Direct comparison of two different strategies to sequence bisulfite-treated genomic DNA for DNA methylation analysis in genetic diseases and cancer samples.

Due to general difficulties for MSP/BSP PCR, special recommendations for different approaches may be useful to maximize sucessful generation of data. Sample tracking is depending on cell type and biological status of isolated cells. It could not be expected that all DNAs isolated will be in the same methylation state. If amplified, a population of molecules with different methylation pattern is very likely. Bisulfite treatment is a very critical step. An incomplete conversion will lead to an arteficial increase of different methylation patterns. Depending on methylation state in general, a very imbalanced nucleotide composition after bisulfite treatment can be expected. This may bias amplification of all of bisulfite treated gDNA with different methylation patterns. If a very C-rich sequence will be converted by bisulfite, long T-stretches will be the result. During amplification Taq Polymerases elongating the primers will slipp over the original template molecules in such areas. Results are populations of product molecules with different length of these T-stretches. Finally this may lead to problems during sequencing. Amplicon length: In general, shorter amplicons will be amplified more robust, bias is not that issue, errors are less frequent. This is an optimal cenario for sequencing PCR products. But with this strategy, it will only be possible to sequence the whole population of amplified molecules. All factors increasing the plurality of the population of molecules (including slippage errors) will directly influence the chance and the results of sequencing. For covering large genomic regions, longer amplicons will be preferred for better coverage/less reactions. For this case, the likelyhood for more inefficient amplification and polymerase errors in A/T-rich regions can be tolerated, if down-stream analysis will be done by cloning. Separation of the population of amplified molecules will allow the sequencing of the individual fragments. For both sequencing strategies application specific recommendations from primer design to analysis of data may be useful to maximize successful generation of results. Abstract: 945/T ABSTRACT methylation on the cytosine of genomic DNA (gDNA) has been shown to correlate with gene expression. Specifically, methylation of cytosines (C) at CpG motifs, usually in the promoter regions of genes in CpG-dense regions known as CpG islands, will shut down expression of the gene in complex biological processes.Biological relevance is given for all developmental processes including aging and tumor growth and methylation pattern may be different between cell types. A well known method to study methylation patterns is to treat gDNA by bisulfite to distinguish methylated cytosine (5mC) from unmethylated C, which is deaminated to uracil (U) and replaced by thymine (T) in subsequent amplification. 5mC still remains as C. Subsequent amplification can focus on selective amplification of methylation patterns in CpG islands (methyation specific PCR, MSP) or on amplification of bisulfite treated (converted) gDNA (Bisulfite treatment specific PCR, BSP). Selection of PCR focus is done by primer design. After PCR, sequencing can clarify the methylation pattern. Due to very imbalanced nucleotide composition after bisulfite treatment bias in amplification of bisulfite treated gDNA as well as in sequencing products may lead to experimental problems. PCR improvements during amplification of bisulfite-converted gDNA by the use of universal tailed primers, combined with changes in thermal cycling and reactant concentrations are described earlier (1,2). These PCR products can be sequenced by using the universal primer tails. This method will be compared to the cloning of BIS PCR products for sequencing the bacterial clones.