Yenilik, British Columbia Bilgi Kalkınma Fonu, Genom British Columbia ve British Columbia Orman Bakanlığı ve Range Kanada Vakfı orman toprak verimliliğini devam eden çalışmaları desteklemek için teşekkür etmek istiyorum. SL Mikrobiyal Çeşitlilik ve Evrim TULA vakıf tarafından finanse edilen Merkezi bir burs ile desteklenmiştir.

Bölüm I: DNA çıkarımı Prosedür 1. 8 saat 6 örnekleri DNA ölçümü hariç işlenmesi için gereklidir. Çıkarma başlamadan önce, ön-chill harç, havaneli ve -20 ° C derin dondurucu içinde spatula veya sıvı azot kullanılarak gerekecektir. Ayrıca, 20 ml kloroform izoamil alkol (24:1) buz üzerinde içeren ön-chill 50 ml tüpler. 65 hibridizasyon fırın ayarlayın ° C ön ısıtma için. ° C kristalleri eritmek için 60 sıcak kristalize ise, CTAB çözüm kontrol edin. Denatüre çözüm ve çıkarma tampon çıkarımı (tarifi aşağıda görebilirsiniz) başlamadan hemen önce son maddeler ekleyerek tamamlayın. Davlumbaz bütün çıkarma işlemleri yürütmek. 2. Denatüre tampon ve tampon ekstraksiyon hazırlayın. Not: DNA verimi en üst düzeye çıkarmak için, kesin talimatları izleyerek tamponlar yapmak için kritik önem taşımaktadır. 3. 2 g toprak önceden soğutulmuş harç yerleştirin. Not: Toprak alanında toplanan taşımacılığı için kuru buz dondurulmuş ve -80 ° C&#39;de saklanan olmalıdır Çözülme ve DNA ekstraksiyonu öncesinde 2 mm elek elek toprak. 10 g. kadar toprak miktarını artırabilir 4. Toprak çözüm denatüre 1 ml ekleyin. 5. Dondurma ve toprak parçacıkları toz ve homojen bir görünüm kadar bir havaneli kullanarak 2 sıvı N toprak öğütmek . Dondurma ve taşlama iki kez tekrarlayın. Not: sırasında donmuş toprak numunesi tutun, taşlama toprak örneği tam olarak karşılamak için yeterli sıvı azot kullanarak. Taşlama sırasında hafif bir erime kabul edilir. 6. 50 ml konik tüp önceden soğutulmuş bir spatula kullanarak bir zemin toprak aktarın. Not: zemin toprak örneği bu noktada -80 ° C, gerekirse saklanabilir. 7. Ekstraksiyon tamponu 9 ml ekleyin. Çözüm ve toprak, vorteks kısa bir süre düşük hızda karıştırın. Not: tampon homojen olduğundan emin olmak için, 60 yaşında sıcak ° C Kullanmadan önce 10-15 dakika. Ekstraksiyon tampon geri kalanı boyunca 60 ° C&#39;de tutun. Vorteks hızı 8 (10 en fazla bir ölçekte). 8. Hibridizasyon fırında 65 ° C&#39;de 40 dakika inkübe edin. İnkübasyon sırasında, sürekli düşük hızda tüpler döndürmek, ya da hafifçe tüpleri her 10 dakikada bir ters. Not: tampon büyük bir yüzey alanı üzerine toprak temas etmesine izin hibridizasyon fırında tüp yatay yerleştirin. Çözüm inkübasyon sırasında biraz ağdalı görünebilir. 