Biomedical subjects

Zachary Kaminsky

Publications and source records attributed to Zachary Kaminsky.

2 recordsLinked to original sources

PubMed2006

Microarray-based DNA methylation profiling: technology and applications.

This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions using microarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340 000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure.

Chromosome Mapping↗

PubMed2006

Whole genome amplification of sodium bisulfite-treated DNA allows the accurate estimate of methylated cytosine density in limited DNA resources.

Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however is that it results in considerable DNA degradation, and large quantities of genomic DNA material are needed if numerous genomic regions are to be profiled. In this study, we examined whether whole genome amplification (WGA) techniques can be applied to sodium bisulfite-treated DNA and whether WGA would bias DNA methylation results. Sodium bisulfite-treated DNA was amplified using a standard WGA method: optimized primer-extension preamplification (PEP) with degenerate primers. Following the PCR of bisulfite-treated DNA, the DNA methylation profiles of specific DNA fragments were assessed using three approaches: (i) direct sequencing of the overall product; (ii) the sequencing of cloned PCR products; and (iii) methylation-sensitive single nucleotide primer extension (MS-SNuPE)--and compared with those obtained from bisulfite-treated DNA not subjected to WGA. Our data indicates that the DNA methylation profiles obtained from WGA of sodium bisulfite-treated DNA are consistent with those obtained from non-WGA DNA. The average difference in methylation percentage calculated from the two sets of template using MS-SNuPE was 4%. If our results are replicated on other genomic loci, WGA may become a useful technique in DNA methylation studies.

CpG Islands↗