Name: synthesis of wavelength-shifting dna hybridization probes by using photostable cyanine dyes
Uploaded: 2016-07-06T13:00:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes at JoVE.com

الدعم المالي من قبل جمعية الألمانية للبحوث (DFG، وا 1386 / 17-1)، والتدريب البحثي المجموعة GRK 2039 (التي تمولها DFG) وKIT ما قدمه من مساعدة.

<ol>
	<li>Kobayashi, H., Ogawa, M., Alford, R., Choyke, P. L., Urano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategies+for+fluorescent+probe+design+in+medical+diagnostic+imaging.">New strategies for fluorescent probe design in medical diagnostic imaging.</a> Chem Rev. 110 (5), 2620-2640 (2010).</li><li>Berezin, M. Y., Achilefu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Lifetime+Measurements+and+Biological+Imaging.">Fluorescence Lifetime Measurements and Biological Imaging.</a> Chem. Rev. 110 (5), 2641-2684 (2010).</li><li>Lee, J. S., Vendrell, M., Chang, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity-oriented+optical+imaging+probe+development.">Diversity-oriented optical imaging probe development.</a> Curr. Opin. Chem. Biol. 15 (6), 760-767 (2011).</li><li>Tyagi, S., Bratu, D. P., Kramer, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+molecular+beacons+for+allele+discrimination.">Multicolor molecular beacons for allele discrimination.</a> Nat. Biotechnol. 16 (1), 49-53 (1998).</li><li>Holzhauser, C., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=#34;DNA+Traffic+Lights&amp;#34;:+Concept+of+Wavelength-Shifting+DNA+Probes+and+Application+in+an+Aptasensor.">#34;DNA Traffic Lights&amp;#34;: Concept of Wavelength-Shifting DNA Probes and Application in an Aptasensor.</a> ChemBioChem. 13 (8), 1136-1138 (2012).</li><li>Holzhauser, C., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+and+RNA+&amp;#34;Traffic+Lights&amp;#34;:+Synthetic+Wavelength-Shifting+Fluorescent+Probes+Based+on+Nucleic+Acid+Base+Substitutes+for+Molecular+Imaging.">DNA and RNA &amp;#34;Traffic Lights&amp;#34;: Synthetic Wavelength-Shifting Fluorescent Probes Based on Nucleic Acid Base Substitutes for Molecular Imaging.</a> J. Org. Chem. 78 (15), 7373-7379 (2013).</li><li>Berndl, S., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Color+Readout+of+DNA+Hybridization+with+Thiazole+Orange+as+an+Artificial+DNA+Base.">Fluorescent Color Readout of DNA Hybridization with Thiazole Orange as an Artificial DNA Base.</a> Angew. Chem. Int. Ed. 48 (13), 2418-2421 (2009).</li><li>Bohl&#228;nder, P. R., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+Photostable+Energy-Transfer+Pair+for+&amp;#34;DNA+Traffic+Lights&amp;#34.">Synthesis of a Photostable Energy-Transfer Pair for &amp;#34;DNA Traffic Lights&amp;#34.</a> Eur. J. Org. Chem. 34, 7547-7551 (2014).</li><li>Walter, H. K., Bohl&#228;nder, P. R., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Wavelength-Shifting+Fluorescent+Module+for+the+Adenosine+Aptamer+Using+Photostable+Cyanine+Dyes.">Development of a Wavelength-Shifting Fluorescent Module for the Adenosine Aptamer Using Photostable Cyanine Dyes.</a> ChemistryOpen. 4 (2), 92-96 (2015).</li><li>Gierlich, J., Burley, G. A., Gramlicj, P. M. E., Hammond, D. M., Carell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry+as+a+reliable+method+for+the+high-density+postsynthetic+functionalization+of+alkyne-modified+DNA.">Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA.</a> Org. Lett. 8 (17), 3639-3642 (2006).</li><li>Matteucci, M. D., Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+deoxyoligonucleotides+on+a+polymer+support.">Synthesis of deoxyoligonucleotides on a polymer support.</a> J. Am. Chem. Soc. 103 (11), 3185-3191 (1981).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids. , 589 (1975).</li><li>Puglisi, J. D., Tinoco, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbance+melting+curves+of+RNA.">Absorbance melting curves of RNA.</a> Meth. Enzymol. 180, 304-325 (1989).</li><li>Johansson, M. K., Fidder, H., Dick, D., Cook, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramolecular+Dimers:+A+New+Strategy+to+Fluorescence+Quenching+in+Dual-Labeled+Oligonucleotide+Probes.">Intramolecular Dimers: A New Strategy to Fluorescence Quenching in Dual-Labeled Oligonucleotide Probes.</a> J. Am. Chem. Soc. 124, 6950-6956 (2002).</li><li>Barrois, S., W&#246;rner, S., Wagenknecht, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Duplex+Stability+for+Wavelength-Shifting+Fluorescent+DNA+Probes:+Energy+Transfer+vs+Excition+Interactions+in+DNA+&amp;#34;Traffic+Lights&amp;#34;,+Photochem.">The Role of Duplex Stability for Wavelength-Shifting Fluorescent DNA Probes: Energy Transfer vs Excition Interactions in DNA &amp;#34;Traffic Lights&amp;#34;, Photochem.</a> Photobiol. Sci. 13, 1126-1129 (2014).</li></ol>