9 - 14 ° C - 10 10 dakika için 1800 x g santrifüjleyin 20 ml kloroform-izoamil alkol (24:1) içeren önceden soğutulmuş 50 ml konik tüp içine supernatant Transferi, buz üzerinde tutun. Not: supernatant aktarırken, organik katmanı (alt tabaka) rahatsız etmemek için dikkatli olun. Sadece temiz süpernatant toplamak için gerekli ise 1-2 ml süpernatant bırakın. Sık sık bej süpernatantı üstünde bir tabaka (kalınlığı yaklaşık 1-3 mm) görebilirsiniz. Süpernatantı toplarken bu katman Rahatsız etmeyin. Bu bej tabakasının kırılması önlemek için tüp duvar boyunca ucu slayt deneyin. Süpernatantı çok bulanık ise, süpernatantı kloroform izoamil alkol çıkarma, bir kez daha tekrarlayın. Kloroform aktarırken kloroform plastik eriyecek gibi, tek kullanımlık plastik pipetler etmeyin. Bir cam pipet kullanın veya cam bir silindir mezun, ya da 50 ml konik tüpler içine doğrudan kloroform dökün. 10 Toprak pelet 2 kez daha ayıkla; 10.1. 5 ml ekstraksiyon tamponu toprak pelet ekleyin, hafifçe, düşük hızda kısa karıştırın 1 ml ucu ile karıştırın. Not: Shake ve tampon kullanmadan önce karıştırın. 10.2. 65 ° C hibridizasyon fırında 10 dakika boyunca inkübe. 14 ° C - 10 10 dakika için 1800 x g santrifüjleyin Adım 9 tüp supernatant aktarın. Tüp toplanan süpernatant ve kloroform-izoamil alkol ile ekstraksiyon sırasında buz üzerinde tutun. Not: süpernatantı örnekleri arasında çapraz karıştırma olmadan aynı tüp eklemek emin olun. 10.3. 9.1 ve 9.2 bir kez daha tekrarlayın. 11. Dönen bir plaka kullanarak, toplanan supernatant (14-19 ml) ve 10 dakika için 1,25 rpm&#39;de kloroform-izoamil alkol içeren tüp çok hafifçe sallayın. Not: kaçağını önlemek için çok sıkı kapağını kapatın. Herhangi bir sızıntı kolaylıkla tespit edilebilir böylece tüp yerleştirmeden önce sallanan plaka üzerinde bir kağıt havlu yatıyordu. 12. 14 ° C - 10, 20 dakika boyunca 1800 x g santrifüjleyin 13. Sulu faz yeni bir tüpe aktarın. Not: sulu faz (üst katman) ve organik katmanı (alt arasında interfaz Rahatsız etmeyintabakası) süpernatant aktarmak. Sadece temiz süpernatant tahsilini sağlamak için yaklaşık 1.5-2 ml supernatant bırakın. 14. Izopropil alkol (21.a adım 15.a) kullanarak DNA kurtarın. Alternatif olarak, Amicon ® Ultra-15 Santrifüj Filtre Aygıtları (adım 15.b 21.b) kurtarmak için zor izopropil alkol yağış zor görünür pelet üretmek örnek biyokütle çok düşük bir miktar bekliyoruz. 15.a. Yeni bir tüp ultrasantrifüjdeki süpernatantı yerleştirin. 16.a. 0.6:1 bir hacim oranı toplanan süpernatant izopropil alkol ekleyin. Tüpler haricindekileri hafifçe bir kaç kez karıştırın. 17.a oda sıcaklığında 30 dakika inkübe edin. Madde 18.A. &#39; 25 ° C - 20 dk 20 16000 x g santrifüjleyin Tüm tüplerin ağırlıkları santrifüj adım önce iyi dengelenmiş olduğundan emin olun. Not: Mark tüp merkezkaç kuvveti yönü, bu nedenle DNA verimi düşük olduğunda daha kolay DNA pelet algılayabilir. 