نود أن نشكر أعضاء فريق وان مختبر لام صفيف BAC خاصة ميوا سوزوكي وتشي بريان لإعداد هذا المقال. وأيد هذا العمل من جانب صناديق من المعاهد الكندية لأبحاث الصحة ، كندا الجينوم / الجينوم كولومبيا البريطانية ، والمعاهد الوطنية للصحة / NIDCR منح RO1 DE15965 - 01.

دقق وصفها ملاحظة : الحد من التعرض للضوء ملونات Cye في جميع الأوقات (ويمكن تحقيق ذلك من خلال العمل في منطقة مظلمة أو عن طريق الأنابيب التدريع بغطاء مثل رقائق الألومنيوم) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الجمع : (1 الإعداد أنبوب رد فعل لتكون مرجعا و1 أنبوب رد فعل لعينة) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحمض النووي (25-400 نانوغرام) </li><li style=";text-align:right;direction:rtl"> 5 ميكرولتر من 5X العازلة الاشعال العشوائي (تركيز النهائي : 5X Promega Klenow العازلة وميكروغرام / 7 octamers عشوائي ميكرولتر) </li><li style=";text-align:right;direction:rtl"> تمييع للحجم الإجمالي 17.0 ميكرولتر مع 2 المقطر O H </li></ul></li><li style=";text-align:right;direction:rtl"> يغلي لمدة 10 دقيقة عند 100 درجة مئوية. نقل على الفور إلى الجليد لمدة 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> إضافة 4 ميكرولتر من مزيج dNTP 10X (2mm وdATP ، dGTP ، dTTP ، dCTP 1.2mM). </li><li style=";text-align:right;direction:rtl"> إضافة CyeDyes : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 2 ميكرولتر (2 nmoles) من CY - 3 dCTP المسمى لعينة الحمض النووي </li><li style=";text-align:right;direction:rtl"> إضافة 2 ميكرولتر (2 nmoles) من قبرصي 5 - dCTP المسمى DNA إلى المراجع (على سبيل المثال ، Novagen الحمض النووي الجينوم البشري) </li></ul></li><li style=";text-align:right;direction:rtl"> إضافة 2.5 ميكرولتر من Klenow (9 U / ميكرولتر ، Promega) ومزيج. </li><li style=";text-align:right;direction:rtl"> احتضان عند 37 درجة مئوية خلال الليل * (~ 18 ساعة). * يمكن ضبط الوقت التهجين بين عشية وضحاها ما بين 14 إلى 24 ساعة دون أن يؤثر ذلك سلبا على عملية وضع العلامات لتناسب لجدول زمني المختبر. </li></ol> SAMPLE نظفوا (الجامع للمسبار التنظيف والتحضير للتهجين) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام YM - 30 Microcon العمود : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 100 كوت - 1 ميكروليتر DNA (1μg/μL) إلى العمود. لا تلمس الغشاء مع تلميح ماصة. </li><li style=";text-align:right;direction:rtl"> تجمع ردود الفعل (المرجعية والعينة) وإضافة إلى العمود. </li><li style=";text-align:right;direction:rtl"> العمود مكان في المقدمة وأنبوب تدور ز 13000 لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> إضافة 200 H 2 0 ميكرولتر المقطر لغشاء تدور وكرر أن يغسل. </li><li style=";text-align:right;direction:rtl"> تجاهل أنبوب وإضافة 45 ميكرولتر حل التهجين : (روش DIG سهلة) </li></ul> اختياري : إضافة 5μL الحيوانات المنوية الرنجة المنفصمة الحمض النووي (وحدة 10μg/μL ، Promega) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عكس Microcon ، ضع في أنبوب الجديد المسمى ، وتدور بسرعة 3000 غرام لمدة 3 دقائق. </li></ul></li></ol> حساب المعلومات المدرجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة 1.5 ميكرولتر وقياس الطيف باستخدام دمج Fluorophore NanoDrop. التهجين باستخدام المخزن الخاص باعتبارها فارغة (DIG سهل) اتبع التعليمات على الشاشة. (يمكنك استعادة عينة من رمى ، إذا لزم الأمر ، وبعد القياس.) (ملاحظة : المعلومات المدرجة أدناه بمول 3.0 / ميكرولتر في أي قناة وأظهرت النتائج متغير) </li><li style=";text-align:right;direction:rtl"> تفسد على 85 درجة مئوية لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> التحقيق في مكان 45 درجة مئوية لمدة 30 دقيقة إلى 1 ساعة (كوت يسمح - 1 الحمض النووي الصلب). </li></ol> ARRAY التهجين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 44 حل مكان التحقيق وساترة على ميكرولتر (22 ملم × 60 ملم) (فيشر العلمية). إذا كانت متوفرة ، ضع ساترة على شريحة أو أكثر دفئا كتلة الحرارة قبل تحسنت إلى 45 درجة مئوية للحفاظ على درجة الحرارة. </li><li style=";text-align:right;direction:rtl"> محاذاة على الشريحة ساترة وتحقيق الحل ، وانخفاض حافة واحدة من الشريحة السماح الاتصال مع الحل التهجين. تواصل انخفاض حتى يتم إرفاق ساترة إلى الشريحة مع التوتر السطحي. عكس الشريحة. </li><li style=";text-align:right;direction:rtl"> ضع الشريحة في الكاسيت التهجين (Telechem) ، قبل أن درجة حرارة 45 درجة مئوية ، وإضافة 10 ميكرولتر من الماء في أسفل الأخدود (للتحكم في نسبة الرطوبة خلال التهجين). </li><li style=";text-align:right;direction:rtl"> وختم الكاسيت احتضان لمدة 36 -- 40 ساعة عند 45 درجة مئوية. </li></ol> ARRAY WASHING ملاحظة : جميع الحلول ويغسل عند pH 7.0 إزالة الشريحة من الكاسيت التهجين أمر بالغ الأهمية. DIG سهل تبلور بسرعة في درجة حرارة الغرفة. يجب أن تكون الشريحة مغمورة فورا ، وإزالة الغطاء زلة في حل يغسل. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة ما يقرب من 60 مل من 0.1XSSC ، 0.1 ٪ SDS (الرقم الهيدروجيني 7.0 ، 45 درجة مئوية) يغسل حل لجرة Coplin قبل افتتاح غرفة التهجين. </li><li style=";text-align:right;direction:rtl"> الغرفة مفتوحة ، إضافة إلى الشريحة غسل حل وإزالة ساترة (يجب أن الشريحة إيقاف). </li><li style=";text-align:right;direction:rtl"> تغسل الشريحة 3 مرات في 0.1XSSC + 0.1 ٪ SDS في 45 درجة مئوية لمدة 1 دقيقة مع كل الانفعالات. </li><li style=";text-align:right;direction:rtl"> شطف الشريحة 3 مرات في 0.1XSSC جديدة *. وينبغي أن يكون هناك فقاعات المتبقية من غسل SDS مرئية. * يمكن الشرائح تبقى في حل غسل النهائي حتى على استعداد لأجهزة الطرد المركزي والفحص (~ 15 دقيقة). </li><li style=";text-align:right;direction:rtl"> الطرد المركزي الشريحة في أنابيب فالكون 50 مل ب 700 غرام لمدة 3 دقائق. </li><li style=";text-align:right;direction:rtl"> تفحص على الفور الشرائح (كثافات إشارة سيتقلص مع مرور الوقت.) </li></ol>

