1. Örnek Toplama <ol><li> Uygun bilgilendirilmiş onam bireylerin kan örnekleri toplayın. Her birey için, bir P100 tüp (BD Diagnostics, Franklin Lake, NY, ABD) içine kan 4 ila 5 ml çizin. Karşılaştırma amacıyla, aynı anda bir EDTA tüp kan toplamak. </li><li> BD P100 tüpler 15.8 mi püskürtülerek kurutulmuş K2EDTA ve pıhtılaşmayı önlemek ve plazma proteinleri stabilize proteaz inhibitörleri arasında, bir liyofilize edilmiş özel bir geniş spektrumlu bir kokteyl içerir. Bunlar, aynı zamanda, mekanik bir ayırıcı içerir. Işleme oluşana kadar tam kan toplanmasından sonra, bir gece için 60 dakika için oda sıcaklığında iki tüp tutun. </li><li> Oda sıcaklığında on beş dakika boyunca 2.500 g BD P100 tüpler santrifüj. Mikro santrifüj tüplerine mekanik ayırıcı üzerinde toplanır Plazma aktarın. </li><li> -80, Ayırıcı altında sıkıştırılmış kan içeren, P100 tüpler saklayın ° C DNA dahili başlamak için hazır olana kadarraction. </li></ol> P100 tüp kan (P100_DNA) itibaren 2.1 <ol><li> P100 tüpler -80 saklanır ° C plazma çekimi sonrası. 3-4 ay içinde, 5 dak (Şekil 1A) için bir su banyosu içinde 37 ° &#39;de dondurucu ve çözülme sıkıştırılmış kanı içeren P100 tüpü alır. Ayırıcı oturur kalan plazma çıkarın. Ayırıcı yukarıdaki P100 tüpleri (Şekil 1B) -% 17 sakaroz çözeltisini (yayınlanmamış veri evde test degrade çözelti) 2 ml ekleyin. 2500 g, 20 dakika boyunca oda sıcaklığında Santrifüj tüpleri, bu süreç tüpün üstüne mekanik ayırıcı iter ve buffy coat (Şekil 1C) gibi çözelti üç tabaka elde edilir. El ile sterilize kanca metal ataç (Şekil 1D) kullanarak ayırıcı ayıklayın. </li><li> Her bir tüp (Şekil 1D) gelen mekanik ayırıcı aldıktan sonra çıkarın ve Discard En iyi sukroz tabaka. Steril bir 15 ml konik bir tüp içine buffy coat (BC) katmanı temsil eden orta tabaka, aktarın. 3 ml buffy coat hacmi artırmak için fosfat tampon tuzu (PBS) ekleyin. Üreticinin tavsiye başı olarak Qiagen Puregene Kan Seti reaktifler kullanarak buffy coat DNA ayıklayın, 2.500 g santrifüj hızı (yerine 2.000 g) hariç. </li><li> Kırmızı kan hücreleri (RBC) lyse ve kaldırmak için, buffy coat 3 ml RBC parçalama 9 ml ekleyin. Her bir tüp birkaç kez ters çevirin ve inkübasyon esnasında zaman zaman Çeviren ile 10 dakika boyunca oda sıcaklığında inkübe edin. 5 dakika boyunca 2.500 g her karışım aşağı Spin ve süpernatant atın. 15 saniye için hücre parçalama çözeltisi ve vorteks 3 ml Her bir tüp içinde kalan topak Treat. 1 proteini çökelme çözeltisi ilave edildi ve 15 saniye boyunca girdap ile lizat tedavi. </li><li> 5 dakika boyunca 2500 g&#39;de santrifüj ihtiva eden bir taze 15 ml konik bir tüp içine süpernatantı transfer% 100 izopropanol, 3 ml. 3 dakika 2500 g yeni tüpler 10 kez ve santrifüj ters. Süpernatant atılır ve% 70 etanol içinde 1 ml. Konik tüpler DNA pelet çıkarmak ve yıkamak için birkaç kez ters. 1 dakika 2.500 g santrifüj. Pelet, oda sıcaklığında 3 dakika (ters tüp yerleştirmek), kuru ve daha sonra 10 dakika karıştırıldı ve bir ön-etiketli 1.7 mL mikrotüp