19.A. Şişeden veya vakum aspirasyon izopropil alkol çıkarın. DNA pelet rahatsız etmemek için dikkatli olun. Not: en kısa sürede santrifüj tamamlanır tamamlanmaz izopropil alkol çıkarın. DNA pelet kaybetmemek için çok dikkatli olun. DNA pelet boyutu zor görünür, açık bir şekilde görünür (~ 9 mm çapında), toprak tipine bağlı olarak büyük ölçüde değişebilir. DNA pelet dışarı emme dikkatle tüp duvar boyunca aspirasyon izopropil alkol artıklarını kaldırmak için tavsiye edilir. Madde 20.A. &#39; 5 oda sıcaklığında kuru DNA pelet - 20 dk. Not: kuru DNA pelet üzerine etmeyin, aksi takdirde DNA çözülür için zor olacak. 21.a. 200 ul TE DNA pelletini tekrar. Hafifçe dokunarak karıştırın. DNA solüsyonu 1.5 ml tüp içine aktarın. 4 tüp tutun ° C DNA tamamen çözünmesi için geceleme. Not: pelet boyutuna bağlı olarak, TE eklemek için ses düzeyini ayarlayabilirsiniz. Genellikle 200 - 400 ul TE iyi çalışır. * Alternatif adımları 15.b takip 21.b. 15.B. Amicon ® Ultra-15 Santrifüj Filtre Cihazları Ön yıkama 10 dk, 3500 x g santrifüj cihazı ve 15 ml TE ekleyin. Flow-through atın. 16.B. DNA çözümü adım 13 ila önceden yıkanmış Amicon filtre yerleştirin. DNA hacmi 200 azalır kadar 3500 x g&#39;de santrifüjleyin - 500 ul. Flow-through atın. 17.B. TE ile DNA iki kez yıkayın; 10 ml TE Amicon filtre cihazları ekleyin. DNA hacmi yaklaşık 200 ul azalır kadar 3500 x g&#39;de santrifüjleyin. Flow-through atın. Bir kez daha tekrarlayın. 18.B. DNA çözümü için filtre üzerinde yeni bir 1.5 ml tüp aktarın. 19.B. 40 ul TE Amicon filtre ekleyin. Filtre üzerinde herhangi bir DNA kalıntı kurtarmak için yukarı ve aşağı pipetleme filtre membranlar iki tarafında yıkayın. Adım 18.B. tüp yıkama solüsyonu 20.B. Gerekirse DNA Microcon ® Ultracel YM-30 Santrifüj Filtre Ünitesi ile daha da yoğun olabilir; 7 dakika 10.000 x g 200 ul TE ve santrifüj ekleyerek filtre ön yıkama. 1 dakika için 5000 x g filtre ve santrifüj DNA çözüm ekleyin. DNA çözüm filtre miktarı yaklaşık 50 ul düşürüldü kadar santrifüj tekrarlayın. Not: Maksimum Microcon filtre için ilk örnek hacmi 500 ul. 14000 x g. üzerindeki hızlarda santrifüj etmeyin Membran tamamen kuruyana kadar santrifüj bitmedi. Bazı sıvı filtre santrifüj sonra hala kapladığından emin olunuz. Üzerinde santrifüj ve membran kuru ise, daha sonra, 15 ul TE eklemek 30 saniye hafifçe ajitasyon ve 21.b. adıma geçin 21.b. DNA kurtarmak için 3 dakika xg 1000 yeni bir Microcon tüp ve santrifüj filtre ünitesi başaşağı yerleştirin. 22. TAE jel DNA (% 0.8, 0.5xTAE, 4 saat 50 V) ile sayısal olarak ve Nanodrop spektrofotometre veya Pico-yeşil tahlil ve plaka okuyucu. Not: DNA miktar jel üzerinde bilinen konsantrasyon (Yüksek moleküler DNA marker veya λHindIII) marker bant için örnek bant karşılaştırarak tahmin edilebilir. 