The authors have nothing to disclose.

<html> ويظهر هذا البروتوكول الإجراء الكامل لتسمية الحمض النووي بعد صناعي عبر CuAAC بواسطة الأصباغ الفلورية المعدلة أزيد. وهذا يشمل تركيب الأصباغ والحمض النووي المعدلة آلكاين فضلا عن إجراءات وضع العلامات. تركيب الأصباغ يتبع أربع خطوا?...

يمثل التصوير الجزيئي تقنية أساسية لفهم العمليات البيولوجية داخل الخلايا الحية. 1-3 تطوير تحقيقات الفلورسنت الحمض النووي على أساس لمثل هذه التطبيقات الكيميائية البيولوجية أصبح حقل البحوث التوسع. وتحتاج هذه تحقيقات الفلورسنت لتلبية بعض المتطلبات لتصبح أدوات مناسبة لتصوير الخلايا. أولا، يجب أن الأصباغ تطبيقها تظهر مضان ذات العوائد العالية الكم، والتحولات الكبيرة ستوكس، والأهم من ذلك، photostabilities مرتفعة للسماح طويلة الأجل في مجال التصوير الجسم الحي. وثانيا، ينبغي أن تظهر قراءات مضان يمكن الاعتماد عليها. وتعتمد أنظمة حامل اللون-فاكهه تقضي التقليدية على قراءات من لون مضان واحد عن طريق تغييرات بسيطة في كثافة مضان. 4 هذا النهج يحمل خطر من النتائج السلبية الإيجابية أو كاذبة كاذبة بسبب تألق ذاتي من مكونات داخل الخلايا أو انخفاض نسب الإشارة إلى الضوضاء بسبب التبريد غير مرغوب فيه من قبل شركات أخرى لponents 4 بلغنا مؤخرا على مفهوم &quot;إشارات المرور الحمض النووي&quot; التي تظهر مزدوجة قراءات اللون مضان باستخدام اثنين من حاملات مختلفة. 5-6 ويستند مفهوم على نقل الطاقة (ET) من الصبغة المانحة إلى صبغ متقبل الذي يتغير مضان اللون (انظر الشكل 1). هذا يسمح للقراءات أكثر موثوقية، وبالتالي يوفر أداة قوية لتحقيقات التصوير الفلورسنت. وضع البطاقات التعريفية على أليغنوكليوتيد مع الأصباغ الفلورية يمكن أن يتحقق من خلال نهجين مختلفين. ويمكن إدراج الأصباغ خلال توليف الحمض النووي الكيميائي في مرحلة الصلبة باستخدام كتل بناء phosphoramidite تعديل تبعا لذلك. 7 يقتصر هذا الأسلوب إلى الأصباغ التي هي مستقرة في ظل phosphoramidite وdeprotection الظروف القياسية. وكبديل لذلك، تم إنشاء منهجيات تعديل آخر الاصطناعية في الكيمياء النوكليوتيد. هنا، علينا أن نظهر تركيب واحدة من الصور الجديدةالطاقة الجدول نقل أزواج 8،9 ووضع العلامات آخر الاصطناعية من الحمض النووي باستخدام المحفز النحاس 1،3-cycloaddition بين أزيدات والألكاينات (CuAAC). 10

