J Genet Med.  2012 Jun;9(1):11-16. 10.5734/JGM.2012.9.1.11.

Application of Hot Start PCR Method in PCR-based Preimplantation Genetic Diagnosis

• Kim SA
• Kang MJ
• Kim HS
• Oh SK
• Ku SY
• Choi YM
• Jun JK
• Moon SY

• KMID: 2055563
• DOI: http://doi.org/10.5734/JGM.2012.9.1.11

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.

Keyword

Hot start PCR; Nested PCR; Preimplantation genetic diagnosis

MeSH Terms

Alleles
Blastomeres
Family Characteristics
Humans
Lymphocytes
Osteogenesis Imperfecta
Patient Dropouts
Polymerase Chain Reaction
Preimplantation Diagnosis
Prostaglandins D
Prostaglandins D