23. (Daha fazla bilgi için Adım II, part2% 1, 1xTAE jel Pulsed Field Jel Elektroforezi (PFGE) kullanarak yüksek molekül ağırlıklı DNA bütünlüğünü denetleyin <a href="https://www.jove.com/index/details.stp?id=1387">https://www.jove.com/index/details.stp?id = 1387</a> , DOI: Taupp ark 10.3791/1387 2009). Not: yerine düşük erime agaroz bu QC amaçla düzenli agaroz jel kullanabilirsiniz. Kısaca, elektroforez koşulları aşağıdaki gibi: 2-250 kb, 6 volt, ilk geçiş süresi: 5 sn, son bir geçiş süresi: 15 sn, 14 yaşında 12 saat çalıştırılan ° C SYBR Altın jel üzerinde DNA görselleştirmek için bir saat boyama. 24. Çıkarılan DNA molekül ağırlıkları tatmin edici, daha sonra aşağıdaki CSCL degrade santrifüj adıma geçin. Alternatif olarak, DNA -20 ° C veya -80 saklanabilir ° C CSCL santrifüj önce. DNA medyan boyutu büyük bir ekleme metagenomic DNA kütüphanesini inşa etmek istiyorsanız, eşit veya 36 Kb aşması gerekir. Bölüm II. Sezyum klorür degrade Ultrasantrifügasyon kullanarak DNA saflaştırılması Santrifüj hazırlanması: 1,5 - 2 saat Santrifüj adım: 18 saat Ethidium bromür (EtBr) Çıkarma ve santrifüj sonra DNA konsantre: 3 saat Prosedür Ayrıntılı bilgi için, Wright ve ark bakın. 2009 <a href="https://www.jove.com/index/details.stp?id=1352">https://www.jove.com/index/details.stp?id=1352</a> , DOI: 10.3791/1352 Temsilcisi Sonuçlar / Sonuç Toplam tutarı 2 orman toprağı organik g (Ç ufuk) ve mineral ufuklar (Ae, AB ve Bt ufuklar) (Uzun Vadeli Toprak Verimlilik (LTSP) Skulow göl, BC, Kanada konumda bulunan numune izole edilen genomik DNA M.Ö. Orman ve Range Bakanlığı tarafından yönetilen) 10 mg ile 180 mg arasında değişmektedir. - Izole edilmiş DNA ortalama büyüklüğü 40 ila 60 kb (Şekil 1) genellikle edildi. 260 nm/280 nm&#39;de absorbans oranı 1,3-1,6 arasında ve genellikle toprak örneklerinde gözlenen hümik asit kirlenme gösterebilir 230 nm dalga boyunda bir tepe vardı. CSCL gradient santrifüj bu kirlenme önemli ölçüde azaltmış ve aynı zamanda 1.8 260/280 oranı arttı - 1.9 Şekil 2a ve 2b de gösterildiği gibi. <img src="/files/ftp_upload/1569/1569fig1.jpg" alt="Şekil 1" /> Şekil 1 CSCL Ultrasantrifügasyon önce çıkarılan genomik DNA PFGE resimler. lane1: 1 kb işaretleyici 100 ng, şerit 2: λHind III işaretleyici 100 ng, şerit 3: λHind III işaretleyici 250 ng, şerit 4: λHind III işaretleyici 500 ng, lane5: Fosmid 36 kb işaretleyici 100 ng, şerit 6 ve 7: genomik mineral toprak ufuk Ae, şerit 8 ve 9 izole edilen DNA: LTSP yerinde Skulow göl, M.Ö. bulunan mineral toprak ufuk AB&#39;dan genomik DNA izole <img src="/files/ftp_upload/1569/1569fig2tmb.jpg" alt="Şekil 2" border="0" /> Genomik DNA Şekil 2 Chromograms önce spektrofotometre tarafından tespit edilen (A) ve (B) sonra CSCL Ultrasantrifügasyon. Genomik DNA kalitesinde düzelme gösterir CSCL Ultrasantrifügasyon sonra 230 nm dalga boyunda absorbans değeri azalma unutmayın. Lütfen Şekil 2&#39;de büyük halini görmek için <a href="http://jove.com/files/ftp_upload/1569/1569fig2.jpg">buraya tıklayın</a> .