transfer etmek için 37 ° C&#39;de rehidrasyon solüsyonu, 250 ml ile DNA askıya izin verin. -80 Tüpler Mağaza ° C kullanana kadar. </li></ol> EDTA Ttube Kan (EDTA_DNA) itibaren 2.2 <ol><li> Immunohematology rutin test için tam kan K2 EDTA, 10.8 mg püskürtülerek kaplanır ve -80 ° C de saklanır vaccutainer plastik tüplerde toplanır Tüpler bir mekanik ayrıştırma içermediklerinden, DNA ekstraksiyon mekanik ayırıcı (2.1.1 ve 2.1.2), ilgili adımları içermez. </li><li> Kan depolama, DN 3 ila 4 ay içindeBir yalıçapkını Flex (Thermo Fisher Scientific, Waltham, MA, ABD) ile elde edilir. Bu manyetik parçacık teknolojisine dayanan ve hücre parçalama / DNA bağlanma, birçok yıkama aşamaları ve DNA elüsyon oluşan bir DNA ekstraksiyon otomasyon sistemidir. </li><li> DNA ekstraksiyonu kullanılan kit, Kingfisher&#39;a Flex 24 DNA&#39;nın kan kiti (Qiagen, Almanya) &#39;dir. </li></ol> 3. DNA Miktar ve Kalite Değerlendirmesi Genomik DNA miktar, kalite ve bütünlüğü PicoGreen, spektroskopi ve elektroforez içeren yönteminin bir kombinasyonu ile değerlendirildi. <ol><li> Genomik DNA&#39;nın konsantrasyonu Nanodrop ve PicoGreen ölçümü ile tespit edilir. </li><li> Saflık NanoDrop spektrofotometresi üzerinde 260/280 oranı ölçümü ile tespit edilir. </li><li> Numune (500 ng), DNA bütünlüğü (bozulma) değerlendirmek için% 1 agaroz jel üzerine yüklenir. </li></ol> 4. Genotyping Tahliller ve Kalite Kontrol <ol><li> Beş P100_DNA ve bunların karşılık gelen EDTA_DNA rastgele seçilen özel Immunochip (Immuno DNA analiz BeadChip) kullanarak, daha fazla analiz için seçilmiştir. Immunochip 196.524 polimorfizmleri içeren bir Infinium genotiplendirme çip ve immünogenetik çalışmaları 5 için tasarlanmıştır. </li><li> Her numune için, DNA 200 ng, parçalanmış güçlendirilmiş çöktürülmüş ve uygun hibridizasyon tamponu içinde tekrar süspanse edilir. </li><li> Denatüre numuneler 16 saat arasında, en az 48 ° C &#39;de Immunochips üzerinde melezlendi. </li><li> Hibritlemeden sonra, Immunochips tek-baz uzatma reaksiyonları ve lekeli için işlenir. </li><li> Immunochips sonra Illumina HiScan Boncuk dizi okuyucu ve normalize boncuk yoğunluğu veri Illumina GenomeStudio yazılım yüklenir ve genotipleri dönüştürülür kullanarak görüntülü. Genotipler arasında GenomeStudio autocalling algoritması kullanılarak adlandırılır. Içinde kalite kontrolBir arama oranı &lt;% 95 ile tek nükleotid polimorfizmlerinin dışlama (SNP) volves. </li><li> SNP arama oranı immunochip tahlilinde etkinliğinin bir ölçüsü olarak kullanılır. Bu otomatik bir genotip atama almak için yeterli sinyal yoğunluğu ve kalitesi elde çip (196.524 SNP her çip üzerinde kodlanmış) üzerindeki deneyleri toplam sayısı yüzdesi olarak hesaplanır. Yüksek kaliteli DNA (260 / 1.8 280 oranı veya daha yüksek ve bozulma olmaması) immunochip testi dahil HapMap kontrol DNA olarak en azından aynı arama oranı elde etmesi bekleniyor. Kalite kontrol bir arama oranı &lt;% 95 her bir veri seti ile SNP dışlanması içerir. </li></ol>

