Method for amplification of nucleic acids of low complexity
OwnerTamas Rujan, Armin Schmitt +2inventors
InventorsTamas Rujan, Armin Schmitt, Peter Adorjan, Christian Piepenbrock
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Abstract
The invention describes a method for amplifying nucleic acids, such as DNA with means of an enzymatic amplification step, such as a polymerase chain reaction, specified for template nucleic acids of low complexity, e.g. pre-treated DNA, like but not limited to DNA pre-treated with bisulfite is disclosed. The invention is based on the use of specific oligo-nucleotide primer molecules to solely amplify specific pieces of DNA. It is disclosed how to optimize the primer design for a PCR if the template DNA is of low complexity.
Claims51
A method for the amplification of nucleic acids comprising the following steps
- 1) isolating a nucleic acid sample,
- 2) treating said sample in a manner that differentiates between methylated and unmethylated cytosine bases within said sample,
- 3) amplifying at least one target sequence, within said treated nucleic acid, by means of enzymatic amplification and a set of primer molecules, wherein said primer molecules are characterized in that
- a) each primer molecule sequence reaches a predefined measure of complexity,
- b) every combination of any two primer molecules in the set has a melting temperature below a specified threshold temperature,
- c) every combination of two primer molecules, under conditions allowing for one or more base mismatches per primer, does not lead to the amplification of an unwanted product when virtually tested using the treated and the untreated sample nucleic acids as template, and
- 4) detecting said amplified target nucleic acid.
Dependencies are inferred from the claim text.