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Haemoglobinopathies including α- and β-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for β-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting β-thalassaemia mutations (codon 41-42 and IVSI-110) and one for α-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of β-globin and δ-globin was also found to be an important issue that necessitated careful design of primers for the β-globin gene. Copyright © 2001 John Wiley & Sons, Ltd. 相似文献
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Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy
Céline Moutou Nathalie Gardes Catherine Rongières Jeanine Ohl Karima Bettahar-Lebugle Christiane Wittemer Pierre Gerlinger Stéphane Viville 《黑龙江环境通报》2001,21(6):498-503
We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMNt) gene. An oligonucleotide was designed to be specific to the SMNt nucleotidic sequence with exonic mismatch G (for SMNt)→A (for SMNc) at its 3′ end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMNt gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre. Copyright © 2001 John Wiley & Sons, Ltd. 相似文献
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M. Kleanthous K. Kyriacou A. Kyrri E. Kalogerou PH. Vassiliades A. Drousiotou I. Kallikas P. Ioannou M. Angastiniotis 《黑龙江环境通报》2001,21(5):413-417
In Cyprus all couples carrying α0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for α-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the ψα1- and α2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two α-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for α-thalassaemia have been reported to date. Copyright © 2001 John Wiley & Sons, Ltd. 相似文献
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水传病原微生物是腹泻等肠胃疾病发病的主要原因,而发展快速、有效的检测方法对于控制和减少该类微生物的传播风险十分重要.因此,本文研究了PCR反应结合不修饰的纳米金颗粒(Gold nano-particles,AuNPs)显色检测方法的最佳盐浓度、探针含量、PCR反应条件等,并从纳米金制备、病原菌种类、检测特异性等方面进行考核,确定了最优检测条件为:10μL PCR扩增产物混合20 pmol探针,95℃反应5 min,55.5℃反应3 min后,40℃杂交10 min;此时,先后加入90μL实验用纳米金和一定浓度的PBS溶液,室温观察颜色变化.结果发现,纳米金溶液由红色变为紫色或近于无色为阳性结果,对应的吸光度值迁移出530 nm,以此可达到快速检测病原微生物的目的. 相似文献