Bu araştırma için genel tıbbi bilim (NIGMS) (5605100122001), bir bileşeni Nebraska Üniversitesi Kearney (UNK) yaz öğrenci araştırma programı (SSRP) ve UNK yanı sıra ulusal kurumları Sağlık (NIH) Ulusal Enstitüsü tarafından finanse edildi Lisans araştırma bursu (URF).

1. hazırlanması boyalar, tamponlar ve özel jel
<ol><li>
Aşağıdaki çözümleri DNA veya lekeli DNA hazım boyama için hazırlayın.
	<ol>
<li>1 x TE (Tris-HCl ve ethylenediaminetetraacetic asit; hazırlamak EDTA) arabelleği 10 mM Tris-HCl ve 1 mM EDTA mezun silindir veya volumetric flask kullanarak. (18-25 ° C) oda sıcaklığında saklayın.</li>
		<li>Her boya aliquots olun: TOTO-1, YOYO-1, POPO-1, BOBO-1, TOTO-3, YOYO-3, POPO-3, BOBO-3 (tablo 1). 1 mM boya koyu amber içine 4 µL pipet veya 1,5 mL tüp siyah ve 1 x TE 36 µL eklemek, pipet kullanarak karıştırın; Bu konsantrasyon 100 µM boya çözüm (40 µL toplam) yapar. Her boya için bu adımı tamamlayın. 4 ° C'de mağaza Not: photobleaching veya boya bozulma önlemek için ışık boya kullanılmasını önlemek.</li>
		<li>Arabellek 1 10 mM Bis-Tris-propan-HCl, 10 mM MgCl2ve 100 µg/mL sığır serum albumin (BSA) kullanarak hazırlayın. Arabellek 2-50 mM NaCl, kullanımı hazırlamak 10 mM Tris-HCl, 10 mM MgCl2ve 100 µg/mL BSA. Tampon 3 50 mM potasyum asetat, 20 mM Tris-asetat, 10 mM magnezyum asetat ve 100 µg/mL BSA kullanarak hazırlayın. Her enzim çalışması için belirli bu arabellekleri birini gerektirir.</li>
	</ol>
</li>
	<li>
Aşağıdaki çözümleri Jel Elektroforez için hazırlayın.
	<ol>
<li>6 yükleme x boya %0.25 bromophenol birleştirerek mavi, olun % 0.25 Ksilen cyanol, % 15 Ficoll dH2O. mağaza oda sıcaklığında.</li>
		<li>Jel, dışarı yüksek jelleşme sıcaklık (HGT) özel bir Erlenmeyer flask 0,07 g tartmak % 0.7 özel yapmak için 1 x TAE arabellek 100 mL ekleyin ve ters bir kabı balonun üstüne yerleştirin.<ol>
<li>Çözüm sıcak tabakta ısı kadar şişesi ve üst için kayan nokta alt formda kabarcıklar başlayacak.</li>
			<li>Çözüm sıcak plaka kaldırmak için balonun ile çıplak bir el dokundu ve jel kuyuları oluşturmak için jel tarak ile 24.5 cm x 21,8 cm jel kalıp içine dökün kadar çözüm soğumaya bırakın.</li>
			<li>Pipet ucu ile haşhaş tarafından baloncuklar tarak ya da jel yüzeyi kaldırmak ve en az 15 dakika boyunca katılaşmaya jel izin. Bu jel veya kirlenme kurutma önlemek 1 hafta için plastik wrap sarılı bir buzdolabı (4 ° C) depolanabilir. Nasıl çalıştırılacağı hakkında daha fazla bilgi için bir jel başvurmak için Lee vd. 33 . Not: 30 bireysel wells ile jel tarak kullanın. Her şey 4.5 mm 2 mm derinliğe ile geniş olmalıdır. Kuyu yüksekliğini jel kutusu dökülür, miktarına bağlı olacaktır.</li>
		</ol>
</li>
		<li>1 x TAE (Tris - asetat - EDTA) arabelleği 40 mM Tris-HCl, 20 mM Asetik asit ve 1 mM EDTA kullanarak hazırlayın. (18-25 ° C) oda sıcaklığında saklayın.</li>
	</ol>
</li>
</ol>2. lekeli DNA hazırlanması
<ol><li>
Lambda DNA (300-800 ng) boyalar farklı aliquots kullanarak leke. Her boya (tablo 1) test, restriksiyon enzimi başına 48 reaksiyonlar olmak üzere toplam altı farklı konsantrasyonlarda olacaktır. Aşağıdaki işlem için 1 tepki (şekil 1) bir küme özetliyor.
	<ol>
<li>1 x TE 100 kullanarak lambda DNA seyreltik ng / µL. mağaza 4 ° c ' Bir çözüm 300 µL hacmi ile olun.</li>
		<li>Bazlar lambda DNA leke. "Denemeler" den beklenen her grup için 75-100 ng/bant tahmin ediyoruz. Boya konsantrasyonları 1.9 µM, 3.8 µM, 19.4 µM, 32.2 µM, 48,3 µM, 63,5 µM üretmek için uygun miktarda ekleyebilirsiniz / 100 ng DNA'ın.</li>
