Detecting Some Kinds Of Gene Changes In Leukemia With Oligonucleotide Microarray

Objective: There are many genetic substance changes in leukemia. Ph chromosome is the first one of the chromosomes that is found to be associated with leukemia. What the Ph chromosome cause is the formation of BCR-ABL fusion gene that can be seen in nearly 90% of CML patients. Ph negative patients also can be divided into two groups based on having or not having the rearrangement of BCR. The two groups have different prognosis.Besides the gene changes directly associated with karyotype, there are many other gene abnormalities in leukemia. For example, we can see P53 and K-ras gene mutations in many species of tumor. They also can be seen in leukemia. The inactivation of P53 is associated with the acute transformation phase and development of CML. Although K-ras mutation is not seen frequently in leukemia, it is also reported to play a role in the pathogenesis and development of leukemia.So we can see the importance of detection of gene changes in diagnosis, disease-typing, therapy, and judgment of prognosis of leukemia. In this experiment, we prepare oligonucleotide microarray to detect the two kinds of gene-changes in leukemia.Methods: After preparing the oligonucleotide microarray, we adopted two ways to detect fusion gene and gene mutations respectively. As for the fusion gene, we chose two cell strains as the detection targets. One was known expressing the BCR-ABL fusion gene and the other was not. Cell total RNA was extracted, and then it was reverse-transcripted and fluorescence labeled. At last the cDNA was hybridized with the microarray. For the gene mutations, the bone marrow samples were collected from the patients and the genome DNA was extracted. We amplified and labeled the target sequences by PCR. Then the PCR product was hybridized with the microarray.Results: The prepared chip had well-distributed background and the probe blotches on it were regular.From the hybridization results, we saw that in lower hybridization temperature, the background signal was high and all the probes signals had no difference. In hightemperature all the signals were very weak and could not get any information. We also found that severe elution condition both decreased the specific and background signal and could not help differ the specific from the non-specific signal. After comparing the various factors that can affect the results, we got a relative ideal detective condition.We screened 39 samples and found out that 8 samples had P53 mutation and 4 samples had K-ras mutation.Conclusion: In this experiment, we checked out the BCR-ABL fusion gene and P53, k-ras gene mutations in samples with the oligonucleotide microarray. We think the oligonucleotide microarray, as a new detective method, has some unique advantages in detecting gene changes of leukemia. For example, it can detect different changes in one gene or in several genes of many samples at the same time. And the time-consuming or the needed equipments are not too much. If we can further improve the sensitivity and accuracy and control the cost, the microarray will have a wild application in clinic.