Development And Validation Of Gene Sequence Capture Chip

Large quantity of data provided and the tools through HGP has enabled a wide range genomics research.Target sequence capture technology is a popular method of human genomics research.Targeted sequence capture combined with high-throughput sequencing is economic and efficient genomic research approach.At present,most of the domestic and foreign labs use Agilent,NimbleGen and other platforms' capture systems to conduct basic research and assist clinical diagnosis.Domestic products are rare or unpopular.Imported products are still too expensive for most lab to do capture sequencing on a regular basis.This work intends to develop convenient and low cost sequence capture reagents.We selected glass slides as solid substrate for the development of gene capture chips.Our effort has resulted in some interesting findings as follows:(1)Gastric cancer FFPE sections were used to extract high quality genomes and explored stable DNA fragmentation conditions.Two short fragment DNA selection libraries were completed,and the DNA library of the specific short fragments was successfully constructed with the gel recovery product,the latter has shown higher consistency.(2)This study determined the PCR amplification conditions of the library DNA,the primer dimer was significantly reduced resulting in more specific amplification producst.We optimized the amount of dUTP insertion without compromising PCR amplification and achieved better dye labeling condition.(3)Highly active Bst polymerase large fragment were successfully expressed and purified,which dramatically lowered the cost of experiments.We found no significant difference in the amplification efficiency between our purified Bst polymerase and the corresponding commercial enzyme.(4)We selected probe groups from our genomic probe library covering the whole human genome for this study.The purified Bst polymerase was used to amplify probe groups and human genomic DNA.Glass powder and slides were selected as probe substrate to explore and verify the probe fixation method.This study realized the preliminary research and development of the capture chip.This thesis provided a new idea and effective method for the gene sequence capture chip preparation,which is simple and inexpensive.It makes it possible for the capture chip to become popular in the laboratory of genomics research.It saves resources for the future large-scale application of basic research related to mutation and disease.It offers hope and support for the early detection of rare diseases,early prevention and early treatment,and guidance of individualized treatment.