dna purification kit  (Zymo Research)


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    Name:
    Quick DNA Microprep Plus Kit
    Description:
    The Quick DNA Plus Kits are the easiest method for high yield total DNA extraction e g genomic mitochondrial viral cell culture solid tissue saliva and any biological fluid sample Innovative reagents and Zymo Spin Technology allow for ultra pure and concentrated genomic DNA 50 kb to be eluted in as little as 10 µl Zymo Spin Columns ensure no buffer retention Purified DNA is RNA free bypassing the need for RNase A treatment and ensuring accurate quantification for applications like library preparations Isolated DNA is suitable for immediate use in sensitive downstream applications including qPCR DNA seq arrays and methylation analysis
    Catalog Number:
    d4074
    Price:
    None
    Applications:
    DNA Purification
    Size:
    50 units
    Category:
    Life Science Reagents and Media
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    Structured Review

    Zymo Research dna purification kit
    Quick DNA Microprep Plus Kit
    The Quick DNA Plus Kits are the easiest method for high yield total DNA extraction e g genomic mitochondrial viral cell culture solid tissue saliva and any biological fluid sample Innovative reagents and Zymo Spin Technology allow for ultra pure and concentrated genomic DNA 50 kb to be eluted in as little as 10 µl Zymo Spin Columns ensure no buffer retention Purified DNA is RNA free bypassing the need for RNase A treatment and ensuring accurate quantification for applications like library preparations Isolated DNA is suitable for immediate use in sensitive downstream applications including qPCR DNA seq arrays and methylation analysis
    https://www.bioz.com/result/dna purification kit/product/Zymo Research
    Average 98 stars, based on 458 article reviews
    Price from $9.99 to $1999.99
    dna purification kit - by Bioz Stars, 2020-10
    98/100 stars

    Images

    1) Product Images from "Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements"

    Article Title: Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.111195

    Comparative analysis of mitochondrial genome structures and annotations of Neospora and Toxoplasma reveals gene fragmentation and reshuffling between species and strains. A. The repetitive nature of the gene structure in a 32kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. B. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. C. Comparative alignment between two NcLiv mitochondrial contigs of 16 and 32kb, respectively. D . Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an NcUru1 mitochondrial contig of 38kb. E . Comparative alignment between two NcUru1 mitochondrial contigs of 16 and 38kb, respectively. F. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of NcUru1 is graphically represented in a YASS plot. G. Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an T. gondii mitochondrial contigs of 39kb.
    Figure Legend Snippet: Comparative analysis of mitochondrial genome structures and annotations of Neospora and Toxoplasma reveals gene fragmentation and reshuffling between species and strains. A. The repetitive nature of the gene structure in a 32kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. B. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. C. Comparative alignment between two NcLiv mitochondrial contigs of 16 and 32kb, respectively. D . Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an NcUru1 mitochondrial contig of 38kb. E . Comparative alignment between two NcUru1 mitochondrial contigs of 16 and 38kb, respectively. F. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of NcUru1 is graphically represented in a YASS plot. G. Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an T. gondii mitochondrial contigs of 39kb.

    Techniques Used:

    2) Product Images from "A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing"

