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 PCRCSGE 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.
| Original language | English |
|---|---|
| Pages (from-to) | 397-407 |
| Number of pages | 11 |
| Journal | Korean Journal of Pediatrics |
| Volume | 53 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2010 Jan 1 |
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Keywords
- DNA mutational analysis
- Electrophoresis
- Hemophilia B
- Polymerase chain reaction
ASJC Scopus subject areas
- Pediatrics, Perinatology, and Child Health
- Pediatrics
Cite this
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.
In: Korean Journal of Pediatrics, Vol. 53, No. 3, 01.01.2010, p. 397-407.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Direct detection of hemophilia B f9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis
AU - Yoo, Ki Young
AU - Kim, Hee Jin
AU - Lee, Kwang Chul
PY - 2010/1/1
Y1 - 2010/1/1
N2 - 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 PCRCSGE 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.
AB - 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 PCRCSGE 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.
KW - DNA mutational analysis
KW - Electrophoresis
KW - Hemophilia B
KW - Polymerase chain reaction
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UR - http://www.scopus.com/inward/citedby.url?scp=79951711635&partnerID=8YFLogxK
U2 - 10.3345/kjp.2010.53.3.397
DO - 10.3345/kjp.2010.53.3.397
M3 - Article
VL - 53
SP - 397
EP - 407
JO - Korean Journal of Pediatrics
JF - Korean Journal of Pediatrics
SN - 1783-1061
IS - 3
ER -