<ol>
	<li>Lockwood, W. W., Chari, R., Chi, B., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+array+comparative+genomic+hybridization+technologies+and+their+applications+in+human+genetics.">Recent advances in array comparative genomic hybridization technologies and their applications in human genetics.</a> European Journal of Human Genetics. 14, 139-1x`48 (2006).</li><li>Ishkanian, A. S., Malloff, C. A., Watson, S. K., deLeeuw, R. J., Chi, B., Coe, B. P., Snijders, A., Albertson, D. G., Pinkel, D., Marra, M. A., Ling, V., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tiling+resolution+DNA+microarray+with+complete+coverage+of+the+human+genome.">A tiling resolution DNA microarray with complete coverage of the human genome.</a> Nature Genetics. 36, 299-303 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Ishkanian, A. S., Malloff, C. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+high+throughput+validation+of+amplified+fragment+pools+of+BAC+DNA+for+constructing+high+resolution+CGH+arrays.">Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays.</a> BMC Genomics. 5, 6 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Horsman, D. E., Squire, J. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetically+balanced+translocations+are+associated+with+focal+copy+number+alterations.">Cytogenetically balanced translocations are associated with focal copy number alterations.</a> Human Genetics. 120, 795-805 (2007).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeGH+--+a+software+tool+for+visualization+of+whole+genome+array+comparative+genomic+hybridization+data.">SeeGH -- a software tool for visualization of whole genome array comparative genomic hybridization data.</a> BMC Bioinformatics. 5, 13 (2004).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., Ng, R. T., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MD-SeeGH:+a+platform+for+integrative+analysis+of+multi-dimensional+genomic+data.">MD-SeeGH: a platform for integrative analysis of multi-dimensional genomic data.</a> BMC Bioinformatics. 9, 243 (2008).</li><li>Coe, B. P., Ylstra, B., Carvalho, B., Meijer, G. A., Macaulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+resolution+of+array+CGH.">Resolving the resolution of array CGH.</a> Genomics. 89, 647-653 (2007).</li><li>Garnis, C., Coe, B. P., Lam, S. L., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+array+CGH+increases+heterogeneity+tolerance+in+the+analysis+of+clinical+samples.">High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.</a> Genomics. 85, 790-793 (2005).</li></ol>

