[Isolation of the target gene from cDNA restriction fragments using 70-mer oligo microarray].
OBJECTIVE
To study the method for using a 70-mer oligo microarray as the probe to isolate target genes from the cDNA restriction fragments.


METHODS
Samples of Saccharomyces cerevisiae mRNA was extracted after heat shock culture and reversely transcribed into the double-stranded cDNAs, which were prepared into restriction cDNA fragments using restriction display (RD) method. The microarray was printed using a single 70-mer specific oligo designed to according to the SSA1 gene of yeast. The cDNA restriction fragments were labeled by PCR method with the Cy5 universal primer before hybridization with the microarray. The microarray was stripped after washing and scanning, and the strip solution was collected for another round of PCR amplification using the universal primer without fluorescence. The PCR product was then cloned into PUC18 T vector and transformed into to E.coli JM109 cells for amplification, and the plasmids were extracted and sequenced for identification.


RESULTS
BLAST results showed that the target gene was cloned successfully.


CONCLUSIONS
The target gene can be isolated directly using the 70-mer oligo microarray as the probe from the cDNA fragments prepared by RD method, without the necessity of building a cDNA library. This method can also be used in further research to acquire the differentially expressed genes after the oligo microarray hybridization.
Shi
Rong
R
Institute of Molecular Biology, First Military Medical University, Guangzhou 510515, China.
Ma
Wen-li
WL
Liu
Cui-hua
CH
Song
Yan-bin
YB
Wu
Qing-hua
QH
Guo
Qiu-ye
QY
Zheng
Wen-ling
WL
D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary