Used Suppressive Subtractive Hybridizationr To Isolate Genes Relevant To Multidrug Resistance In Leukemiat

Objective: With Suppression subtractive hybridization (SSH) to isolate and clone the expression of different genes between K562 and K562/DOX cells.Methods: Used SHH in order to isolate and clone the expression of different genes between K562 and K562/DOX cells.Total RNA were extracted between the two cells and then blended into DNA respectively . In our study, we divided into test group (K562/DOX) and the control group (K562) which treated with the same restriction enzyme RsaI digestion. Then the CDNA of K562/DOX cells were divided into two groups, they respectively connected with adopter1 and adopter2R.With the cDNA of K562, the three groups were detected by twice SSH and PCR , then the products were purified and linked with pMD19-T Vector.Finally they were tansferred to E.coli.Form the subtracted cDNA library,we randomly selected clone gengs and verified by PCR. At the same time the positive clones were picked in order to sequence and analyze homologous sequence. At last, the different genes isolated were determined.Results: Cloned the different cDNA fragmengts, then picked 24 monoclonal colonies to identify by PCR. Insert fragment were obtained in 15 monoclonal colonies . Blast sequence analysis were applied after sequencing these fragments .To determine whethder these fragments were subracted specific gene fragment.Conclusions:The subtractive cDNA library about K562/DOX and K562 was on the basis of using suppression subtractive hybridization and T / A cloning. It is a new foundation for further selecting and cloning of leukemia cell lines with different expression of multidrug resistance gene.