Application of Hot Start PCR Method in PCR-based Preimplantation Genetic Diagnosis.
- Author:
Sung Ah KIM
1
;
Moon Joo KANG
;
Hee Sun KIM
;
Sun Kyung OH
;
Seung Yup KU
;
Young Min CHOI
;
Jong Kwan JUN
;
Shin Yong MOON
Author Information
- Publication Type:Original Article
- Keywords: Hot start PCR; Nested PCR; Preimplantation genetic diagnosis
- MeSH: Alleles; Blastomeres; Family Characteristics; Humans; Lymphocytes; Osteogenesis Imperfecta; Patient Dropouts; Polymerase Chain Reaction; Preimplantation Diagnosis; Prostaglandins D
- From:Journal of Genetic Medicine 2012;9(1):11-16
- CountryRepublic of Korea
- Language:English
- Abstract: PURPOSE: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. MATERIALS AND METHODS: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. RESULTS: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. CONCLUSION: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.