<table><tbody><tr><td colspan="4">&lt;strong&gt;synthesis&lt;/strong&gt;</td></tr><tr><td>4-Picoline</td><td>Sigma Aldrich</td><td>239615</td><td></td></tr><tr><td>1,3-Diiodopropane</td><td>Sigma Aldrich</td><td>238414</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>10660131</td><td>HPLC grade</td></tr><tr><td>Ethyl acetate</td><td>Fisher Scientific</td><td>10456870</td><td>technical grade</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td>p.a. grade</td></tr><tr><td>Dichloromethane</td><td>Fisher Scientific</td><td>10626642</td><td>technical grade</td></tr><tr><td>Indole-3-carboxaldehyde; 98%</td><td>ABCR</td><td>AB112969</td><td></td></tr><tr><td>Potassium carbonate, 99+%</td><td>Acros</td><td>424081000</td><td></td></tr><tr><td>dimethylcarbonate</td><td>Sigma Aldrich</td><td>517127</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide, 99.8%, Extra Dry over Molecular Sieve</td><td>Acros</td><td>348435000</td><td></td></tr><tr><td>Sodium sulfate</td><td>Bernd Kraft</td><td>12623.46</td><td></td></tr><tr><td>Ethanol, 99.5%</td><td>Acros</td><td>397690010</td><td></td></tr><tr><td>Piperidine, 99%</td><td>Acros</td><td>147181000</td><td></td></tr><tr><td>Diethylether</td><td>Fisher Scientific</td><td>10407830</td><td>technical grade</td></tr><tr><td>2-Phenylindole-3-carboxaldehyde; 97%</td><td>ABCR</td><td>AB125050</td><td></td></tr><tr><td>4-Methylquinoline</td><td>ABCR</td><td>AB117222</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;DNA synthesis&lt;/strong&gt;</td></tr><tr><td>Expedite 8909 Nucleic Acid Synthesizer</td><td>Applied Biosystems</td><td>&amp;nbsp;-</td><td></td></tr><tr><td>DMT-dA(bz) Phosphoramidite</td><td>Sigma Aldrich</td><td>A111081</td><td></td></tr><tr><td>DMT-dT Phosphoramidite</td><td>Sigma Aldrich</td><td>T111081</td><td></td></tr><tr><td>DMT-dG(dmf) Phosphoramidite</td><td>Sigma Aldrich</td><td>G11508</td><td></td></tr><tr><td>DMT-dC(bz) Phosphoramidite</td><td>Sigma Aldrich</td><td>C11108</td><td></td></tr><tr><td>Amidite Diluent for DNA synthesis</td><td>Sigma Aldrich</td><td>L010010</td><td></td></tr><tr><td>Ultrapure Acetonitrile for DNA synthesis</td><td>Sigma Aldrich</td><td>L010400</td><td></td></tr><tr><td>Cap A</td><td>Sigma Aldrich</td><td>L840000</td><td></td></tr><tr><td>Cap B</td><td>Sigma Aldrich</td><td>L850000</td><td></td></tr><tr><td>CPG dT Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>T461010</td><td></td></tr><tr><td>CPG dA(bz) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>A461010</td><td></td></tr><tr><td>CPG dG(ib) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>G461010</td><td></td></tr><tr><td>CPG dC(bz) Column 1.0&amp;nbsp;&amp;micro;mole</td><td>Proligo Reagents</td><td>C461010</td><td></td></tr><tr><td>ammonia (aqueous solution)&amp;nbsp;</td><td>Fluka Analytical</td><td>318612</td><td></td></tr><tr><td>centrifugal devices nanosep 0.45&amp;nbsp;&amp;micro;m</td><td>Pall</td><td>ODGHPC34</td><td></td></tr><tr><td>5-(Benzylthio)-1H-tetrazole (Activator)</td><td>Sigma Aldrich</td><td>75666</td><td></td></tr><tr><td>2&#039;-O-propargyl deoxyuridinephosphoramidite</td><td>Chem Genes</td><td>ANP-7754</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;workup&lt;/strong&gt;</td></tr><tr><td>vacuum concentrator</td><td>Christ</td><td></td><td></td></tr><tr><td>&lt;strong&gt;clicking procedure&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tetrakis(acetonitrile)copper(I) hexafluorophosphate</td><td>Sigma Aldrich</td><td>346276</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma Aldrich</td><td>S2889</td><td></td></tr><tr><td>(+)-Sodium L-ascorbate</td><td>Sigma Aldrich</td><td>A7631</td><td></td></tr><tr><td>EDTA disodium salt</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>TBTA-ligand</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>synthesized according to a literature procedure&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;HPLC&lt;/strong&gt;</td></tr><tr><td>HPLC-system</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>MALDI-Biflex-IV spectrometer</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>LC-318 C18 column</td><td>Supelcosil via Sigma Aldrich</td><td>58368</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;determination of concentration&lt;/strong&gt;</td></tr><tr><td>ND 1000 Spectrophotometer</td><td>nanodrop</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;sample preparation and spectroscopy&lt;/strong&gt;</td></tr><tr><td>Cary 100 Bio</td><td>Varian</td><td></td><td></td></tr><tr><td>Fluoromax-3 fluorimeter</td><td>Jobin-Yvon</td><td></td><td></td></tr><tr><td colspan="4">&lt;sup&gt;1&lt;/sup&gt; R. Chan Timothy, R. Hilgraf, K. B. Sharpless, V. Fokin Valery, &lt;em&gt;Org Lett&lt;/em&gt; 2004, &lt;strong&gt;6&lt;/strong&gt;, 2853-2855.</td></tr></tbody></table>

synthesis of wavelength-shifting dna hybridization probes by using photostable cyanine dyes