		<li>Gecede kuluçkaya (15 h - 18 h) 4 ° C'de</li>
		<li>Denetim25hareket günahı bir tepki bırak. Not: bazlar DNA sindirim tepki için kullanın. Metilasyon duyarlı enzimler DNA metillenmiş bölgelerde kesemezsin ve eksik bir sindirim bir görünüm verecektir.</li>
	</ol>
</li>
</ol>3. restriksiyon enzimi tahlil hazırlanması
<ol><li>
Özet Bu enzim prestained DNA (şekil 1) sindirmek olabilir belirlemek için bir restriksiyon enzimi ile DNA lekeli.
	<ol>
<li>Ertesi gün 20 U restriksiyon enzimi, 3 µL uygun arabellek (Tablo 2) ve toplam 30 µL distile, autoclaved ve süzülmüş su ekleyin. Her enzim reaksiyon için arabellek üreticinin Web sitesinde bulunabilir en yüksek kesme verimliliği ile seçin.</li>
		<li>Maschmann ve arkiçinde kullanılan 6 enzimler için Tablo 2'de listelenen en iyi enzim aktivitesinin sıcaklığında su banyosu tepkiler yer., 2-4 h (Tablo 2)25.</li>
		<li>Reaksiyon 0,5 M EDTA pH 8.0 2 µL ekleyerek durdurmak.</li>
	</ol>
</li>
</ol>4. bir özel jel DNA'da lekeli Electroeluting
<ol><li>Taraf (Adım 1.2.2) 24.5 cm x 21,8 cm jel kalıptan çıkarın ve jel Jel Elektroforez kutusuna yerleştirin. 1 x TAE arabelleği 2.5 L kutusu dökün. Dikkatli bir şekilde böylece kuyu erişilebilir33jel tarak jel çıkarın.</li>
	<li>Restriksiyon enzimi tepkiler üzerine bir plastik film pipette ve her reaksiyon boya yükleme x 6 ile birleştirir. Her reaksiyon çözüm için boya başına 5-6 µL DNA çözüm (1 x) yükleme x 6 1 µL ekleyin. Öyleyse karşılıklar pipette (yükleme boya içeren < 18 µL) kuyu içine.</li>
	<li>1 kb merdivenin 1 µL, 1 x TE 9 µL ve boya yükleme x 6 2 µL karıştırın. Çözüm başka çözümler kanattan kuyu yükleyin.</li>
	<li>Koyu mavi veya siyah kağıt veya kumaş ile jel kutu örtmek. Jel kutusu 30 V güç kaynağı ayarlayın ve gecede çalıştırın bir güç kaynağı için (15-20 h) bağlayın. Jel, photocleavage etiketli DNA'ın önlemek için çalışırken ışıkları kapatın.</li>
	<li>Ertesi sabah güç kaynağını kapatmak. Tepsi kullanarak jel al ve bir konteyner ile 45 µL etidyum bromür ve 1 L 1 x TAE arabelleği aktarmak. Karanlık oda sıcaklığında veya kapak konteyner folyo ışığa maruz kalma önlemek için saklayın. Leke, oda sıcaklığında en az 45 dakika için jel izin. Dikkat: etidyum bromür kullanım ile eldiven ve koruyucu gözlük giymek. Konteynerin etidyum bromür solüsyon içeren ve jel atmadan temizlerken, kullanılan etidyum bromür ile başa çıkmak konusunda bilgi almak senin Birleşik elden çıkarma yönergelere başvurun; Ayrıca, diğer lekeleri etidyum bromür yerine yararlı.</li>
</ol>5. özel jel görüntüleme
<ol><li>Jel 400-500 nm arasında en fazla ışık verimi yayar mavi bir ışık transilluminator üstüne yerleştirin. Merkeze bağlı kamera ile fotoğraf çekmek. Dikkat: kapak ışığı için kullanın ve ışık kaynağında dönüm önce koruyucu gözlük koymak emin olun. Not: Bu adımı da herhangi bir UV ışık kaynağı veya standart jel tarayıcı kullanılarak tamamlanabilir.</li>
	<li>Dosyayı ImageJ durum çubuğuna sürükleyerek ImageJ içinde resimleri görüntüleme ve görüntü çıkacaktır.</li>
	<li>Sindirim oranı deneme için beklenen silico jel desen jöleye bands karşılaştırarak belirle. Sindirim oranı belirlemek için deneysel grup dizi DNA bantları24,25beklenen numarasına göre bölünür. Deneysel sindirilir DNA bant deseni beklenen desen varsa, sindirim oranı 1'dir. Desen beklenenden daha fazla parçaları varsa, sindirim 1'den büyük oranıdır. Desen beklenenden daha az parçaları varsa, sindirim birden oranıdır.</li>
</ol>