    Article Title: A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

    Journal: bioRxiv

    doi: 10.1101/663195

    The cf-RRBS method is optimized to obtain a sequencing library enriched in MspI/MspI – fragments when starting from highly fragmented circulating cell-free DNA (cfDNA). (a) Capillary gel electropherograms of white blood cell (WBC) genomic DNA (gDNA) and cfDNA, either undigested or MspI-digested. The undigested WBC gDNA runs above the upper marker (UM) and is not observed on the gel image. After MspI digestion, a smear of shorter DNA appears. Also three characteristic satellite DNA bands can be observed resulting from the digestion of high-abundant DNA repeats in the genome. The undigested cfDNA sample shows the expected apoptotic DNA pattern of a multiple of nucleosome repeats. After MspI digestion, especially the DNA fragment of three nucleosome repeats is not observed anymore. In addition, a smear of shorter DNA fragments is observed beneath the one nucleosome repeat. In classic RRBS, the MspI/MspI-fragments ranging from 30 to 160 bp (indicated with a red rectangle) would be extracted from gel. Starting from cfDNA, extracting these fragments without co-extracting input cfDNA is impossible as these completely overlap in size range. (b) In the cf-RRBS workflow the cfDNA sample is first dephosphorylated prior to MspI digestion. Only MspI/MspI-fragments yield unnicked DNA molecules without free ends upon dA-tailing and ligation of hairpin-shaped adapters. Those ‘circular’ molecules are resistant to exonuclease digestion, whereas all other DNA is degraded using a combination of exonucleases. All these steps are performed on the nanogram quantities of cfDNA typically obtainable from patient’s blood samples, necessitating thorough development of these reactions so that they can now be performed without any tube transfers or purifications by subsequent reagent addition and incubation. (c) The graph shows that a large portion of the uniquely mapping reads originate from MspI/MspI-fragments between 20 to 165 bp when performing cf-RRBS on cfDNA, while this is not the case when performing classical RRBS on the same cfDNA (AUC(cf-RRBS) = 90.67% vs AUC(RRBS) = 13.73%).
    Figure Legend Snippet: The cf-RRBS method is optimized to obtain a sequencing library enriched in MspI/MspI – fragments when starting from highly fragmented circulating cell-free DNA (cfDNA). (a) Capillary gel electropherograms of white blood cell (WBC) genomic DNA (gDNA) and cfDNA, either undigested or MspI-digested. The undigested WBC gDNA runs above the upper marker (UM) and is not observed on the gel image. After MspI digestion, a smear of shorter DNA appears. Also three characteristic satellite DNA bands can be observed resulting from the digestion of high-abundant DNA repeats in the genome. The undigested cfDNA sample shows the expected apoptotic DNA pattern of a multiple of nucleosome repeats. After MspI digestion, especially the DNA fragment of three nucleosome repeats is not observed anymore. In addition, a smear of shorter DNA fragments is observed beneath the one nucleosome repeat. In classic RRBS, the MspI/MspI-fragments ranging from 30 to 160 bp (indicated with a red rectangle) would be extracted from gel. Starting from cfDNA, extracting these fragments without co-extracting input cfDNA is impossible as these completely overlap in size range. (b) In the cf-RRBS workflow the cfDNA sample is first dephosphorylated prior to MspI digestion. Only MspI/MspI-fragments yield unnicked DNA molecules without free ends upon dA-tailing and ligation of hairpin-shaped adapters. Those ‘circular’ molecules are resistant to exonuclease digestion, whereas all other DNA is degraded using a combination of exonucleases. All these steps are performed on the nanogram quantities of cfDNA typically obtainable from patient’s blood samples, necessitating thorough development of these reactions so that they can now be performed without any tube transfers or purifications by subsequent reagent addition and incubation. (c) The graph shows that a large portion of the uniquely mapping reads originate from MspI/MspI-fragments between 20 to 165 bp when performing cf-RRBS on cfDNA, while this is not the case when performing classical RRBS on the same cfDNA (AUC(cf-RRBS) = 90.67% vs AUC(RRBS) = 13.73%).

    Techniques Used: Sequencing, Marker, Ligation, Incubation

    Related Articles

    DNA Extraction:

    Article Title: Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements
    Article Snippet: .. High Molecular Weight DNA extraction and sequencing For “Oxford Nanopore” and Illumina sequencing, DNA was extracted from N. caninum Liverpool, N. caninum Uru1 and T. gondii RH ΔKu80 strain, using a DNA purification kit from Zymo Research (#D4074). .. Minion Oxford Nanopore sequencing was done In house as described in .

    RNA Extraction:

    Article Title: Insertion of a chimeric retrotransposon sequence in mouse Axin1 locus causes metastable kinky tail phenotype
    Article Snippet: .. DNA, RNA extraction and cDNA synthesis DNA and RNA were extracted from kidneys and tail snips from penetrant and silent mice using the Quick-DNA Plus Kits (D4074, Zymo Research) and mirVana miRNA Isolation Kit (AM1560, Thermo Fisher Scientific), respectively, according to the manufacturer’s instructions. .. Briefly, kidney or tail samples were homogenized in 600 μL of Lysis/Binding Buffer with homogenizer (D1000, Benchmark), followed by centrifugation to remove cell debris.