<ol>
	<li>Hurt, R. A., Qiu, X., Wu, L., Roh, Y., Palumbo, A. V., Tiedje, J. M., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recovery+of+RNA+and+DNA+from+soils+and+sediments.">Simultaneous recovery of RNA and DNA from soils and sediments.</a> AEM. 67 (10), 4495-4503 (2001).</li><li>Wright, J. J., Lee, S., Zaikova, E., Walsh, D. A., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+extraction+from+0.22+&#956;M+Sterivex+filters+and+cesium+chloride+density+gradient+centrifugation.">DNA extraction from 0.22 &#956;M Sterivex filters and cesium chloride density gradient centrifugation.</a> JoVE. , (2009).</li><li>Taupp, M., Lee, S., Hawley, A., Yang, J., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+insert+environmental+genomic+library+production.">Large insert environmental genomic library production.</a> J Vis Exp. , (2009).</li></ol>

Reçete * Çözüm denatüre 4 M Guanidin izotiyosiyanat 10 mM Tris-HCl (pH 7.0) 1 mM EDTA 0.5% 2-mercaptoethanol <table border="0" style="font-size:9pt; width:400px;"><tr><td colspan="2"> Denatüre çözüm: toplam 10 ml </td></tr><tr><td> Guanidin izotiyosiyanat (MW 118,16) </td><td> 4.73 g </td></tr><tr><td> 1M Tris-HCl (pH 7.0) </td><td> 100 ul </td></tr><tr><td> 0,5 M EDTA </td><td> 20 ul </td></tr><tr><td colspan="2"> Toplam 9.95 ml su ekleyin. </td></tr><tr><td colspan="2"> Otoklav. </td></tr><tr><td colspan="2"> Kullanmadan önce 50 ul 2-mercaptoethanol ekleyin. (1 ml denatüre Çözüm başına 2-mercaptoethanol 5 ul) Not: 4 denatüre çözüm tutun ° C Bir hafta daha büyük bir tampon kullanmayın. Mümkünse, taze tampon kullanımı yapmak. </td></tr></table> ** Ekstraksiyon Tampon 100 mM sodyum fosfat tamponu pH 7,0] 100 mM Tris-HCl pH 7,0] 100 mM EDTA pH 8.0] 1.5 M NaCl % 1 hekzadesiltrimetilamonyum bromür (STAB) % 2 SDS <table border="0" style="font-size:9pt; width:400px;"><tr><td colspan="2"> Toplam 1 L Ekstraksiyon Tampon </td></tr><tr><td> 1M sodyum fosfat tamponu pH 7.0] * </td><td> 100 ml. </td></tr><tr><td> 1M Tris-HCl pH 7,0] </td><td> 100 ml. </td></tr><tr><td> 0,5 M EDTA pH 8,0] </td><td> 200 ml </td></tr><tr><td> 5 M NaCl </td><td> 300 ml </td></tr><tr><td colspan="2"> Otoklav ve oda sıcaklığında saklayın. </td></tr><tr><td colspan="2"> 200ml,% 5 hekzadesiltrimetilamonyum bromür (STAB, otoklava) ve 100 ml,% 20 SDS (otoklava) kullanmadan hemen önce ekleyin. CTAB kristalize ise, 60 eritebilir ° C * 1M sodyum fosfat tamponu: 57.7 ml 1M sodyum fosfat tekbazlı (NaH 2 PO 4) ve 42.3 ml, 1M sodyum fosfat dibazik (NaH 2 PO 4). PH 7.0 &#39;a ayarlayın Not: 20% SDS yağış varsa kullanmayın. Çözüm için SDS eklediğinizde sütlü süspansiyon görmek normaldir. Bir kere, SDS eklemek ekstraksiyon tamponu yere 60 ° C SDS iyi askıya sağlamak için. </td></tr></table>

