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Reverse transcription polymerase chain reaction (RT-PCR) is an RNA reverse transcription and PCR to combine the establishment of a RNA- polymerase chain reaction , the advantage of quick , simple, and highly sensitive . Can be used : ① analysis of gene transcription product for detecting mutations and gene expression strength ; ② cDNA cloning and cDNA probe synthesis , cDNA sequences, etc. transformation . This topic will be by RT-PCR and nested PCR method combined for fragile X syndrome (FXS), hemophilia A (HA) and Duchenne / Becker muscular dystrophy (DMD) patients with genetic testing . FXS is second only to Down's syndrome incidence of hereditary mental retardation X-linked recessive genetic disease , X-linked mental retardation accounted for 40 %, 95 % or more of the Fragile X Syndrome and is based on the molecular genetics of FMR1 gene (CGG) n structure extended dynamic mutations , when n> 200 when , FMR1 gene 5 '(CGG) n -side CpG island hypermethylation , so no FMR1 gene expression , and therefore can not detect cDNA product . DMD / BMD is a common human X-linked recessive lethal neuromuscular disease , manifested as due to mutations caused by skeletal muscle weakness and calf pseudo-hypertrophy of the gastrocnemius . DMD gene due about 2500kb, DNA level is detected from the latest MLPA technique can only detect about 65 % of the loss, and small change only by one by exons sequenced some cases, even if it is still unable to detect the DMD gene change , while only 0.5% of the length of gene cDNA of about 14kb analysis can detect not only deletions, insertions , but also can detect the abnormal RNA processing , the remaining small mutations with cDNA for mutation detection are more easily gDNA line . HA is due to plasma coagulation factor Ⅷ (F Ⅷ) due to the lack of X-linked recessive genetic blood clotting deficiency diseases , including intron 22 and intron 1 inversion about genetic changes in patients with severe hemophilia A , 50 % , the application of long PCR is difficult, by RT-PCR method can be performed on the inverted F Ⅷ gene mutation was detected , but also the coding region for the detection of small mutations , improve the detection efficiency . Meanwhile, with the RT-PCR method for the establishment and techniques become more sophisticated , is expected to be applied villi and amniotic fluid cells transcript detection, prenatal diagnosis to help.
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