    Isolation:

    Article Title: Bone marrow adipose tissue-derived stem cell factor mediates metabolic regulation of hematopoiesis
    Article Snippet: .. DNA for verifying knockout was isolated using the Quick-DNA Plus Kit (Zymo Research, #D4074). .. Total RNA from tissues was isolated using TRIzol (Invitrogen, 15596018).

    Article Title: Insertion of a chimeric retrotransposon sequence in mouse Axin1 locus causes metastable kinky tail phenotype
    Article Snippet: .. DNA, RNA extraction and cDNA synthesis DNA and RNA were extracted from kidneys and tail snips from penetrant and silent mice using the Quick-DNA Plus Kits (D4074, Zymo Research) and mirVana miRNA Isolation Kit (AM1560, Thermo Fisher Scientific), respectively, according to the manufacturer’s instructions. .. Briefly, kidney or tail samples were homogenized in 600 μL of Lysis/Binding Buffer with homogenizer (D1000, Benchmark), followed by centrifugation to remove cell debris.

    Knock-Out:

    Article Title: Bone marrow adipose tissue-derived stem cell factor mediates metabolic regulation of hematopoiesis
    Article Snippet: .. DNA for verifying knockout was isolated using the Quick-DNA Plus Kit (Zymo Research, #D4074). .. Total RNA from tissues was isolated using TRIzol (Invitrogen, 15596018).

    Mouse Assay:

    Article Title: Insertion of a chimeric retrotransposon sequence in mouse Axin1 locus causes metastable kinky tail phenotype
    Article Snippet: .. DNA, RNA extraction and cDNA synthesis DNA and RNA were extracted from kidneys and tail snips from penetrant and silent mice using the Quick-DNA Plus Kits (D4074, Zymo Research) and mirVana miRNA Isolation Kit (AM1560, Thermo Fisher Scientific), respectively, according to the manufacturer’s instructions. .. Briefly, kidney or tail samples were homogenized in 600 μL of Lysis/Binding Buffer with homogenizer (D1000, Benchmark), followed by centrifugation to remove cell debris.

    DNA Purification:

    Article Title: Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements
    Article Snippet: .. High Molecular Weight DNA extraction and sequencing For “Oxford Nanopore” and Illumina sequencing, DNA was extracted from N. caninum Liverpool, N. caninum Uru1 and T. gondii RH ΔKu80 strain, using a DNA purification kit from Zymo Research (#D4074). .. Minion Oxford Nanopore sequencing was done In house as described in .

    Sequencing:

    Article Title: Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements
    Article Snippet: .. High Molecular Weight DNA extraction and sequencing For “Oxford Nanopore” and Illumina sequencing, DNA was extracted from N. caninum Liverpool, N. caninum Uru1 and T. gondii RH ΔKu80 strain, using a DNA purification kit from Zymo Research (#D4074). .. Minion Oxford Nanopore sequencing was done In house as described in .

    FACS:

    Article Title: Caspase-11 Contributes to Oviduct Pathology during Genital Chlamydia Infection in Mice
    Article Snippet: .. A Zymo Quick-DNA microprep kit was used to extract genomic DNA from the chlamydial stock to generate a standard curve or from 100 μl of tissue homogenate (1/10 of the FACS homogenate). .. Standard curves were generated by dilution of genomic DNA and converting the concentration to copy number, which was equal to (the amount [in nanograms] of genomic DNA · 6.022 × 1023 )/(length · 1 × 109 [a conversion factor] · 650 [molecular weight, in base pairs]).

    Purification:

    Article Title: Start codon disruption with CRISPR/Cas9 prevents murine Fuchs’ endothelial corneal dystrophy
    Article Snippet: .. Genomic DNA from the corneal endothelium/stroma was purified by Quick-DNA Microprep Plus Kit (Zymo research). ..