والفقراء لا توفر نوعية الحمض النووي لمحة تهجين جيدة. فمن الضروري التأكد من أن عينة الحمض النووي ، ومرجعية تكون خالية من الملوثات ، مثل الحمض النووي الريبي ، والفينول ، والملح ، وغيرها التي قد تتداخل مع الخطوة العلامات رئيس عشوائي قبل البدء في تجربة التهجين. على سبيل المثال ، إعادة تعليق من الحمض النووي في تريس -- يمكن أن لا ينصح EDTA (TE) بدلا من الماء والملح تركيز عالية تمنع رد الفعل التوسيم. نوصي معايرة الجودة باستخدام الحمض النووي Nanodrop معمل لقياس كل من كمية الحمض النووي فضلا عن الشكل العام للمنحنى الامتصاصية و260/280 ، 260/230 النسبة. الغسيل : إزالة الشريحة من الكاسيت التهجين ونقل الى حل غسل ساخنة هو خطوة حاسمة في عملية التهجين كحل DIG التهجين سهل تبلور بشكل سريع جدا. من المهم أن الحلول تكون في غسل محايد الرقم الهيدروجيني ودرجة الحرارة من الحل غسل المهم بالنسبة الصرامة في حين إزالة التحقيق غير منضم. بعد تجفيف شرائح من المهم الاحتفاظ بها في الظلام إذا لم المسح على الفور ، أو بعد المسح إذا كنت ترغب في إعادة تفحص لهم في وقت لاحق. الأوزون : الأوزون كانت مصدر قلق في جميع أنحاء العالم عند تنفيذ CGH الصفيف. وقد تبين أن مستويات الأوزون فوق 5ppm تؤثر سلبا على الأصباغ Cye. 5 Cye حساسة بشكل خاص للتدهور طبقة الأوزون. التقليل من التعرض الأوزون هو المفتاح لمنع تدهور وفقدان صبغة Cye إشارة قبل وأثناء المسح. المسح الضوئي ليلا على سبيل المثال ، عندما الأوزون البيئية أقل عادة ، هو أحد الخيارات الممكنة. مرفقات الأوزون مجانا للشريحة الغسيل الآلي والمسح الضوئي ومختلف العلاجات الكيميائية الشريحة متاحة تجاريا. الأصباغ Cye مقاومة الأوزون كما تم مؤخرا تطوير.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5X Klenow Buffer</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Random Octamers </td>
<td>Reagent</td>
<td>Alpha DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X dNTP mix</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>2mM dATP, dGTP, dTTP, 1.2mM dCTP</td>
</tr>
<tr>
<td>Cy-3 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Human Genomic DNA Reference</td>
<td>Reagent</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>Example of a possible reference</td>
</tr>
<tr>
<td>Klenow</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>YM-30 Column</td>
<td>Reagent</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cot-1 DNA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DIG Easy</td>
<td>Reagent</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sheared Herring Sperm DNA</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>22mm x 60mm</td>
</tr>
<tr>
<td>Hybridization Cassette</td>
<td>Tool</td>
<td>Telechem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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technical demonstration of whole genome array comparative genomic hybridization

