scholarly journals Whole-Genome Amplification by Adaptor-Ligation PCR of Randomly Sheared Genomic DNA (PRSG)

2008 ◽  
Vol 2008 (2) ◽  
pp. pdb.prot4922-pdb.prot4922 ◽  
Author(s):  
N. Arneson ◽  
S. Hughes ◽  
R. Houlston ◽  
S. Done
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Genome Amplification

187 EMBRYO GENOTYPING FROM IN VIVO BIOPSIED BOVINE EMBRYOS AFTER WHOLE GENOME AMPLIFICATION

2009 ◽  
Vol 21 (1) ◽  
pp. 192 ◽  
Author(s):  
D. Le Bourhis ◽  
Y. Amigues ◽  
F. Charreaux ◽  
S. Lacaze ◽  
M. Tissier ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Detection Rate ◽  
Whole Genome Amplification ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Whole Genome ◽  
Bovine Embryos ◽  
Genome Amplification ◽  
Number Of Cells

Genomic tools are now available for most livestock species and used routinely for marker-assisted selection (MAS) in cattle. The detection of a large number of markers that are widespread over the genome is generally limited by the amount of genomic DNA available in an embryo biopsy of a small size not to be detrimental to embryonic survival. Amplification of DNA from such a biopsy is then necessary. In this study, the efficiency of embryo genotyping for 45 microsatellites (MS) following whole-genome amplification (WGA) was evaluated from samples of a variable number of cells isolated from cattle embryos. In a second part, this work aims to test the reliability of the MAS method for 45 MS and 13 single nucleotide polymorphisms (SNP) from bovine embryo biopsies under field conditions. In experiment 1, in vitro bovine morulae (n = 10) were produced, and 1, 5, and 10 embryonic cells were removed from each morula. Cells were dry frozen in tubes before further processing. Whole-genome amplification was performed using the commercial Qiagen REPLI-g® Mini Kit according to the manufacturer instructions (Qiagen, Valencia, CA, USA). WGA solution was then diluted, processed by PCR with 45 markers, and the resulting data were genotyped with GeneMapper software® (Applied Biosystems Europe). Accuracy and reliability of genotyping were assessed using different samples of cells from the same embryo. In experiment 2, after superovulation (10 cows), bovine embryos were in vivo-produced and collected at day 6 or day 7 of pregnancy. Only grade 1 embryos were washed and biopsied using a microblade. Biopsied embryos were either frozen or transferred back to synchronized recipients. Individual biopsies were transferred as dry samples to the laboratory. Genomic DNA was amplified using WGA, and embryos were genotyped. The results of experiment 1 clearly indicate that a conventional biopsy of 5 to 10 cells was sufficient for multi-markers detection after whole-genome amplification as 98% of the 45 markers were detected compared to 45% of marker detection using 1 cell (P < 0.01). In experiment 2, from 123 collected embryos, 79 were classified as grade I or II transferable embryos (64.2%) and 57 were biopsied (34 were classified as stage 4–5 and 23 as stage 5–6, according to the IETS criteria). Using the stereomicroscopic analysis, 44 biopsies had a number of cells ranging from 4 to 7 (5.6 ± 1.4) and 13 biopsies from 8 to 10 (8.4 ± 1.6). Overall, at least 95% of markers (MS + SNP) were detected in 49.1% of biopsies (28/57). The total detection rate for SNP was significantly higher than for MS; 70.2% (40/57) v. 31.6% (18/57), respectively, (chi-square, P < 0.01). The detection rate of the markers was not significantly affected by the embryo stage or the biopsy size. Our results confirm that genotyping a large number of markers from biopsy samples after whole-genome amplification is possible under field conditions. A larger number of biopsies is required to assess the reliability of this method that may allow the development of MAS from early embryo. This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) with the financial support of FRT/ANR and Apis-Genes.

