Determination of the risk of breast and stomach cancer by identifying variants of the 5mC/T polymorphism in the intron of the KIF26B gene at position xp1: 245618129

We used GlaI/FatI-PCR assay for determination of 5mC/T polymorphism frequencies at position xp1: 245618129 (according to the GRCh38.p13 genomic assembly) in DNA preparations isolated from blood cells of 51 breast cancer (BC) patients and 63 gastric cancer patients (GC). The data obtained were compared with those previously obtained for 92 healthy blood donors. It was shown that the cytosine in this position is predominantly is methylated in all DNA samples. The incidence of a diploid C/C set in this position in patients with breast cancer and gastric cancer is 9.8% and 7.94%, respectively. This is significantly different from the incidence of C homozygotes in healthy women (26.09%) and healthy donors of both sexes (22.83%). At the same time, the incidence of C/T heterozygotes and T/T homozygotes in patients with gastric cancer and breast cancer exceeds similar indicators for control groups of healthy people by 5.5-10.7%. Thus, the substitution of a methylated cytosine base for a…
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GLAD PCR Assay

Slide De novo DNA methylation Slide Step 1: DNA hydrolysis Slide Step 2: adapter ligation Universal adapter Slide Step 3: Real time PCT Universal adapter Genomic primer Genomic primer TaqMan probe TaqMan probe Hybrid primer

GlaI PCR Assay

Slide De novo DNA methylation Slide Step 1: DNA hydrolysis Slide Step 2: Real time PCT Genomic primer Genomic primer TaqMan probe TaqMan probe

Study of the methylation of RCGY sites in the regulatory region of the FAM19A4 gene in human cell line dna preparations by GLAD-PCR assay

The method of GLAD-PCR analysis was used to determine the methylation of RCGY sites in the regulatory region of the FAM19A4 gene (Xp3: 68932451 - 68932800) in the DNA preparations of the malignant human cell lines Raji, Jurkat, HeLa, U-937 and the control DNA of the lung fibroblast cell line L68. It is shown that the octanucleotide GCGCGCGC (Xp3: 68932500), located in the first exon of the FAM19A4 gene, is cleaved approximately equally in the DNA of all 4 malignant human cell lines, whereas in the DNA of L68 this site is hydrolyzed 10-15 times weaker. These results are consistent with data of epigenomic sequencing of L68, Raji, and U-937 DNA. Methylation of CG-dinucleotides at positions 68932727 and/or 68932740 in the promoter of the FAM19A4 gene occurs only in tumor DNA and is not observed in L68 DNA, whereas methylation of CG-dinucleotide at position 68932704 is observed only in HeLa DNA.

Determination of SNP 5mC/T in position Chr1: 245618129 in DNA samples from the human blood by GlaI- and FatI-PCR assay

SNP 5mC/T (Chr1: 245618129) 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 Chr1: 245617889 - 245618464, c) determination of 5mC (Chr1: 245618129) and thymine in DNA preparations and d) comparative analysis of the obtained results. It was shown that in position (Chr1: 245618129) 43 donors (46,74%) have a heterozygote (5mC/T), 28 donors (30,43%) have homozygote (T/T) and 21 donors (22,83%) have homozygote (5mC/5mC). Thus, taking into account a diploid set of chromosomes in the blood cells, SNP 5mC/T is observed in 99 positions from 184 (53,8%). The results obtained have shown that cytosine in position Chr1: 245618129 in the most of the blood DNA is methylated.

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.

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