Determination of SNP 5mC/T in AluSx repeat (Chr16: 75033884) in DNA samples from the human blood by GlaI- and FatI-PCR assay
SNP 5mC/T in AluSx repeat (Chr16: 75033884) in DNA samples from the human blood of 92donors was studied by GlaI and FatI-PCR assay. The work includes a) isolation of DNA from the blood cells, b) GlaI and FatI-PCR assay of DNA fragment Chr16: 75033860 – 75033957, c) determination of 5mC (Chr16: 75033884) and thymine (Chr16: 75033884) в in DNA preparations and d) comparative analysis of the obtained results. It was shown that in position (Chr16: 75033884) 53 donors (57,61%) have a heterozygote (5mC-T), 19 donors (20,65%) have homozygote (T-T) and 20 donors (21,74%) have homozygote (5mC-5mC). Thus, taking into account a diploid set of chromosomes in the blood cells, SNP 5mC/T is observed in 70 positions from 156 (49,46%). The results obtained have shown that cytosine in position Chr16: 75033884 in the most of the blood DNA is methylated in accordance with a literature data about a significant methylation of CG-dinucleotides in Alu-repeats
GlaI-PCR analysis of methylation of ACGC site in Chr11: 65647266 in DNA samples from the blood cells of healthy donors and early stage breast cancers
Methylation of ACGC site in position Chr11: 65647266 (according to GRCh38 PrimaryAssembly) in DNA preparations from blood cells of healthy donors and early stage breast cancers was studied with GlaI-PCR assay. The work includes a) preparation of light fraction of the blood cells, b) isolation of genomic DNA, c) determination of genomic DNA concentration by real-time PCR, d) determination of concentration of unmethylated ACGC site in position Chr11: 65647266 with GlaI-PCR assay and e) calculation of percent of DNA molecules with unmethylated ACGC site in position Chr11: 65647266. GLAD-PCR analysis showed that in more than 82% of donors, the level of ACGC methylation in the SIPA1 gene is 3% or less, while in approximately 70% of patients with breast cancer, the level of ACGC site methylation in SIPA1 gene is in the range of 3.2 to 6.4%. These data suggest that at early stages of breast cancer, demethylation of A(5mC)GC site in the SIPA1 gene at position 65647266 takes place in 2/3 of patients. The data of this work may be used for a development of PCR test-system for exclusion of diagnosis “early stage breast cancer”. GlaI-PCR assay doesn’t require special and expensive equipment. The study may be carried out in a standard PCR laboratory when a blood analysis is taken place.