Whole genome amplification of buccal cell DNA: genotyping concordance before and after multiple displacement amplification

2005 ◽  
Vol 43 (2) ◽  
Author(s):  
Miles D. Thompson ◽  
Raffick A. R. Bowen ◽  
Betty Y. L. Wong ◽  
Joan Antal ◽  
Zhanqin Liu ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Buccal Cell ◽  
Whole Genome ◽  
Buccal Cells ◽  
Genome Amplification ◽  
Dna Yield ◽  
Epidemiological Surveys ◽  
Before And After

AbstractWhile buccal cells provide an easily accessible source of genomic DNA, the amount extracted may be insufficient for many studies. Whole genome amplification (WGA) using multiple displacement amplification (MDA) may optimize buccal cell genomic DNA yield. We compared the usefulness, in epidemiological surveys, of DNA derived from buccal cells collected by alcohol mouthwash and amplified by WGA protocol and standard protocols. Buccal cell collection kits were mailed to 300 randomly selected members of a large cohort study, and 189 subjects returned buccal cell samples. We determined: (i) which QIAamp

Whole genome amplification/hybridization following ooplasmic aspiration reveals no evidence of genomic DNA disturbance

2003 ◽  
Vol 80 ◽  
pp. 118
Author(s):  
Henry E. Malter ◽  
Dagan Wells ◽  
Mercedes Garcia Bermudez ◽  
Jacques Cohen
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Genome Amplification

Amplification of haplo-separated genomic DNA by haplotype-specific extraction (HSE) and whole-genome amplification (WGA)

2004 ◽  
Vol 65 (9-10) ◽  
pp. S25
Author(s):  
Cynthia Turino ◽  
Johannes Dapprich ◽  
Colleen Murphy ◽  
Dylan Membrino ◽  
Nancy Murphy
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Genome Amplification

scholarly journals Whole-genome amplification in double-digest RADseq results in adequate libraries but fewer sequenced loci

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5089 ◽  
Author(s):  
Bruno A. S. de Medeiros ◽  
Brian D. Farrell
Keyword(s):  
Population Genetics ◽  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Sequence Data ◽  
Multiple Displacement Amplification ◽  
Whole Genome ◽  
Promising Technique ◽  
Genome Amplification ◽  
Template Dna

Whole-genome amplification by multiple displacement amplification (MDA) is a promising technique to enable the use of samples with only limited amount of DNA for the construction of RAD-seq libraries. Previous work has shown that, when the amount of DNA used in the MDA reaction is large, double-digest RAD-seq (ddRAD) libraries prepared with amplified genomic DNA result in data that are indistinguishable from libraries prepared directly from genomic DNA. Based on this observation, here we evaluate the quality of ddRAD libraries prepared from MDA-amplified genomic DNA when the amount of input genomic DNA and the coverage obtained for samples is variable. By simultaneously preparing libraries for five species of weevils (Coleoptera, Curculionidae), we also evaluate the likelihood that potential contaminants will be encountered in the assembled dataset. Overall, our results indicate that MDA may not be able to rescue all samples with small amounts of DNA, but it does produce ddRAD libraries adequate for studies of phylogeography and population genetics even when conditions are not optimal. We find that MDA makes it harder to predict the number of loci that will be obtained for a given sequencing effort, with some samples behaving like traditional libraries and others yielding fewer loci than expected. This seems to be caused both by stochastic and deterministic effects during amplification. Further, the reduction in loci is stronger in libraries with lower amounts of template DNA for the MDA reaction. Even though a few samples exhibit substantial levels of contamination in raw reads, the effect is very small in the final dataset, suggesting that filters imposed during dataset assembly are important in removing contamination. Importantly, samples with strong signs of contamination and biases in heterozygosity were also those with fewer loci shared in the final dataset, suggesting that stringent filtering of samples with significant amounts of missing data is important when assembling data derived from MDA-amplified genomic DNA. Overall, we find that the combination of MDA and ddRAD results in high-quality datasets for population genetics as long as the sequence data is properly filtered during assembly.

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 Improved DOP-PCR (iDOP-PCR): a robust and simple WGA method for efficient amplification of low copy number genomic DNA

2017 ◽  
Author(s):  
Konstantin A. Blagodatskikh ◽  
Vladimir M. Kramarov ◽  
Ekaterina V. Barsova ◽  
Alexey V. Garkovenko ◽  
Dmitriy S. Shcherbo ◽  
...  
Keyword(s):  
Copy Number ◽  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Dropout Rate ◽  
Whole Genome ◽  
Genome Amplification ◽  
Dna Libraries ◽  
Degenerate Oligonucleotide ◽  
Low Copy Number ◽  
Cell Genome

AbstractWhole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

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