Method for the quantitative analysis of nucleic acid fragmentation and amplificability

摘要

The present invention relates to a method for the quantitative analysis of complex nucleic acids (NA), i.e. their fragmentation/degradation and amplificability as a marker of biomolecular quality and integrity of a biosample. Said method comprises the steps of subjecting said NA to a multiplex polymerase chain reaction using primers to generate different-size amplicons (referred to as indicator PCR). For simplicity, a duplex PCR using one primer pair for the generation of a longer PCR product and a second primer pair for the generation of a shorter PCR product is being described as the most simple variant of this test. Following the duplex PCR amplification, the ratio between the yield of the longer PCR product and the yield of the shorter PCR product generated during duplex PCR is determined using a read-out that allows relative quantification between the two (e.g. Pyrosequencing). The ratio is proportional to the nucleic acids quality, because the larger fragment tends to be under-represented with increased fragmentation impeding with its amplificability. The invention further relates to the generation and use of reference high-molecular weight DNA samples subjected to degradation under controlled conditions (e.g. by inflicting heat for specified periods of time) to generate a degradation calibration curve. The fragmentation of a query NA sample previously prepared from a liquid or solid biosource can then be quantified by use of the duplex indicator PCR after direct comparison to the calibrator DNA fragmentation curve. The present invention further relates to a comprehensive kit containing all specific components required to apply said method.