| 摘要 |
The invention disclose the method of identification of DNA or RNA fragments pairs that are initially present in the same living or fixed cells, in particular the method of identification of native pairs of antibody heavy and light chains genes, as well as native pairs of T-cell receptor (TCR) alpha and beta chains genes. The method is based on emulsion PCR or RT-PCR that is performed on living or fixed cells, that enables the PCR-amplification of DNA or RNA fragments pairs with subsequent physical fusion of products of PCR-amplification. All described reactions are performed within the emulsion with separated cells, where each cell is enclosed into separate emulsion droplet. The new feature of the method is innovative new type of PCR-suppression that allows selectively amplify the library of gene fragments that were fused in the emulsion after the emulsion reaction. The character of the invention is that selective PCR-suppression of unfused fragments allows predominant amplification of target fragment pairs, which were successfully fused within the emulsion droplets previously, as compared with amplification of fragment pairs capable of non target fusion after the isolation of nucleic acids mixture from the emulsion. The generated amplified fused libraries may be further analyzed using massive sequencing methods, e.g. high throughput Illumina sequencing method. Using the present method the pairs of native TCR chains genes in the human donor blood sample were found and the accuracy of identification was independently confirmed. The described approach can be effectively applied for native antibody chains pairs, native TCR chains pairs and for search of any RNA molecules that are co-expressed in the same cells, or the DNA fragments that are contained within the same cells. |
