Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2007-12-28
2010-06-29
Myers, Carla (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S023720, C536S024300, C536S024330
Reexamination Certificate
active
07745131
ABSTRACT:
A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from aB. rapaBAC library and a BAC clone anchoring Bn-FAE1.2 from aB. oleraceaBAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome specific primers that could amplify the corresponding genome in allotetraploidB. napus. The two-base deletion in the C genome gene was detected as a sequence characterized sequence region (SCAR) marker. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously, making it possible to analyze over half a million of samples per day using a medium capacity ABI 3100 Genetic Analyzer. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5′ primer end to increase SNP detection throughput through sample pooling. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.
REFERENCES:
patent: 2004/0106122 (2004-06-01), Wilmer et al.
patent: WO 2005/058019 (2005-06-01), None
NCBI Database, National Center for Biotechnology Information, National Library of Medicine, NIH (Bethesda, MD,USA) GenBank Accession No. BZ499865, Dec. 16, 2002.
Katavic et al. European Journal of Biochem. 2002. 269: 5625-5631.
Fourmann et al. Theor Appl Genet. 1998. 96: 852-858.
Applied Biosystems. Protocol for ABI PRISM® SNaPshot Multiplex Kit. 2000, Applied Biosystems, Foster City, California, USA.
Ade & Company Inc
Myers Carla
Williams MIchael R.
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