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Çıkar çatışması ilan etti.

Birçok insan hastalıkları genetik nedenleri ve insan hastalığın genetik temeli keşfetmek giderek artan kaliteli DNA gerektirir. Genetik çalışmalar kullanılan DNA&#39;nın en yaygın kaynağı tam kan antikoagüle edilir. En klinik laboratuvarlar fenotipik sonuç soruşturma bir ilk adım olarak, biyokimyasal serolojik ve viral test gerçekleştirmek için, kan genellikle plazma tüp toplanır. Plazma toplama sonra, sıkıştırılmış kan genellikle atılır. Bu nedenle, DNA genetik çalışmalar veya test y...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>BD P100 tubes</td>
 <td>BD Diagnostics</td>
 <td>366448</td>
 </tr>
 <tr>
 <td>EDTA tubes</td>
 <td>BD Diagnostics</td>
 <td>367863</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378</td>
 </tr>
 <tr>
 <td>Paper clip</td>
 <td>Office product</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Corning</td>
 <td>430052</td>
 </tr>
 <tr>
 <td>Red blood cells (RBC)</td>
 <td>Qiagen</td>
 <td>158389 (kit)</td>
 </tr>
 <tr>
 <td>Phosphate Buffer Saline (PBS)</td>
 <td>Thermo Scientific</td>
 <td>SH30256.01</td>
 </tr>
 <tr>
 <td>BioSprint 96 DNA Blood Kit (48) </td>
 <td>Qiagen</td>
 <td>940054</td>
 </tr>
 <tr>
 <td>1.7 ml microtubes</td>
 <td>Axygen</td>
 <td>MCT-175-C</td>
 </tr>
 <tr>
 <td>Human Immuno DNA Analysis BeadChip Kit</td>
 <td>Illumina</td>
 <td>WG-352-1001</td>
 </tr>
 <tr>
 <td>Bead array reader</td>
 <td>Illumina</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>GenomeStudio Software</td>
 <td>Illumina</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

a practical and novel method to extract genomic dna from blood collection kits for plasma protein preservation

Laboratuvar testleri, kanın hücresel veya sıvı kısımlar üzerinde yapılabilir. Farklı kan toplama tüpleri kullanımı analiz edilebilir, kan (tam kan, plazma veya serum) kısmını belirler. Insan hastalıkların genetik temeli eğitim dahil Laboratuvarları genomik / genetik analiz için DNA kaynağı olarak EDTA içeren vacutainer toplanan antikoagüle tam kan güveniyor. En klinik laboratuvarlar fenotipik sonuç soruşturma bir ilk adım olarak biyokimyasal, serolojik ve viral test gerçekleştirmek için, antikoagüle kan da heparin içeren tüp (plazma tüp) toplanır. DNA ve plazma genomik ve proteomik her iki veri aynı anda ve paralel analizler için gerekli olan bu nedenle zaman, EDTA ve heparin tüpleri hem de kan toplamak için gelenektir. Kan tek bir tüp içerisinde toplanır ve plazma DNA ve hem de bir kaynağı olarak hizmet olabilir, bu yöntem, mevcut meth bir gelişme olarak düşünülebilirods. Plazma çıkarma sonra sıkıştırılmış kan kullanımı, böylece işlenmiş kan örnekleri miktarını en aza indirmek ve her bir hastanın gerekli olan örnek sayısını azaltarak, genomik DNA için alternatif bir kaynak oluşturmaktadır. Bu sonuçta zaman ve kaynak tasarrufu olur. Plazma proteinlerine korunması için BD P100 kan toplama sistemi insan kanından daha iyi protein biyolojik keşif ve proteomik deney sağlayan, kan protein içeriği stabilize etmek, önceki plazma veya serum toplama tüpleri 1 üzerinde geliştirilmiş bir yöntem olarak yaratılmıştır. BD P100 tüpler 15.8 püskürtülerek kurutulmuş K2EDTA ml ve pıhtılaşmayı önlemek ve plazma proteinleri stabilize proteaz inhibitörleri arasında, bir liyofilize edilmiş özel bir geniş spektrumlu bir kokteyl içerir. Ayrıca, uygulanan santrifüj işlemi sonrasında serum ve hücre topakları arasında fiziksel bir engel, mekanik ayırıcı, içerir. Birkaç yöntem pıhtılaşmış kan sa DNA elde etmek için icat edilmiştirmples eski plazma tüpler 2-4 toplandı. Bu yöntemleri Zorluklar özellikle tüpler (jel ayırıcı) içinde ayırıcı türü ile ilişkili ve pıhtılaşmış kan kurtarma zorluk, parçalanan veya pıhtı dağıtma rahatsızlık, ve ayırma jel ile pıhtı çıkarma tıkanıklığı dahil edildi. Biz yeni BD P100 tüplerde alınan kan genomik DNA ayıklar ve temizler ilk yöntem mevcut. Biz P100 tüplerinden EDTA tüpleri edilene DNA örneği kalitesini karşılaştırın. Yaklaşımımız basit ve etkilidir. ) Kan toplama, 2 mekanik ayırıcı ile sakaroz bir arada kullanarak mekanik ayırıcı çıkarılması ve 1) bir plazma BD P100 (BD Diagnostics, Sparks, MD, ABD) kullanımı tüp: Aşağıdaki gibi dört ana adımdan oluşur steril ataç metal kanca, 3) beyaz hücreleri ve 4 içeren buffy coat tabakasının ayrılması) gelen genomik DNA izolasyonudüzenli bir ticari DNA ekstraksiyon kiti ya da benzer bir standart protokolü kullanarak buffy coat.