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Fluorescently etiketli DNA (şekil 1)sindirmek amacıyla bir dizi adım gereklidir. İlk olarak, DNA ile bir fluorochrome gecede lekeli. DNA daha kısa bir süre için siyanür dimer ile inkübe; Ancak, Carlsson vd. DNA çift kişilik gruplar her DNA boyutu nedeniyle eksik boyama20için oluşturulan bulundu. Bu sorunu gidermek için DNA çift kişilik grup oluşmasını önlemek için gece Boyanabilen. Bu protokol kritik bir adımdır. Boya i...

TOTO serisi (TOTO-1, YOYO-1, POPO-1, BOBO-1, TOTO-3, YOYO-3, POPO-3 ve BOBO-3; Tablo 1) çeşitli deneyler DNA görselleştirme gerekli1,2,3,4,5,6,7, nerede olarak kullanılmaktadır 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17. siyanür dimer aile onların kuantum verimi, ışık hassasiyeti ve DNA molekülleri18,19,20için yüksek ilgi nedeniyle yaygın olarak kullanılır. Siyanür dimer boyalar Floresans21100-1000 kat artış var büyük seçicilik çift iplikçikli DNA için ve ne zaman ara var. Pyridinium boya maddeler, araçlar (YOYO-1, TOTO-1, YOYO-3 ve TOTO-3) onların quinolium boya daha karşılıkları (Tablo 1)22(BOBO-1, POPO-1, BOBO-3 ve POPO-3) bir daha kısa emisyon dalga boyu var. Aynı zamanda, DNA'ya ara siyanür dimer kuantum verimi yüksektir (0.2 - 0,6)22. Ancak, bir enzim bir DNA molekülü2 metilasyonu profili belirleme veya23 zaten ile floresan boya lekeli bir DNA molekülünün germek için kullanmak ne enzimler lekeli DNA sindirmek belirlemek için bir metod gerekir. DNA'ya intercalates boya herhangi bir tür veya DNA substrat discernable örnek verir herhangi bir enzim için bu yöntemi kullanabilirsiniz.
Meng vd. ilk prestained DNA yoluyla farklı boya24çeşitli kullanarak Jel Elektroforez sindirim oranı belirlenir. Maschmann ve ark. delved derin boyalar TOTO aileye bakmak için. Her ikisi de belirli bir boya ile lekeli DNA restriksiyon enzimi25ile sindirmek Eğer görmek için lekeli DNA sindirim oranı belirlenir. Diğer yöntemler optik cımbız26 veya NMR27kullanarak DNA ile ara boya etkileri bağlama çalışma. Her iki yöntem özel ekipman gerektirir; ise bu yöntem çoğu moleküler biyoloji laboratuarları bir boya olup olmadığını belirlemek zorunda ekipman sağlar, restriksiyon enzimi sindirim ile etkilemektedir.
Ayrıca, belirli bir molekül uzunluğunu ölçmek için diğer yöntemleri, optik eşleme günahı DNA molekülleri bir yüzey üzerinde uzamış ve streç ve parçaları boyutunu belirlemek için DNA sindirilir. Boya enterkalasyon kontur uzunluğu fluorescently lekeli DNA moleküllerinin ve bağlı olarak kullanılan boya artırmak için gösterilmiştir, kontur uzunlukları farklı21. Bu yöntem genleri1,3,4,6,13,28,29,30, çeşitli kullanıldığında vardır 31. molekülleri, boya ve enzim bağlı olarak önceden lekeli olsaydı, ancak, enzim DNA ile belirli bir boya lekeli kesmek mümkün olmayabilir. Bu nedenle, bu yöntem ile belirli bir boya lekeli DNA bir enzim ile sindirilmiş Eğer belirler. Ayrıca, boya ve boya DNA hareketliliğini kullanıldığında, konsantrasyon bağlı olarak bir jel bantlarında daha yavaş boya baz çifti32 arasında eklemek yer açmak için yerel DNA kısmi DNA omurgası gevşemek nedeniyle göç edecek .
Ancak, bazen bu boyalar olabilir kısmen veya tamamen belirli enzimleri7,24eylem inhibe. Bu enzim, belirli sıra tanımasını engelleyebilir floresan boyalar ve eki neden olduğu DNA yapısal bir değişiklik nedeniyle düşünülmektedir. Bu boyalar enzimleri etkilemesi anlamak metilasyonu profil veya uzatma lekeli DNA'ın gerekli olduğu deneylerde yardımcı olabilir.
Bizim yöntem, DNA faiz fluorochrome ile lekeli ve restriksiyon enzimi ile sindirilir. O zaman DNA görüntüsü, bir jel üzerinde electrophoresed ve restriksiyon enzimi sindirim oranı ölçüldü. Enzimleri dayalı bir jel üzerinde kesme desen olarak seçilmiştir. Çok fazla grup örtüşme DNA gruplarından neden ve çok az bantları DNA molekülünün tam bir resim vermek değil. Profil sindirilir DNA molekülünün belirlemek için tatlı bir nokta var; Bu nedenle, kullanılan DNA ve enzim bağlı olacaktır. Bu yöntemin bir avantajı basitliğidir; Sadece bir kısıtlama sindirim ve Jel Elektroforez cihazları gerektirir.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Unmethylated lambda DNA</td>
