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Preimplantation Genetic Testing for Monogenic Disorders
Published in Darren K. Griffin, Gary L. Harton, Preimplantation Genetic Testing, 2020
Martine De Rycke, Pieter Verdyck
Many WGA protocols have been published over time and some of them are available as commercial kits. The first WGA methods were PCR-based and suffered from very incomplete genome coverage and amplification bias. The use of Taq DNA polymerase yielded an average fragment length of 400–500 bp (with a maximum size of 3 kb) and introduced many DNA sequence errors. A multiple displacement amplification (MDA) method relying on isothermal strand displacement amplification was established at the single-cell level more than a decade ago [19]. In an MDA reaction, random exonuclease-resistant primers anneal to the denatured target DNA and a DNA polymerase with strand-displacement activity such as Phi29 elongates the primers in an isothermal reaction at 30°C. Additional priming events can occur on each displaced strand leading to a network of branched DNA strands over 10 kb. Because of the proofreading activity of the Phi29 polymerase, the error rate of MDA-based WGA is much lower compared with Taq DNA polymerase−based methods, but the non-linear amplification yields over- or underrepresentation of genomic regions [20].
Genetic analysis of the embryo
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Yuval Yaron, Liran Hiersch, Veronica Gold, Sagit Peleg-Schalka, Mira Malcov
Multiple displacement amplification (MDA) is a recently developed non-PCR-based method that has been utilized for clinical samples with limited DNA content, providing high yields of relatively long fragments (>10 kb) with uni- form and reliable representation across the genome (40). In MDA, annealing of exonuclease-resistant random hexam- ers to a DNA template is followed by strand-displacement DNA synthesis at a constant temperature of 30°C, without the need for cyclic DNA denaturation (40, 41). The strand- displacing mechanism is accomplished by the Φ29 DNA polymerase (40) or the Bacillus stearothermophilus DNA polymerase large fragment (42). This mechanism allows increasing random priming events that form a network of hyper-branched DNA structures that generate thousands of copies of the original DNA in only a few hours (42, 43). It appears that MDA is more advantageous due to decreased rates of unspecific amplification artifacts (44), incomplete coverage of loci (33), strong amplification bias (40), and the short length of the DNA products (45).
Detection Techniques for Single Nucleotide Polymorphisms
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
W. Mathias Howell, Johan Stenberg, Chatarina Larsson, Mats Nilsson, Ulf Landegren
For some purposes, generation of immortalized cell lines from patients can be a suitable means of ensuring access to biological material. A number of in vitro approaches can extend the amount of genomic DNA starting from a minimal amount of genetic material. The first is the whole genome amplification (WGA) method. Although several different strategies have been proposed (reviewed in Reference 127), one method that has been successfully implemented into SNP genotyping is multiple displacement amplification (MDA).128,129 In this isothermal technique, a series of random hexamers (six-base oligonucleotides) are mixed with genomic DNA along with ϕ29 DNA polymerase. When the hexamers anneal to the genome, they serve as primers for replication by the polymerase.
The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia–haemoglobin E disease and chromosome balance using karyomapping
Published in Journal of Obstetrics and Gynaecology, 2022
Sirivipa Piyamongkol, Suchada Mongkolchaipak, Pimlak Charoenkwan, Rungthiwa Sirapat, Wanwisa Suriya, Tawiwan Pantasri, Theera Tongsong, Wirawit Piyamongkol
Biopsied cells were washed thoroughly in phosphate-buffered saline (PBS, Cell Signaling Technology, Theera Trading Co. Ltd., Bangkok, Thailand) with 0.1% polyvinyl alcohol (PVA, Sigma-Aldrich, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) before transference to microcentrifuge tubes. DNA extraction was performed using an alkaline lysis buffer protocol (Sermon et al. 1995). 2.5 μL of lysis buffer (0.75 μL of water, 1.25 μL of 0.1 M DTT and 0.5 μL of 1 M NaOH) was added, mixtures were incubated at 60 °C for 10 min. After that, a neutralisation buffer (2.5 μL of 0.4 M tricine) was added. Whole genome amplification with multiple displacement amplification (MDA, REPLI-g® Single Cell Kit, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) was then carried out by manufacturer’s instructions. A mixture of 12.5 μL of water, 29 μL of reaction buffer and 1 μL of DNA polymerase (REPLI-g® Single Cell Kit) was added to extracted DNA, making a total volume of 50 μL. The mixtures were incubated at 30 °C for 2 h then at 65 °C for 5 min to inactivate the reaction.
Pre-implantation genetic testing for Marfan syndrome using mini-sequencing
Published in Journal of Obstetrics and Gynaecology, 2022
Sirivipa Piyamongkol, Krit Makonkawkeyoon, Vorasuk Shotelersuk, Opas Sreshthaputra, Tawiwan Pantasri, Rekwan Sittiwangkul, Theera Tongsong, Wirawit Piyamongkol
Pre-implantation genetic testing (PGT) (Handyside et al. 1992) is an alternative to traditional PND giving the couples the chance of starting pregnancy with the confidence that the baby will be unaffected. Multiplex nested PCR protocol analysing intragenic microsatellite and amelogenin genes via denaturing polyacrylamide gel electrophoresis with [35S]-dATP labelling was successfully performed (Harton et al. 1996). PGT-M for MFS1 employing single-cell nested PCR with restriction fragment length polymorphism (RFLP) and traditional agarose gel electrophoresis has been reported (Blaszczyk et al. 1998). Automated laser fluorescence DNA sequencing was employed for PGT of Marfan syndrome on the linkage analysis of an mts1 locus basis in one family (Sermon et al. 1999). More intragenic (mts-1, mts-2, mts-3, mts-4) and extragenic (D15S103, D15S123, D15S161) microsatellite markers were introduced for PGT in two families (Loeys et al. 2002). Sequencing exon 41 was included for mutation identification in one family. A protocol using an intragenic marker (mts-2, mts-4) following whole genome amplification (WGA) with multiple displacement amplification (MDA) has been reported (Lledo et al. 2006). In a study a few years later, PGT of Marfan syndrome in 10 families used the combination of RFLP with microsatellites-based linkage analysis (Spits et al. 2006). One protocol employed the combination of mini-sequencing with microsatellite-based linkage analysis. However, the details of mini-sequencing techniques were not mentioned. PGT-M for Marfan syndrome using single nucleotide polymorphism genotyping and karyomapping analysis has been reported (Thornhill et al. 2015). However, the techniques are sophisticated and very expensive. Recent reports performed PGT for Marfan syndrome by mutation analysis alone without mentioning details of the protocol (Vlahos et al. 2013; Wang et al. 2017).
The clinical application of single-sperm-based SNP haplotyping for PGD of osteogenesis imperfecta
Published in Systems Biology in Reproductive Medicine, 2019
Linjun Chen, Zhenyu Diao, Zhipeng Xu, Jianjun Zhou, Guijun Yan, Haixiang Sun
Twelve metaphase II oocytes were inseminated with ejaculated sperm by intracytoplasmic sperm injection (ICSI) and nine had been fertilized normally as shown by the presence of two pronuclei. Nine embryos developed into 7–9 cells on day 3 post-insemination. Three blastocysts (B3, B11 and B12) were used for the TE biopsy on day 5/6 post-insemination. Whole-genome amplification (WGA) was successfully performed on the biopsied TE cells by multiple displacement amplification (MDA).