The present work comprised molecular genetic analysis of four types of leukemia, Acute Lymphoid leukemia (ALL), Acute Myeloid Leukemia (AML), hronic Lymphoid Leukemia (CLL) and Chronic Myeloid Leukemia (CML). Samples of blood were obtained from 65 Syrian patients affected with one of the four types of leukemia, admitted to Al-Bairuni University Hospital and Al-Assad University Hospital. The Wizard Promega kit was found suitable for DNA isolation from leukemia patients and normal individuals. By this method suitable quantities of DNA approximately (100 – 600 ìg) were obtained from 300 ìl of whole blood. The purity of isolated DNA ranged from (1.7 - 1.9). However only 45 patients of the total number of leukemia cases gave good yield of DNA. The rest of cases were distributed between ten cases showed clotted blood, and ten cases showed low W.B.C count with low DNA yield. RAPD-PCR analysis was carried out with 22 different arbitrary primers of decamer oligonucleotides. These primers were evaluated for their usefulness in detecting DNA polymorphism among leukemia patients. The study Chapter Three Results and Discussion involved optimization for RAPD reaction for DNA template, primer concentration and run program. The obtained results were as following : A- Thirteen primers showed no amplified products in all normal individuals and leukemia patients. B- Nine primers amplified genomic DNA in the normal individuals and leukemia patients with different efficiencies. The generated DNA fragments (when using these primers) were scored by their presence or absence and the differences in their molecular weight across the normal individuals and leukemia patients. The total number of amplified bands generally ranged between (1 - 15) and the molecular weights varied between (95 – 3162) bp according to the primer used. The minimum number of amplified bands was one band which was observed when using primers OPB-18 and OPZ- 02; on the other hand the highest number of bands were (15) bands which were observed when using primer OPJ-05. The lowest molecular weight of amplified band was 95 bp which was obtained with primers OPB-15 and OPJ-05, and the highest molecular weight of amplified band was 3162 bp which was obtained with primer OPA-09. C- In addition it was possible to observe decrease or increase in the relative intensities of the bands amplified from the genomic DNA of leukemia patients as compared with normal DNA. D- Among the leukemia patients tested, 37.8% of patients showed the highest DNA polymorphism because they were detected by nine primers used in this study. 48.8% of patients were detected by eight primers, 8.9% of patients were detected by seven primers and 4.5% of patients were detected by six primers. E- The ability to detect genetic alteration by each primer was variable, the highest value of efficiency was shown by primer OPB-17 (0.059), whereas primer OPE-05 showed the lowest value (0.016). F- The study had shown that genetic instabilities were frequent in genomic DNA of leukemia patients and could be detected by using suitable RAPD primers, e.g. primer OPA-09 could be used as a marker for detection of ALL and CLL patients. Primers OPB-17 and OPW-17 gave a specific band in all AML patients which was absent in normal individuals and other leukemia patients. On other hand, primer OPW-17 gave another specific band in AML and CLL patients, while, it was absent in normal individuals and other leukemia patients. Whereas, primers OPB- 15 and OPW-17 showed another specific band which was present in normal individuals and in other leukemia patients, but was lost in AML patients. The result of primer OPZ-02 showed a specific band was present in normal individuals, and in all leukemia patients, however in CML patients was lost . G- Primer OPJ-05 could amplify specific band in normal male individuals but it could not amplify this band in normal female individuals. RAPD-PCR analysis was applied to investigate polymorphism in DNA methylation of CML leukemia patients and normal individuals. The results of digested genomic DNA with HpaII and MspI showed clear differences in the methylation of the genomic DNA of the CML patients and normal individuals, suggesting variation in the proportion and pattern of methylated and unmethylated CGs. The results revealed differences in banding patterns which include: banding shifts , missing bands and / or banding intensity changes.