			<td style="width:111px;">NEB</td>
			<td style="width:155px;">N3013S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">YOYO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>Y3601</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">TOTO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>T3600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">POPO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>P3580</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">BOBO-1</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>B3582</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">YOYO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>Y3606</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">TOTO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>T3604</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">POPO-3</td>
			<td style="width:111px;">ThermoFisher Scientific</td>
			<td>P3584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">BamHI</td>
			<td style="width:111px;">NEB</td>
			<td>R0136S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">PmlI</td>
			<td style="width:111px;">NEB</td>
			<td>R0532S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">ScaI</td>
			<td style="width:111px;">NEB</td>
			<td>R3122S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">EcoRI&#160;</td>
			<td style="width:111px;">NEB</td>
			<td>R0101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">HindIII</td>
			<td style="width:111px;">NEB</td>
			<td>R0104S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">SmaI</td>
			<td style="width:111px;">NEB</td>
			<td>R0141S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Tris</td>
			<td style="width:111px;">Fisher</td>
			<td>BP152-1</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">EDTA</td>
			<td style="width:111px;">Fisher</td>
			<td style="width:155px;">S316-212</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">HCl</td>
			<td style="width:111px;">Fisher</td>
			<td>S25358</td>
			<td>Corrosive and irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 1</td>
			<td style="width:111px;">NEB</td>
			<td>B7201S</td>
			<td>NEB 1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 2</td>
			<td style="width:111px;">NEB</td>
			<td>B7202S</td>
			<td>NEB 2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Buffer 3</td>
			<td style="width:111px;">NEB</td>
			<td>B7204S</td>
			<td>NEB Cutsmart</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">High gelling temperature agarose gel</td>
			<td style="width:111px;">Lonza</td>
			<td>50041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Acetic Acid</td>
			<td style="width:111px;">Fisher</td>
			<td>S25118</td>
			<td>Hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Blue light transilluminator</td>
			<td style="width:111px;">Dark Reader</td>
			<td>DR196</td>
			<td>Wear correct eyewear&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Ethidium bromide</td>
			<td style="width:111px;">Fisher</td>
			<td>15585011</td>
			<td style="width:167px;">Carcinogen; other alternatives are Nancy 520, Gel red, etc.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Cannon EOS Rebel T3I camera</td>
			<td style="width:111px;">Cannon</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apogee Model H4</td>
			<td style="width:111px;">Biorad</td>
			<td>1704510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Power Pac Basic Power Supply</td>
			<td style="width:111px;">Biorad</td>
			<td>1645050&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Ficoll</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:155px;">BP525-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Bromophenol blue</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:155px;">B-392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Xylene Cyanol</td>
			<td style="width:111px;">Eastmen</td>
			<td style="width:155px;">T1579</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining if dna stained with a cyanine dye can be digested with restriction enzymes

