![]() ![]() This is a simple nonradioactive labeling technique that can be used in number of universal labeling practices. 6 A DIG-based Southern blot 4 was used to hybridize large Arabidopsis thaliana DNA fragments. 5 DIG-based dot blot hybridization has been reported to provide a dependable, easy, and cost-efficient tool for plant molecular breeders, in which a large number of single nucleotide polymorphisms can be analyzed for breeding purposes. 3 The genome of the primitive land plant Selaginella kraussiana was analyzed using a DIG-based Southern blot 4 method to identify nine xyloglucan endotransglycosylase/hydrolase genes. 2 DIG-labeled probes have also been used successfully in detecting transgenes in other transgenic plants, such as rice, potato, sugarbeet, maize, and wheat. 1 DIG polymerase chain reaction (PCR)-labeled probes were used to detect transgenes in barley, rice, and Nicotiana benthamiana. 1 DIG-labeled probes and chemiluminescent substrates have proved to be a popular method for nucleic acid detection because of susceptibility, quicker results, and dependability. 1 Digoxigenin (DIG)-labeled probes are not as expensive as radioisotope-labeled probes, they last longer, and there is no environmental hazard caused by radiation. When comparing radioactive and nonradioactive in situ hybridization, nonradioactive methods have shown to have many advantages, including shorter development time, higher histological resolution, and higher detection sensitivity. This paper describes a nonradioactive in situ hybridization technique to detect nucleic acids in plants. To test the efficacy of our optimization protocol, we used 1 μL of labeled plasmid DNA pU16893 harboring an Arabidopsis housekeeping gene elongation factor-1 and showed that the elongation factor-1 gene could be detected in Arabidopsis genome under various environmental conditions. We also examined the sensitivity of labeled probe and showed that 2 μL of labeled probe was not able to hybridize with 1 μg of target DNA, although 2 μL of labeled probe was able to detect target DNA ranging from 2 to 10 μg. We showed that 1 μL of labeled probe was sufficient to hybridize onto 1–10 μg of target plasmid DNA. In this experiment we optimized minimum probe requirement for a nonradioactive digoxigenin-based gene detection system in the model plant Arabidopsis thaliana. Digoxigenin has been used successfully in labeling nucleic acids. DNA samples from 16 children with deafness gene mutation were corroborated by Sanger sequencing,and the compliance rate was 100%.Conclusions For 20 hot-spot mutations of 4 common deafness pathogenic genes,the matc-hing PCR-RDB gene membroine chip technology was designed and the susceptible gene of congenital deafness children was detected.This technique has some advantages like high detection rate,fast,accurate and economical.It is an ideal method for gene screening on hereditary non- syndrome deafness children and has good clinical application prospects.Digoxigenin is derived from a plant steroid hormone digoxin found in the plants Digitalis sp. Results Among 38 subjects, deafness gene mutations were detected in 16 cases,with a detection rate of 42.11%,and they were all verified by family study.Among 16 cases,6 cases of GJB2 gene mutation(3 cases of homozygote,3 cases of heterozygous),4 cases of SLC26A4 mutation,2 cases of MTRNR (m.1555A>G) mutation,4 cases of compound muta-tion,but none of GJB3 gene mutations.And their detection rates were 15.79%,10.53%,5.26%,10.53%,and 0,re-spectively. Methods The blood samples(2 mL)were collected from 38 children with congenital deafness,excluding high- risk factors for deaf-ness at Dongguan Rehabilitation School,and then genomic DNA extracted.By using self-designed multiplex- PCR combined with PCR-RDB gene chip technology,20 hot-spot mutations of 4 pathogenic genes of common deafness in Chinese population were detected.Sanger sequencing was used as the gold standard to corroborate the positive samples. Objective To explore the application value of PCR-reverse dot blot hybridization ( PCR-RDB) gene membrane chip technique in genetic diagnosis of hereditary non- syndrome deafness in children. ![]()
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