مجموعة التهجين الجينومي المقارن (CGH الصفيف) هو وسيلة للكشف عن المكاسب والخسائر في قطاعات الحمض النووي أو الجينات الدواء في الجينوم 1. وقد مكنت التطورات الحديثة في هذه التكنولوجيا العالية مقارنة الجينوم حل كامل لتحديد تغيرات جينية في حالات السرطان والأمراض الوراثية الأخرى 2. اللجنة الفرعية Megabase قرار تعيين مجموعة التبليط ، (أو SMRT) وتتألف مجموعة من مجموعة من حوالي 30000 تداخل الصبغي الاصطناعي البكتيري (BAC) التي تمتد استنساخ الجينوم البشري في ~ الزوج kilobase 100 (كيلوبايت) شرائح 2. وقد تم تجميع هذه الأهداف بشكل فردي ورصدت في BAC مكررة على شريحة زجاجية واحدة 2-4. وتستند مجموعة CGH على مبدأ التهجين تنافسية. وصفت وتفاضليا عينة الحمض النووي مع الإشارة 3 Cyanine والأصباغ الفلورية Cyanine - 5 ، وشاركت في المهجنة إلى الصفيف. بعد فترة حضانة المرض هي غسل العينات غير منضم من الشريحة والتقط الصفيف. يتم استخدام حزمة البرامج المتاحة بحرية مخصصة دعا SeeGH (www.flintbox.ca) لمعالجة حجم كبير من البيانات التي تم جمعها -- تجربة واحدة يولد 53892 نقاط البيانات. SeeGH يتصور قوة إشارة نسبة log2 بين عينات 2 في كل هدف BAC التي تتماشى مع موقف عموديا الكروموسومات 5،6. يمكن الكشف عن مجموعة SMRT تعديلات صغيرة مثل 50 كيلو بايت في حجم 7. لا يمكن للمجموعة SMRT كشف مجموعة متنوعة من الأحداث إعادة ترتيب الحمض النووي DNA بما في ذلك الأرباح والخسائر والتكبير والحذف متماثل. وهناك ميزة فريدة من الصفيف SMRT هو أن واحدة يمكن استخدام الحمض النووي المعزولة من عينات الفورمالين البارافين الثابتة المدمجة. عند دمجها مع متطلبات مدخلات منخفضة من الحمض النووي unamplified (25 100ng) يسمح هذا التنميط من العينات الثمينة مثل تلك التي تنتجها microdissection 7،8. ويعزى ذلك إلى حجم كبير من كل هدف التهجين BAC التي تسمح للربط ما يكفي لانتاج عينات المسمى إشارات للكشف. ميزة أخرى لهذا المنبر هو التسامح مع عدم تجانس الأنسجة ، وتقليل الحاجة إلى microdissection الأنسجة مملة 8. هذا البروتوكول الفيديو هو برنامج تعليمي خطوة بخطوة من وضع العلامات على الحمض النووي عن طريق إدخال إشارة إلى اقتناء لتبليط الجينوم SMRT مجموعة المسار.

Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

هذا الفيديو هو تظاهرة فنية للبروتوكول لتهجين كامل الجينوم تبليط الطريق CGH الصفيف ، الذي مسح الجينوم البشري بأكمله فقط باستخدام 25-100 نانوغرام من الحمض النووي يمكن أن يكون معزولا عن مجموعة متنوعة من المصادر ، بما في ذلك المواد الأرشيفية الثابتة الفورمالين.

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 ...

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Nanyang Technological University

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12.7K Views.  BC Cancer Research Centre. *The slides can remain in the 

Video: Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

الجامعة جبل مزدوجة في الموقع التهجين لافراخ الدجاج في وقت مبكر

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

الزرد الجامع جبل الرفيع قرار مزدوج نيون في الموقع التهجين

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

جبل به الجامعة في الموقع تهجين ارتباط البيولوجيا الجزيئية والعضوي

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

ميكروأرس] DNA : نموذج مراقبة الجودة ، وتهجين صفيف والمسح الضوئي

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

جبل كله<em&gt; في الموقع</em&gt; التهجين من E8.5 لأجنة الفئران E11.5

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

الجينوم شاشات التنافسية للمكتبات الخميرة Barcoded

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

الكشف عن المتغيرات الجينية النادرة من تسلسل المجمعة عن طريق سبلينتر

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

كله جبل RNA نيون<em&gt; في الموقع</em&gt; التهجين من<em&gt; ذبابة الفاكهة</em&gt; الأجنة

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

مجموعة المقارن الجينوم التهجين (صفيف [ك]) عن الكشف عن الجينوم نسخ عدد المتغيرات

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

تنقية الجينوم على نطاق خارج الصبغي التعميم الحمض النووي من خلايا حقيقية النواة

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...