DNA kalite değerlendirme ve konsantrasyon ölçüm P100_DNAs bir verim EDTA_DNAs (Tablo 1 ve 2) göre daha düşüktü. Onun 260/280 oranı değeri temsil DNA saflık P100_DNA ve EDTA_DNA örnek seti (Tablo 1 ve 2) her ikisi için de benzer. DNA kalitesi örneğin her resim grubu bazı bozulma EDTA_DNA örneklerde görüldüğü halde, olarak (Şekil 2) ışık yayma ile gösterilen içinde oldukça düzenlidi...

Watch this Scientific Journal Video about A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation at JoVE.com

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Bu plazma / seroloji alınan tam kandan genomik DNA&#39;nın izole edilmesi için yeni bir yöntem tarif edilmektedir. Plazma toplama sonra, sıkıştırılmış kan genellikle atılır. Bizim yeni bir yöntem mevcut yöntemlere göre önemli bir gelişme gösteren ve ek kan almadan, DNA ve tek bir koleksiyon edinilebilir plazma yapar.

Plazma Protein Koruma Kan Alma Kitleri Genomik DNA Ayıklama Pratik ve Yeni Bir Yöntem

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

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41.9K Görüntüleme.  Emory University School of Medicine and Children's Health Care of Atlanta. Bu plazma / seroloji alınan tam kandan genomik DNA'nın izole edilmesi için yeni bir yöntem tarif edilmektedir. Plazma toplama sonra, sıkıştırılmış kan genellikle atılır. Bizim yeni bir yöntem mevcut yöntemlere göre önemli bir gelişme gösteren ve ek kan almadan, DNA ve tek bir koleksiyon edinilebilir plazma yapar.

Video: A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Isolation of Genomic DNA from Mouse Tails

Fare Tails Genomik DNA izolasyonu

Biyoloji

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Amerikan Horseshoe Yengeç, Limulus Polyphemus&#39;un Kan Toplama

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Topraklar ve tortulları Yüksek Moleküler Ağırlık Genomik DNA çıkarımı

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Manyetik Aktif Hücre Ayırma (LIMACS) kullanarak Lipid veziküllü aracılı Afinite Kromatografisi: Protein-lipit Etkileşim Analiz Roman Yöntemi

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

MR Genomik - Sıra Desenler Genomik DNA içinde incelenmesi için Kamu Kaynak

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Genomik DNA 5-hydroxymethylcytosine seçici Yakalama

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Nonexperts için Phylogenetics İçin Pratik Kılavuz

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Kinaz İnhibitörleri Karşı Dayanıklı Mutasyonlarının Tarama ve Doğrulama için Bir Yöntem

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Yetişkin Zebra balığı Kan Testleri için Tekrarlanan Kan Alma

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviral Entegrasyon Siteleri Amplifikasyon, Yeni Nesil Sıralama ve Genomik DNA Haritalama

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...