The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders.
نویسندگان
چکیده
We have applied a new method of genetic analysis, called 'minisequencing', to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer-assisted mutation analysis, which allows exact base identity determination and computer-assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, we analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, beta-thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing was found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the blastomeres tested. Fifteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 10 healthy babies have already been born. Its applicability to PGD could be helpful, particularly in cases in which the mutation(s) involved are difficult to assess by restriction analysis or other commonly used methods.
منابع مشابه
O-27: Preimplantation Genetic Diagnosis in Prevention of Genetic Diseases -Diagnostic of Spinal Muscular Atrophy (SMA)
Background: Preimplantation genetic diagnosis - PGD is currently an established procedure allowing genetic research of oocyte or embryo before implantation to the uterus. Spinal muscular atrophy (SMA) is a neurodegenerative disorder, being the second most common lethal autosomal recessive disease in Caucasians, after cystic fibrosis. There are three clinically different types of which type I (W...
متن کاملO-36: Genome Haplotyping and Detection of Meiotic Homologous Recombination Sites in Single Cells, A Generic Method for Preimplantation Genetic Diagnosis
Background: Haplotyping is invaluable not only to identify genetic variants underlying a disease or trait, but also to study evolution and population history as well as meiotic and mitotic recombination processes. Current genome-wide haplotyping methods rely on genomic DNA that is extracted from a large number of cells. Thus far random allele drop out and preferential amplification artifacts of...
متن کاملPreimplantation diagnosis by whole-genome amplification, PCR amplification, and solid-phase minisequencing of blastomere DNA.
We have developed a new method for preimplantation diagnosis of inherited diseases. Our procedure for the identification of point mutations in single cells combines whole-genome amplification using 15-mer random primers (primer extension preamplification, PEP) with a single locus-specific PCR amplification, followed by detection of the mutation by solid-phase minisequencing. The procedure was e...
متن کاملI-13: Transcriptome Dynamics of Human and Mouse Preimplantation Embryos Revealed by Single Cell RNA-Sequencing
Background: Mammalian preimplantation development is a complex process involving dramatic changes in the transcriptional architecture. However, it is still unclear about the crucial transcriptional network and key hub genes that regulate the proceeding of preimplantation embryos. Materials and Methods: Through single-cell RNAsequencing (RNA-seq) of both human and mouse preimplantation embryos, ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Molecular human reproduction
دوره 9 7 شماره
صفحات -
تاریخ انتشار 2003