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Methylation-dependent melting polymorphisms in genomic fragments of deoxyribonucleic acid
R H Reindollar1, K W Fusaris, M R Gray
1Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School 02215, USA.
Objectives:
Denaturing gradient gel electrophoresis can detect single base sequence differences in deoxyribonucleic acid and methylation differences in small cloned fragments of deoxyribonucleic acid. We previously detected cell type-specific melting differences by denaturing gradient gel electrophoresis in paired leukocyte and sperm cell samples of deoxyribonucleic acid. We proposed that these differences were caused by differential methylation and that blotting strategies using denaturing gradient gel electrophoresis might be useful in detecting in vivo variations in methylation patterns.
Study Design:
Genomic deoxyribonucleic acid from leukocytes and sperm cells of 35 male subjects was analyzed by denaturing gradient gel electrophoresis after digestion by 4-bp site enzymes and Msp I and its methylation-sensitive isoschizomer Hpa II. Some fragments were amplified by polymerase chain reaction.
Results:
Cell type-specific melting polymorphisms were detected in all genes from all subjects. Analysis of Msp I/Hpa II sites demonstrated that differences noted correlated with the methylation state. Cell type-specific differences were absent in fragments amplified by polymerase chain reaction.
Conclusions:
The denaturing gradient gel electrophoresis blotting technique is a fast and comprehensive method for comparing in vivo methylation differences.