    Molecular Weight:

    Article Title: Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements
    Article Snippet: .. High Molecular Weight DNA extraction and sequencing For “Oxford Nanopore” and Illumina sequencing, DNA was extracted from N. caninum Liverpool, N. caninum Uru1 and T. gondii RH ΔKu80 strain, using a DNA purification kit from Zymo Research (#D4074). .. Minion Oxford Nanopore sequencing was done In house as described in .

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  • 98
    Zymo Research dna purification kit
    Comparative analysis of mitochondrial genome structures and annotations of Neospora and Toxoplasma reveals gene fragmentation and reshuffling between species and strains. A. The repetitive nature of the gene structure in a 32kb mitochondrial <t>DNA</t> contig of Nc Liverpool is graphically represented in a YASS plot. B. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. C. Comparative alignment between two NcLiv mitochondrial contigs of 16 and 32kb, respectively. D . Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an NcUru1 mitochondrial contig of 38kb. E . Comparative alignment between two NcUru1 mitochondrial contigs of 16 and 38kb, respectively. F. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of NcUru1 is graphically represented in a YASS plot. G. Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an T. <t>gondii</t> mitochondrial contigs of 39kb.
    Dna Purification Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 98/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna purification kit/product/Zymo Research
    Average 98 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    dna purification kit - by Bioz Stars, 2020-10
    98/100 stars
      Buy from Supplier

    98
    Zymo Research quick cfdna serum plasma kit
    The cf-RRBS method is optimized to obtain a sequencing library enriched in MspI/MspI – fragments when starting from highly fragmented circulating cell-free <t>DNA</t> <t>(cfDNA).</t> (a) Capillary gel electropherograms of white blood cell (WBC) genomic DNA (gDNA) and cfDNA, either undigested or MspI-digested. The undigested WBC gDNA runs above the upper marker (UM) and is not observed on the gel image. After MspI digestion, a smear of shorter DNA appears. Also three characteristic satellite DNA bands can be observed resulting from the digestion of high-abundant DNA repeats in the genome. The undigested cfDNA sample shows the expected apoptotic DNA pattern of a multiple of nucleosome repeats. After MspI digestion, especially the DNA fragment of three nucleosome repeats is not observed anymore. In addition, a smear of shorter DNA fragments is observed beneath the one nucleosome repeat. In classic RRBS, the MspI/MspI-fragments ranging from 30 to 160 bp (indicated with a red rectangle) would be extracted from gel. Starting from cfDNA, extracting these fragments without co-extracting input cfDNA is impossible as these completely overlap in size range. (b) In the cf-RRBS workflow the cfDNA sample is first dephosphorylated prior to MspI digestion. Only MspI/MspI-fragments yield unnicked DNA molecules without free ends upon dA-tailing and ligation of hairpin-shaped adapters. Those ‘circular’ molecules are resistant to exonuclease digestion, whereas all other DNA is degraded using a combination of exonucleases. All these steps are performed on the nanogram quantities of cfDNA typically obtainable from patient’s blood samples, necessitating thorough development of these reactions so that they can now be performed without any tube transfers or purifications by subsequent reagent addition and incubation. (c) The graph shows that a large portion of the uniquely mapping reads originate from MspI/MspI-fragments between 20 to 165 bp when performing cf-RRBS on cfDNA, while this is not the case when performing classical RRBS on the same cfDNA (AUC(cf-RRBS) = 90.67% vs AUC(RRBS) = 13.73%).
    Quick Cfdna Serum Plasma Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quick cfdna serum plasma kit/product/Zymo Research
    Average 98 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    quick cfdna serum plasma kit - by Bioz Stars, 2020-10
    98/100 stars
      Buy from Supplier