في هذا البروتوكول، ونحن يبرهن على وجود طريقة لتركيب 2&#39;-آلكاين تعديل النووي الريبي منقوص الأكسجين حامض (DNA) خيوط من قبل الآلي توليف المرحلة الصلبة باستخدام الكيمياء phosphoramidite القياسية. وصفت [أليغنوكليوتيد ما بعد صناعي من قبل اثنين من cyanine الأصباغ صامد جديدة باستخدام المحفز النحاس النقر الكيمياء. ووصف تركيب كل من الجهات المانحة ومتقبل صبغ ويتم تنفيذ في ثلاث خطوات متتالية. مع الحمض النووي كما أن الهيكل المحيطة بها، وهذه الأصباغ اثنين من الخضوع لنقل الطاقة عندما يتم تقديمهم الى مقربة من التهجين. لذلك، هو تصور الصلب اثنين واحد جدائل الحمض النووي تقطعت بهم السبل بسبب تغيير اللون مضان. يتميز هذا التغيير اللون عن طريق التحليل الطيفي مضان ولكن يمكن أيضا أن نلاحظ مباشرة باستخدام مصباح الأشعة فوق البنفسجية المحمولة (الأشعة فوق البنفسجية). مفهوم مزدوج قراءات اللون مضان يجعل هذه التحقيقات النوكليوتيد أدوات ممتازة للتصوير الجزيئي خصوصا عندما هواتف وصفهوتستخدم الأصباغ tostable. وبالتالي، يتم منع photobleaching من لتحقيقات التصوير، والعمليات البيولوجية يمكن ملاحظتها في الوقت الحقيقي لفترة زمنية أطول.

وتسجل امتصاص ومضان أطياف من الحمض النووي مفردة ومزدوجة الذين تقطعت بهم السبل كما هو مبين في الشكل (4). أطياف الامتصاص سجلت (الشكل 4 يمين) تظهر امتصاص ماكسيما λ كحد أقصى في ...

Watch this Scientific Journal Video about Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes at JoVE.com

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

تجميع لتحويل الطول الموجي تهجين الحمض النووي تحقيقات باستخدام صامد Cyanine الأصباغ

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

synthesis-wavelength-shifting-dna-hybridization-probes-using

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Organic Chemistry

Biology

Unit 1

Alkynes

SCIENTISTS IN ACTION

11.1K Views.  Karlsruhe Institute of Technology. Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Video: Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

بروتوكول الأمثل لالكهربي التنقل التحول الفحص عن طريق أليغنوكليوتيد] وصفت صبغ الأشعة تحت الحمراء نيون

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Biochemistry

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

تظاهرة فنية من الجامعة التهجين الجينومي المقارن صفيف الجينوم

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

التفريد الحمض النووي باستخدام أورانج Thiazole بدلاً من اثيديوم بروميد أو الأصباغ البديلة

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

الجمع بين QD - الحنق و microfluidics الحمض النووي لرصد Nanocomplex الذاتي الجمعية في الوقت الحقيقي

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

مجسات توتر الحمض النووي لرسم خريطة لقوى مستقبلات بيكونيوتن العابرة بواسطة الخلايا المناعية

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

Bioengineering

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

التوقيت كروموسوم النسخ المتماثل جنبا إلى جنب مع نيون<em&gt; في الموقع</em&gt; التهجين

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

قوية FISH DNA 3D استخدام مجسات إعتبر مباشرة

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

DNA ستراند المهجرين غيتس لشبكات التفاعل التنفيذية الكيميائية المشتقة من البلازميد

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

فحص حركية الميرنا في المكان والزمان في الإشريكية القولونية باستخدام الفلورس أحادي الجزيء بلونين في التهجين الموقعي

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

The ability of proteins&#160;involved in eukaryotic DNA replication to&#160;overcome obstacles &#8212; such as protein and DNA &#39;roadblocks&#39;&#160;&#8212; is critical for ensuring faithful&#160;genome duplication.&#160;G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase &#948;, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.

This protocol outlines a fluorescence microscopy-based single-molecule DNA replication assay, enabling real-time visualization of interactions between DNA polymerases and obstacles such as G-quadruplex structures.

تجميع لتحويل الطول الموجي تهجين الحمض النووي تحقيقات باستخدام صامد Cyanine الأصباغ

Summary

Explore More Videos

Chapters in this video