extraction of high molecular weight genomic dna from soils and sediments

Toprak mikrobiyomları, karasal ekosistemler içinde besin ve enerji akışı ile yakından ilişkili genomik çeşitlilik ve metabolik yenilik ve nispeten keşfedilmemiş geniş bir rezervuar. Metagenomic olarak da bilinen Yetiştiriciliği bağımsız çevre genomik yaklaşımlar, bu genetik bilginin değeri yüksek tedavi ve biyokütle dönüşüm süreçleri için yolağı yeniden yapılanma ve fonksiyonel taraması ile ilgili benzeri görülmemiş bir erişim söz veriyorum. Ancak, toprak mikrobiyomları, hala büyük ekleme kütüphane üretimi için yeterli kalitede yüksek molekül ağırlıklı DNA temininde zorluk büyük ölçüde bir sorun olmaya devam etmektedir. Burada toprak ve tortular yüksek molekül ağırlıklı, mikrobiyal topluluk genomik DNA ayıklamak için bir protokol tanıtmak. Izole genomik DNA kalitesi downstream sıralama ve tarama uygulamaları için büyük ekleme çevre genomik kütüphaneler oluşturmak için idealdir. Bu prosedür, hücre parçalama ile başlar. Mikropların hücre duvarlarını ve membranlar (β-mercaptoethanol) hem mekanik (taşlama) ve kimyasal güçleri tarafından parçalanmış. Genomik DNA ekstraksiyon tamponu, kloroform-izoamil alkol ve izopropil alkol kullanarak izole edilmiştir. Parçalama ve çıkarma adımları istihdam tamponlar guanidin izotiyosiyanat ve yüksek molekül ağırlıklı genomik DNA bütünlüğünü korumak için hekzadesiltrimetilamonyum bromür (STAB) içerir. Alt uygulanmasına ilişkin olarak, izole genomik DNA sezyum klorür (CSCL) hümik asitler de dahil olmak üzere kirleri azaltır degrade Ultrasantrifügasyon kullanarak saflaştırılmış olabilir. Ilk yordamı, ekstraksiyon, DNA sayısallaması adım hariç, yaklaşık 8 saat sürer. CSCL degrade Ultrasantrifügasyon, iki gün bir süreçtir. Bütün bu prosedür sırasında, genomik DNA kesme önlemek için nazikçe tedavi edilmelidir, ciddi vorteks önlemek ve tekrarlayan ağır pipetleme.

Watch this Scientific Journal Video about Extraction of High Molecular Weight Genomic DNA from Soils and Sediments at JoVE.com

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

Mikrobiyal topluluk yüksek molekül ağırlıklı ve yüksek kalitede genomik DNA izole etmek için bir metodoloji tarif edilir.

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 ...

extraction-high-molecular-weight-genomic-dna-from-soils

Research

JoVE Journal

Biology

25.5K Görüntüleme.  University of British Columbia - UBC. Mikrobiyal topluluk yüksek molekül ağırlıklı ve yüksek kalitede genomik DNA izole etmek için bir metodoloji tarif edilir.

Video: Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

Isolation of Genomic DNA from Mouse Tails

Fare Tails Genomik DNA izolasyonu

Biyoloji

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

0.22 mcM Sterivex Filtreler ve Sezyum Klorür Yoğunluk Gradient Santrifüj DNA Ekstraksiyon

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

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 ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Genetik ve epigenetik Analizleri Parafin Gömülü Malzeme DNA Ekstraksiyon

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Mikrobiyal Paspas Yüksek Moleküler Ağırlıklı DNA çıkarımı

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

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

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 blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

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 ...

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 ...

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 ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Musin Agaroz jel elektroforezi: Yüksek molekül ağırlıklı glikoproteinler için Western Blot


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Yüksek İşlemli DNA Çıkarma Plakalarının Yüklenmesinde Modifiye Yöntemler Kontaminasyon Potansiyelini Azaltır

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...