    94
    Zymo Research quick gdna midiprep kit
    The cf-RRBS method is optimized to obtain a sequencing library enriched in MspI/MspI – fragments when starting from highly fragmented circulating cell-free <t>DNA</t> <t>(cfDNA).</t> (a) Capillary gel electropherograms of white blood cell (WBC) genomic DNA (gDNA) and cfDNA, either undigested or MspI-digested. The undigested WBC gDNA runs above the upper marker (UM) and is not observed on the gel image. After MspI digestion, a smear of shorter DNA appears. Also three characteristic satellite DNA bands can be observed resulting from the digestion of high-abundant DNA repeats in the genome. The undigested cfDNA sample shows the expected apoptotic DNA pattern of a multiple of nucleosome repeats. After MspI digestion, especially the DNA fragment of three nucleosome repeats is not observed anymore. In addition, a smear of shorter DNA fragments is observed beneath the one nucleosome repeat. In classic RRBS, the MspI/MspI-fragments ranging from 30 to 160 bp (indicated with a red rectangle) would be extracted from gel. Starting from cfDNA, extracting these fragments without co-extracting input cfDNA is impossible as these completely overlap in size range. (b) In the cf-RRBS workflow the cfDNA sample is first dephosphorylated prior to MspI digestion. Only MspI/MspI-fragments yield unnicked DNA molecules without free ends upon dA-tailing and ligation of hairpin-shaped adapters. Those ‘circular’ molecules are resistant to exonuclease digestion, whereas all other DNA is degraded using a combination of exonucleases. All these steps are performed on the nanogram quantities of cfDNA typically obtainable from patient’s blood samples, necessitating thorough development of these reactions so that they can now be performed without any tube transfers or purifications by subsequent reagent addition and incubation. (c) The graph shows that a large portion of the uniquely mapping reads originate from MspI/MspI-fragments between 20 to 165 bp when performing cf-RRBS on cfDNA, while this is not the case when performing classical RRBS on the same cfDNA (AUC(cf-RRBS) = 90.67% vs AUC(RRBS) = 13.73%).
    Quick Gdna Midiprep Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quick gdna midiprep kit/product/Zymo Research
    Average 94 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    quick gdna midiprep kit - by Bioz Stars, 2020-10
    94/100 stars
      Buy from Supplier

    Image Search Results


    Comparative analysis of mitochondrial genome structures and annotations of Neospora and Toxoplasma reveals gene fragmentation and reshuffling between species and strains. A. The repetitive nature of the gene structure in a 32kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. B. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. C. Comparative alignment between two NcLiv mitochondrial contigs of 16 and 32kb, respectively. D . Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an NcUru1 mitochondrial contig of 38kb. E . Comparative alignment between two NcUru1 mitochondrial contigs of 16 and 38kb, respectively. F. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of NcUru1 is graphically represented in a YASS plot. G. Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an T. gondii mitochondrial contigs of 39kb.

    Journal: bioRxiv

    Article Title: Reevaluation of the Toxoplasma gondii and Neospora caninum genomes reveals misassembly, karyotype differences and chromosomal rearrangements

    doi: 10.1101/2020.05.22.111195

    Figure Lengend Snippet: Comparative analysis of mitochondrial genome structures and annotations of Neospora and Toxoplasma reveals gene fragmentation and reshuffling between species and strains. A. The repetitive nature of the gene structure in a 32kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. B. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of Nc Liverpool is graphically represented in a YASS plot. C. Comparative alignment between two NcLiv mitochondrial contigs of 16 and 32kb, respectively. D . Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an NcUru1 mitochondrial contig of 38kb. E . Comparative alignment between two NcUru1 mitochondrial contigs of 16 and 38kb, respectively. F. The repetitive nature of the gene structure in a 16kb mitochondrial DNA contig of NcUru1 is graphically represented in a YASS plot. G. Comparative alignment between an NcLiv mitochondrial contigs of 32kb and an T. gondii mitochondrial contigs of 39kb.

    Article Snippet: High Molecular Weight DNA extraction and sequencing For “Oxford Nanopore” and Illumina sequencing, DNA was extracted from N. caninum Liverpool, N. caninum Uru1 and T. gondii RH ΔKu80 strain, using a DNA purification kit from Zymo Research (#D4074).

