The Application Of RT-PCR To Characterization Of Mutations In Monogenic Diseases

RT-PCR (reverse transcription-polymerase chain reaction) is a method used to amplify cDNA by the combination of the reverse transcription of RNA and polymerase chain reaction. It's the most sensitive and effective technique for mRNA detection and quantitative analysis. RT-PCR can be applied to the following aspects: (1) To analyze the transcripts, then to detect mutations and to quantitate gene expression. (2) To clone cDNA, to make cDNA probe and to modify the sequence of cDNA.Fragile X syndrome (FXS) is an X-linked recessive inherited disease. About 40% of X-linked mental retardation is caused by FXS, second only to Down's syndrome. In most cases(>95%), the etiology of the disorder is the unstable expansion of a CGG-repeat((CGG)n) within the FMR1 gene and the abnormal methylation of the nearby CpG island, which results in suppression of FMR1 transcription and decreasing of protein levels in the brain.Hemophilia A is an X-linked, recessive, bleeding disorder caused by a deficiency in the activity of coagulation factor VIII, mapped to Xq28. Int1h-related and int22h-related inversions account for about 50% of patients with severe hemophilia A. Compared with amplification gDNA, RT-PCR is more effective in detecting the inversion mutations and examining the coding region of FVIII gene to detect all types of mutations including micromutation.Duchenne and Becker muscular dystrophies (DMD and BMD respectively) are caused by mutations in the dystrophin gene, which is spread over 2,500 kb of the X chromosome. A large proportion of mutations are partial deletions or duplications (65% and 6-10%, respectively) of varying sizes. It is defined as progressive deterioration and resultant weakness of muscle tissue; high resting serum levels of creatine kinase and well-developed musculature with calf hypertrophy. For DNA level examination, even with the application of the latest developed MLPA technique, detecting all of deletions and insertions, micromutation can only be realized by the sequencing of all exons one by one. In spite employed the methods mentioned above, there are still some mutations remaining unknown. Advantage of RT-PCR in charactering the mutations in DMD is very obvious, since not only deletions, insertions and splicing mutations caused by single base substitution can be detected directly with this technique, but also the other micro-mutations can be detected by sequencing more effectively, since there is only 14kb of cDNA, instead of 79 exons.The RT-PCR approach has a potential to apply in prenatal diagnosis by the examination the transcription of chorionic villi and amniotic fluid cells.