scholarly journals Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): II. Second-Strand Synthesis and In Vitro Transcription

2008 ◽  
Vol 2008 (6) ◽  
pp. pdb.prot5003-pdb.prot5003 ◽  
Author(s):  
C. L. Liu ◽  
B. E. Bernstein ◽  
S. L. Schreiber
Keyword(s):  
Whole Genome Amplification ◽  
In Vitro Transcription ◽  
Whole Genome ◽  
Linear Amplification ◽  
Genome Amplification

266 WHOLE GENOME AMPLIFICATION ON BLASTOMERS OF POST-BIOPSY BOVINE EMBRYO

2009 ◽  
Vol 21 (1) ◽  
pp. 230
Author(s):  
J. Polisseni ◽  
M. A. Machado ◽  
A. L. Souza ◽  
R. Domingues ◽  
M. O. Guerra ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Gene Profiling ◽  
Proteinase K ◽  
Chromosomal Mosaicism ◽  
Bovine Embryo ◽  
Whole Genome ◽  
Bovine Embryos ◽  
Genome Amplification

Biopsy of embryos is very useful for choosing the desired sex and for production of cloned and transgenic livestock. However, only a small amount of genomic DNA is available to perform genetic studies. Alternatively, methodologies using whole genome amplification (WGA) have been developed. The aims of this study were to evaluate the effect of WGA on blastomeres removed from 8- to 16-cell bovine embryos and to determine the sex of blastomeres. Oocytes obtained from slaughterhouse ovaries were in vitro matured and fertilized. On the fourth day after fertilization, 8- to 16-cell bovine embryos were biopsied, and one-fourth of an embryo was removed. The blastomeres (n = 56) were submitted to WGA followed by PCR. Prior to the whole genome amplification, male and female bovine DNA samples were serially diluted (30 ng μL–1, 3.0 ng μL–1, 0.3 ng μL–1, 0.03 ng μL–1, 0.003 ng μL–1, 0.0003 ng μL–1) and embryos of various development stages (2, n = 6; 4–7, n = 5; ≥8-cell, n = 5; blastocyst n = 27) were used to standardize PCR protocols and set the amplification limits. To digest the cellular cytoplasm and release the genomic DNA, embryos and blastomeres were submitted a 3 mg mL–1 proteinase K before PCR. Next, blastomeres were submitted to the GenomiPhi DNA Amplification Kit (GE Healthcare) according to manufacturer’s instructions. The product (1 μL) was electrophoresed on a 1% agarose gel stained with 3.0 μg mL–1 ethidium bromide. The reaction mixture was added to the material to be amplified (2 mm MgCl2, 5X PCR buffer, 0.2 mm each dNTPs, 0.05 U μL–1 GoTaq DNA polymerase, 0.25 μm of primer). The products were submitted to electrophoresis on 8% polyacrilamide gel and stained with silver nitrate procedure. The chi-square test was used for statistic evaluation of the results to test the WGA efficiency and to determine the sex rates of bovine embryos and biopsied samples submitted to PCR. It was possible to achieve 98% efficiency in amplifying blastomeres using the WGA kit. Amplified samples showed approximately 400 ng of DNA generated from an estimated initial amount of 12 pg of DNA resulting from two cells per embryo. In whole embryos from different stages, no diffference was detected in the proportion of sexes (P > 0.05). However, a greater number of female samples was noted in biopsied material (76%, 25/33) (P < 0.05). PCR efficiency in blastocysts (93%, 2/27) was statistically greater (P < 0.05) than embryos in early stages of development (83%, 5/6), and biopsied material to 2, 4–7 and ≥8-cell (40%, 2/5; 60%, 3/5; and 59%, 33/56; respectively). These differences could be related to a sex-chromosomal mosaicism or absence of a nucleus in biopsied samples. The WGA creates a DNA stock sample that could be used for various gene profiling and sex determination analyses. Financial support: Fapemig, CNPq.

scholarly journals Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): Overview

2008 ◽  
Vol 2008 (6) ◽  
pp. pdb.top42-pdb.top42 ◽  
Author(s):  
C. L. Liu ◽  
B. E. Bernstein ◽  
S. L. Schreiber
Keyword(s):  
Whole Genome Amplification ◽  
Whole Genome ◽  
Linear Amplification ◽  
Genome Amplification

scholarly journals Structure of chromosomal abnormalities in the cycles of IVF-PGS

2019 ◽  
pp. 47-54
Author(s):  
Д.И. Жигалина ◽  
Н.А. Скрябин ◽  
О.Р. Канбекова ◽  
А.Н. Марошкина ◽  
Е.О. Чуркин ◽  
...  
Keyword(s):  
Genetic Screening ◽  
Whole Genome Amplification ◽  
Chromosomal Abnormalities ◽  
Preimplantation Genetic Screening ◽  
Human Embryos ◽  
Whole Genome ◽  
Chromosomal Imbalance ◽  
Genome Amplification ◽  
Wide Range

