knockout<br />
mouse models.<br />
SERVICES<br />
This facility provides UM/<strong>Sylvester</strong> investigators,<br />
in support of their peer-reviewed research, with<br />
the following services:<br />
Genetic Analysis using the Beckman CEQ 2000<br />
Genetic Analyzer<br />
1) Capillary-based DNA sequencing of plasmids<br />
and PCR fragments (800 base pairs in 90 minutes),<br />
using standard (T3, T7, Sp6, M13, etc.)<br />
or custom-designed primers.<br />
2) Shotgun sequencing of large cDNA and genomic<br />
DNA inserts (transgenic and knockout/targeting<br />
vectors) using transposon technology for<br />
fast and easy insertion of sequencing primer<br />
binding sites and kanamycin resistance markers<br />
into target DNA in vitro. Selection of different<br />
clones with transposon integrated only<br />
into the genomic insert (not the plasmid<br />
backbone) by digest restriction and size difference.<br />
Based on the insert size, 20 to 40 different<br />
clones are subjected to automated sequencing<br />
and assembled into contigs using sequence utility<br />
software.<br />
UM/<strong>Sylvester</strong> <strong>Comprehensive</strong> <strong>Cancer</strong> <strong>Center</strong> Scientific Report <strong>2004</strong> 153