    Techniques:

    The cf-RRBS method is optimized to obtain a sequencing library enriched in MspI/MspI – fragments when starting from highly fragmented circulating cell-free DNA (cfDNA). (a) Capillary gel electropherograms of white blood cell (WBC) genomic DNA (gDNA) and cfDNA, either undigested or MspI-digested. The undigested WBC gDNA runs above the upper marker (UM) and is not observed on the gel image. After MspI digestion, a smear of shorter DNA appears. Also three characteristic satellite DNA bands can be observed resulting from the digestion of high-abundant DNA repeats in the genome. The undigested cfDNA sample shows the expected apoptotic DNA pattern of a multiple of nucleosome repeats. After MspI digestion, especially the DNA fragment of three nucleosome repeats is not observed anymore. In addition, a smear of shorter DNA fragments is observed beneath the one nucleosome repeat. In classic RRBS, the MspI/MspI-fragments ranging from 30 to 160 bp (indicated with a red rectangle) would be extracted from gel. Starting from cfDNA, extracting these fragments without co-extracting input cfDNA is impossible as these completely overlap in size range. (b) In the cf-RRBS workflow the cfDNA sample is first dephosphorylated prior to MspI digestion. Only MspI/MspI-fragments yield unnicked DNA molecules without free ends upon dA-tailing and ligation of hairpin-shaped adapters. Those ‘circular’ molecules are resistant to exonuclease digestion, whereas all other DNA is degraded using a combination of exonucleases. All these steps are performed on the nanogram quantities of cfDNA typically obtainable from patient’s blood samples, necessitating thorough development of these reactions so that they can now be performed without any tube transfers or purifications by subsequent reagent addition and incubation. (c) The graph shows that a large portion of the uniquely mapping reads originate from MspI/MspI-fragments between 20 to 165 bp when performing cf-RRBS on cfDNA, while this is not the case when performing classical RRBS on the same cfDNA (AUC(cf-RRBS) = 90.67% vs AUC(RRBS) = 13.73%).

    Journal: bioRxiv

    Article Title: A versatile method for circulating cell-free DNA methylome profiling by reduced representation bisulfite sequencing

    doi: 10.1101/663195

    Figure Lengend Snippet: The cf-RRBS method is optimized to obtain a sequencing library enriched in MspI/MspI – fragments when starting from highly fragmented circulating cell-free DNA (cfDNA). (a) Capillary gel electropherograms of white blood cell (WBC) genomic DNA (gDNA) and cfDNA, either undigested or MspI-digested. The undigested WBC gDNA runs above the upper marker (UM) and is not observed on the gel image. After MspI digestion, a smear of shorter DNA appears. Also three characteristic satellite DNA bands can be observed resulting from the digestion of high-abundant DNA repeats in the genome. The undigested cfDNA sample shows the expected apoptotic DNA pattern of a multiple of nucleosome repeats. After MspI digestion, especially the DNA fragment of three nucleosome repeats is not observed anymore. In addition, a smear of shorter DNA fragments is observed beneath the one nucleosome repeat. In classic RRBS, the MspI/MspI-fragments ranging from 30 to 160 bp (indicated with a red rectangle) would be extracted from gel. Starting from cfDNA, extracting these fragments without co-extracting input cfDNA is impossible as these completely overlap in size range. (b) In the cf-RRBS workflow the cfDNA sample is first dephosphorylated prior to MspI digestion. Only MspI/MspI-fragments yield unnicked DNA molecules without free ends upon dA-tailing and ligation of hairpin-shaped adapters. Those ‘circular’ molecules are resistant to exonuclease digestion, whereas all other DNA is degraded using a combination of exonucleases. All these steps are performed on the nanogram quantities of cfDNA typically obtainable from patient’s blood samples, necessitating thorough development of these reactions so that they can now be performed without any tube transfers or purifications by subsequent reagent addition and incubation. (c) The graph shows that a large portion of the uniquely mapping reads originate from MspI/MspI-fragments between 20 to 165 bp when performing cf-RRBS on cfDNA, while this is not the case when performing classical RRBS on the same cfDNA (AUC(cf-RRBS) = 90.67% vs AUC(RRBS) = 13.73%).

    Article Snippet: Circulating cell-free DNA was extracted from 5 mL plasma using the Quick-cfDNA™ Serum & Plasma Kit (ZymoResearch, Cat. No. D4076) following the manufacturer’s instructions.

    Techniques: Sequencing, Marker, Ligation, Incubation