Актуальность: Ограниченный репродуктивный потенциал у человека и прогрессирующее ухудшение репродуктивного здоровья населения стали причиной развития вспомогательных репродуктивных технологий в последние десятилетия. С целью повышения вероятности имплантации бластоцисты, снижения частоты спонтанных абортов у семейных пар, которые имеют проблемы репродукции, в клиническую практику был введен преимплантационный генетический скрининг (ПГС). Культивирование эмбрионов человека in vitro в циклах экстракорпорального оплодотворении (ЭКО), а также возможность получения генетического материала при проведении ПГС позволяют оценить частоту и спектр хромосомных нарушений в бластоцистах человека. Цель: Анализ частоты и спектра числовых хромосомных аномалий в бластоцистах, полученных в рамках циклов ЭКО-ПГС. Материалы и методы. Проведен ретроспективный анализ молекулярных кариотипов 113 бластоцист, полученных в рамках циклов ЭКО-ПГС от 47 женщин. Полногеномная амплификация (ПГА) ДНК из клеток трофэктодермы проводилась с использованием набора реактивов PicoPlex (Rubicon Genomics, США). Анализ образцов ДНК, полученных после ПГА, был проведен методом микроматричной сравнительной геномной гибридизации (aCGH) с использованием микрочипа GenetiSure Pre-Screen, 8×60K (Agilent Technologies, США). Результаты: Эффективность ПГА составила 97,3% (110/113). Сбалансированный кариотип был установлен в 31% (34/110) бластоцист. Частота бластоцист с хромосомным дисбалансом в группе женщин моложе 35 лет оказалась значимо ниже (46,9 %) по сравнению с частотой бластоцист с хромосомным дисбалансом в группе женщин старше 35 лет (81,0 %) (р < 0,001). Хромосомные аномалии были представлены анеуплоидиями (74 %), в 26 % - структурными нарушениями хромосом. Распределение анеуплоидий имело следующую структуру: трисомии аутосом составили 41 %, моносомии аутосом - 48 %, анеуплоидии половых хромосом - 7%, тетрасомии аутосом - 3 %, нуллисомии аутосом - 1 %. Наиболее часто отмечались анеуплоидии хромосом 5, 15, 16, 17, 19, 21 и 22. Выводы: Анализ хромосомных аберраций в бластоцистах продемонстрировал высокую частоту хромосомного дисбаланса (69%) и широкий спектр как числовых, так и структурных нарушений хромосом. ПГС методом aCGH позволяет отобрать бластоцисты со сбалансированным набором хромосом. По результатам переносов бластоцист в циклах ЭКО-ПГС клиническая беременность наступила в 32% случаев. Introduction: Limited reproductive potential in humans and the progressive decline of the reproductive health of the population have led to the development of assisted reproductive technologies in recent decades. In order to improve pregnancy rates in couples with reproduction problems, preimplantation genetic screening was introduced into clinical practice. Cultivation of human embryos in vitro in in vitro fertilization cycles (IVF), as well as the possibility of obtaining genetic material during preimplantation genetic screening and diagnosis (PGS / PGD), allow us to estimate the frequency and spectrum of chromosomal abnormalities in human blastocysts. Aim: Analysis of the rate and spectrum of aneuploidies in human blastocysts obtained in the IVF-PGD cycles. Material and methods: A retrospective analysis of the molecular karyotypes of 113 blastocysts obtained in the cycles of assisted reproductive technology IVF-PGD from 47 women was carried out. The whole genomic amplification of DNA from trophectoderm cells was performed using the PicoPlex reagent kit (Rubicon Genomics, USA). Analysis of DNA samples obtained after whole genome amplification was carried out by array comparative genomic hybridization (aCGH) using a GenetiSure Pre-Screen microchip, 8×60K (Agilent Technologies, USA). Results: The efficiency of whole genome amplification was 97.3% (110/113). A balanced karyotype was established in 31% (34/110) blastocysts. The rate of a blastocyst with chromosomal imbalance in the group of women under 35 years old was lower (46.9%) compared to the rate of blastocyst with chromosomal imbalance in the group of women over 35 years old (81.0%) (p < 0.001). 74% of the identified chromosomal abnormalities were aneuploidy, 26% - structural chromosomal abberations. The distribution of aneuploidies had the following structure: autosomal trisomies (41%), autosomal monosomies (48%), aneuploidies of sex chromosomes (7%), autosomal tetrasomies (3%), autosomal nullisomies (1%). Aneuploidies of chromosomes 5, 15, 16, 17, 19, 21 and 22 were noted with the greatest frequency. Conclusions: Analysis of chromosomal aberrations in human embryos at the blastocyst stage showed a high frequency of chromosomal imbalance (69%) and a wide range of both numerical and structural abnormalities of chromosomes. PGS with aCGH allows the selection of blastocysts with a balanced karyotype. According to the results of blastocyst transfers in IVF-PGD cycles, clinical pregnancy occurred in 32 % of cases.

