KMID : 1130320100530030397
|
|
Korean Journal of Pediatrics 2010 Volume.53 No. 3 p.397 ~ p.407
|
|
Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis
|
|
Yoo Ki-Young
Kim Hee-Jin Lee Kwang-Chul
|
|
Abstract
|
|
|
Purpose: The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs) and conformation sensitive gel electrophoresis (CSGE) to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption.
Methods: A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA) was conducted.
Results: With direct sequencing, the mutations could be identified from 26 patients (96.3%), whereas for multiplex PCR- CSGE screened sequencing, the mutations could be detected in 23 (85.2%). One patient¡¯s mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients.
Conclusion: Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.
|
|
KEYWORD
|
|
Hemophilia B, DNA mutational analysis, Polymerase chain reaction, Electrophoresis
|
|
FullTexts / Linksout information
|
|
|
|
Listed journal information
|
|
|
|