DNA'ın görselleştirme floresan mikroskopi için boyalar siyanür boyalar gibi çeşitli kullanır. Bu boyalar onların yüksek benzeşme ve duyarlılık nedeniyle DNA için kullanılmaktadır. DNA molekülleri deney tamamlandıktan sonra tam uzunlukta olup olmadığını belirlemek için bir yöntem enzimleri ile DNA sindirerek tarafından lekeli molekülleri tam uzunlukta olup olmadığını belirlemek için gereklidir. Bir yöntem için fluorochrome ihtiyacım ne enzimler DNA lekeli belirlemek için gerekli değildir ancak, lekeli DNA enzimleri inhibe. Bu yöntemde, DNA boya ve DNA equilibrate izin veren bir siyanür boya ile bir gecede lekeli. Sonraki, lekeli DNA restriksiyon enzimi ile sindirilmiş, bir jel yüklenen ve electrophoresed. Deneysel DNA Özet bantları restriksiyon enzimi etkinliğini belirlemek için bir silico sindirimi karşılaştırılır. Beklendiği gibi grup aynı dizi Eğer reaksiyon tamamlandıktan. Kısmi sindirim belirtmek beklenenden daha fazla grup ve daha az bantları eksik sindirim gösterir. Bu yöntemin avantajı basitliğidir ve bir bilim adamı için bir restriksiyon enzimi gerekir ekipman kullanır tahlil ve Elektroforez jel. Bu yöntemin bir sınırlama enzimler için çoğu bilim adamı mevcut piyasada bulunan enzimler vardır; Ancak, herhangi bir enzimleri kullanılabilir.

İntercalating bir boya sindirerek DNA restriksiyon enzimi etkilerse, adımları doğru sırada olmalıdır belirlemek için (şekil 1) takip. Bir kez DNA lekeli ve belirli bir enzim ile sindirilmiş, jel resmini parçaları ve büyüklükleri (Şekil 2) sayısını belirlemek için alınabilir. Enzim verimlilik, beklenen görünür bant görünür grup numarasına göre bölünmüş toplam sayısı belirlemek için. Enzim veriml...

Watch this Scientific Journal Video about Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes at JoVE.com

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

DNA molekülleri floresan mikroskopi için boyama bir deney sırasında görüntüleyebilmesi için bir bilim adamı sağlar. Burada sunulan yönteminde DNA molekülleri ile floresan boyalar önceden lekeli ve metilasyonu ve sigara metilasyonu hassas enzimleri ile sindirilir.

DNA ile bir siyanür boya olabilir olmak sindirilmiş enzimleri ile lekeli belirleme

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and ...

determining-if-dna-stained-with-cyanine-dye-can-be-digested-with

Research

JoVE Journal

Biochemistry

8.7K Görüntüleme.  University of Nebraska - Kearney. DNA molekülleri floresan mikroskopi için boyama bir deney sırasında görüntüleyebilmesi için bir bilim adamı sağlar. Burada sunulan yönteminde DNA molekülleri ile floresan boyalar önceden lekeli ve metilasyonu ve sigara metilasyonu hassas enzimleri ile sindirilir.

Video: Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Taşınabilir FRET Analyzer ile son derece hassas ve hızlı Floresan Algılama

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Biyokimya

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

Biyolojik Numune metabolik profil için Sheathless Kapiler Elektroforez-Kütle Spektrometre

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Amiloid doku Imaging ile Işıksaçan konjuge Oligothiophenes Hyperspectral tarafından Confocal mikroskobu ve floresan ömür boyu görüntüleme lekeli

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

Çift DNA cetvelleri tek Nükledotid çözünürlüğe sahip Ribosome translocation mekanizmasını Incelemek için

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

Hücre İçi Hızlı Fotokimyasal Oksidasyon ile Hücresel Proteinlerin Karakterizasyonu

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

Restriksiyon endonükleaz aracılı DNA bölünmesinin tek moleküllü bekleme süresi analizi

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Cryo-EM and Single-Particle Analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

Scipion ile Kriyo-EM ve Tek Parçacık Analizi

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA Metiltransferaz İnhibitörlerini Taramak için Sürekli Floresan Bazlı Endonükleaz Bağlantılı DNA Metilasyon Testi

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase &#946; and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

Kriyo-Elektron Mikroskobu Yapısal Tayini için DNA Onarım Faktörleri ile Komplekslenmiş Nükleozom Çekirdek Parçacıklarının Hazırlanması

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

İzotermal Titrasyon Kalorimetrisi Kullanılarak DNA Aptamer ve Tetrasiklinin Termodinamik ve Kinetik İlişkisinin Belirlenmesi

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...