102 ACCURACY OF PRE-IMPLANTATION GENETIC DIAGNOSIS USING CELLS BIOPSIED FROM EQUINE BLASTOCYSTS

2012 ◽  
Vol 24 (1) ◽  
pp. 163 ◽  
Author(s):  
Y. H. Choi ◽  
M. C. T. Penedo ◽  
P. Daftari ◽  
I. C. Velez ◽  
K. Hinrichs
Keyword(s):  
Whole Genome Amplification ◽  
Genetic Diagnosis ◽  
Coat Colour ◽  
Drop Out ◽  
Biopsy Sample ◽  
Whole Genome ◽  
Genome Amplification ◽  
Allele Dropout

There is growing interest in pre-implantation genetic diagnosis (PGD) for management of inherited genetic disease in the horse. In a previous study (Choi et al. 2010 Reproduction 140, 893–902), we demonstrated normal viability of equine blastocysts after biopsy. However, genome amplification was only moderately successful and only 1 of 2 analyses of heterozygous loci accurately detected both alleles. In the current study, we investigated different methods for amplification of DNA to improve the efficiency of PGD. To evaluate allele drop-out, multiple commonly-heterozygous gene loci were evaluated. In Experiment 1, using a piezo drill, 3 to 5 biopsy samples of 20 to 30 cells each were obtained from each of 4 in vitro-produced blastocysts. The samples and embryos were stored at –20°C, then shipped to the Veterinary Genetics Laboratory at the University of California, Davis. Whole genome amplification was done with an Illustra Genomiphi V2 kit (GE Healthcare, Waukesha, WI) before PCR for specific markers. Two disease-related (SCN4A and PPIB), one gender (AME) and 17 microsatellite identification markers were genotyped, for a total of 20 loci. Results for biopsy samples were compared with those for the corresponding embryo. A DNA signal was obtained from 14/15 biopsy samples, but for only 59.6% of the 280 total genotypes. Of 40 heterozygous loci, the signal from the corresponding biopsy sample showed only one allele (underwent allele dropout) in 60/80 instances (75%). In Experiment 2, 4 biopsies were obtained from each of 4 additional in vitro-produced blastocysts, then all samples were stored at –20°C. The Repli-G Mid kit (Qiagen, Valencia, CA) was used for whole genome amplification. Two disease-related (SCN4A and PPIB), 2 gender (AME and eSRY), 10 coat colour and 17 identification markers (total of 31 loci) were examined in each biopsy sample and were compared with results for the embryos. One biopsy sample was lost. Signal was obtained from 14/15 of the remaining biopsy samples and gave a 100% match at the 2 gender loci, 2 disease-related loci and 10 coat colour loci. One identification locus, LEX33, amplified in only 8 of 22 analyses. At the remaining 16 identification loci, 223/224 biopsy results matched those for the embryos. Overall, of 51 heterozygous loci among the 4 embryos, biopsy samples exhibited allele dropout in 1/180 instances (0.6%). In conclusion, results obtained using piezo-driven embryo biopsy and whole genome amplification using the Qiagen Repli-G kit have high accuracy and this technique may be suitable for use in a clinical setting. Further studies are needed with in vivo-derived embryos and to optimize accuracy of PCR of some identification markers. This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University and by Ms Kit Knotts.

scholarly journals Quantifying genome DNA during whole-genome amplification via quantitative real-time multiple displacement amplification

RSC Advances ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 4617-4621
Author(s):  
Jing Tu ◽  
Yi Qiao ◽  
Yuhan Luo ◽  
Naiyun Long ◽  
Zuhong Lu
Keyword(s):  
Real Time ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Whole Genome ◽  
Genome Amplification

Monitoring multiple displacement amplification by fluorescence signals.

scholarly journals Species Identification of Pinus Pollen Found in Belukha Glacier, Russian Altai Mountains, Using a Whole-Genome Amplification Method

Forests ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 444
Author(s):  
Fumio Nakazawa ◽  
Yoshihisa Suyama ◽  
Satoshi Imura ◽  
Hideaki Motoyama
Keyword(s):  
Species Identification ◽  
Whole Genome Amplification ◽  
Pollen Grains ◽  
Pollen Grain ◽  
Pinus Sibirica ◽  
Whole Genome ◽  
Accurate Identification ◽  
Closely Related Species ◽  
Genome Amplification ◽  
Amplification Method

Pollen taxa in sediment samples can be identified based on morphology. However, closely related species do not differ substantially in pollen morphology, and accurate identification is generally limited to genera or families. Because many pollen grains in glaciers contain protoplasm, genetic information obtained from pollen grains should enable the identification of plant taxa at the species level. In the present study, species identification of Pinus pollen grains was attempted using whole-genome amplification (WGA). We used pollen grains extracted from surface snow (depth, 1.8–1.9 m) from the Belukha glacier in the summer of 2003. WGA was performed using a single pollen grain. Some regions of the chloroplast genome were amplified by PCR, and the DNA products were sequenced to identify the pollen grain. Pinus includes approximately 111 recognized species in two subgenera, four sections, and 11 subsections. The tree species Pinus sibirica and P. sylvestris are currently found at the periphery of the glacier. We identified the pollen grains from the Belukha glacier to the level of section or subsection to which P. sibirica and P. sylvestris belong. Moreover, we specifically identified two pollen grains as P. sibirica or P. cembra. Fifteen species, including P. sibirica, were candidates for the remaining pollen grain.

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