Abstract:
:Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Tusnády GE,Simon I,Váradi A,Arányi Tdoi
10.1093/nar/gni012keywords:
subject
Has Abstractpub_date
2005-01-13 00:00:00pages
e9issue
1eissn
0305-1048issn
1362-4962pii
33/1/e9journal_volume
33pub_type
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