Two-step method of dna amplification for maldi-tof measurement

Abstract

The invention referres to a method for the preparation and selective replication of deoxyribonucleic acid (DNA) from biomaterial using PCR (polymerase chain reaction) for the analysis in time-of-flight mass spectrometers (TOF) with matrix-assisted laser desorption and ionization (MALDI) for determining specific genetic features in biomaterial. The invention consists, in a first step, of replicating the selected DNA segments by the PCR method in the usual unmodified fashion, and in a further enzymatic replication process, to replicate the DNA segments using modified substrates (the nucleic acids used for PCR) and specially prepared primers to such DNA analogs as are especially suitable for ionization by MALDI. Preparation of the primers particularly consists of introducing a charged group, which improves the ionization, and modification of the substrates is used to neutralize the negative charge on the phosphoric acid group of the DNA backbone. It is especially practical to immobilize the DNA on a surface for the fmal enzymatic reduplication process, for example on a prepared MALDI layer located on magnetic beads. In the final reduplication process, extremely little reagent is used, thus it is also possible in terms of low cost to use isotope-enriched material here in order to increase the mass resolving power.