**4. Chimerism evaluation by two different DNA marker sets and molecular methods 'FA' and 'RQ PCR'**

#### **4.1 Introduction**

The aim of our study was to compare patient's chimerism monitoring by two different DNA marker type sets and molecular methods 'FA' and 'RQ PCR' measured in parallel after allo-hSCT [40, 42]. The subject matter of the first PCR-STR method 'FA' is explained in chapter '2—PCR-STR DNA markers in chimerism monitoring by "FA"'. Informative STR DNA markers were amplified with special fluorescent

labelled primers by PCR. Amplicons were separated by the capillary electrophoresis on genetic analyser and then evaluated by fragment analyses GeneMapper software. The basis of the second method 'RQ PCR' (SYBR green-based relative quantification by real-time PCR) to be compared is discussed in the chapter "3—SNP and NPs DNA markers in chimerism monitoring by RQ PCR". Informative SNP and biallelic NP (including DIPs—IN/DEL polymorphic markers) DNA markers were obtained from donor's and recipient's DNA before transplantation by SYBR green-based real-time PCR amplification following dissociation curves analysing screening. Post allo-hSCT chimerism monitoring is provided by relative quantification (RQ-PCR) of SYBR green-based real-time PCR of SNP or NPs informative DNA markers and evaluated by gene expression software. The relative quantity of the donor's and recipient's DNA markers is proportional to total leukocytes and can be expressed as mixed (MC) or complete chimerism (CC) in post-transplant patients.
