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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

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