Name: chromosome replicating timing combined with fluorescent in situ hybridization
Uploaded: 2012-12-10T13:00:00
Description: Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

यह काम राष्ट्रीय कैंसर संस्थान, CA131967 से एक अनुदान द्वारा समर्थित किया गया.

1. BrdU निगमन (टर्मिनल लेबल) <ol><li> बारे में एक 150 मिमी ऊतक संस्कृति डिश 24 BrdU के अलावा पहले घंटा में 70% संगम प्लेट कोशिकाओं. </li><li> ताजा पूरा उपयुक्त समय बिंदुओं पर 20 ग्राम / एमएल BrdU (सिग्मा) फसल के लिए पहले वाले मीडिया के...

<ol>
	<li>Payer, B., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu. Rev. Genet. 42, 733-772 (2008).</li><li>Breger, K. S., Smith, L., Turker, M. S., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+radiation+induces+frequent+translocations+with+delayed+replication+and+condensation.">Ionizing radiation induces frequent translocations with delayed replication and condensation.</a> Cancer Research. 64, 8231-8238 (2004).</li><li>Smith, L., Plug, A., Thayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+Replication+Timing+Leads+to+Delayed+Mitotic+Chromosome+Condensation+and+Chromosomal+Instability+of+Chromosome+Translocations.">Delayed Replication Timing Leads to Delayed Mitotic Chromosome Condensation and Chromosomal Instability of Chromosome Translocations.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 13300-13305 (2001).</li><li>Stoffregen, E. P., Donley, N., Stauffer, D., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+autosomal+locus+that+controls+chromosome-wide+replication+timing+and+mono-allelicexpression.">An autosomal locus that controls chromosome-wide replication timing and mono-allelicexpression.</a> Hum. Mol. Genet. 20, 2366-2378 (2011).</li><li>Breger, K. S., Smith, L., Thayer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+translocations+with+delayed+replication:+evidence+for+cis+control+of+chromosome+replication+timing.">Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing.</a> Hum. Mol. Genet. 14, 2813-2827 (2005).</li><li>Camargo, M., Cervenka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+DNA+replication+of+human+chromosomes.+II.+Replication+map+and+replication+model.">Patterns of DNA replication of human chromosomes. II. Replication map and replication model.</a> Am. J. Hum. Genet. 34, 757-780 (1982).</li><li>Cohen, S. M., Cobb, E. R., Cordeiro-Stone, M., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+chromosomal+bands+replicating+early+in+the+S+phase+of+normal+human+fibroblasts.">Identification of chromosomal bands replicating early in the S phase of normal human fibroblasts.</a> Exp. Cell Res. 245 (98), 321-329 (1998).</li><li>Diaz-Perez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+element(s)+at+the+nontranscribed+Xist+locus+of+the+active+X+chromosome+controls+chromosomal+replication+timing+in+the+mouse.">The element(s) at the nontranscribed Xist locus of the active X chromosome controls chromosomal replication timing in the mouse.</a> Genetics. 171, 663-672 (2005).</li><li>Diaz-Perez, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+deletion+at+the+mouse+Xist+gene+exposes+trans-effects+that+alter+the+heterochromatin+of+the+inactive+X+chromosome+and+the+replication+time+and+DNA+stability+of+both+X+chromosomes.">A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.</a> Genetics. 174, 1115-1133 (2006).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2415-2420 (2008).</li><li>Schlesinger, S., Selig, S., Bergman, Y., Cedar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+inactivation+of+rDNA+loci.">Allelic inactivation of rDNA loci.</a> Genes Dev. 23, 2437-2447 (2009).</li><li>Branzei, D., Foiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+checkpoint+response+to+replication+stress.">The checkpoint response to replication stress.</a> DNA Repair (Amst). 8, 1038-1046 (2009).</li></ol>

गुणसूत्र फैलता की तैयारी यहाँ वर्णित सफल परख प्रतिकृति - समय के लिए एक महत्वपूर्ण कदम है. एक colcemid pretreatment कदम का समावेश hypotonic उपचार से पहले आवृत्ति में और mitotic कोशिकाओं के प्रसार में सहायता कर सकते हैं. हम आम तौर ...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> विरोधी BrdU - FITC </td><td> Roche Millipore </td><td> 11202593001 MAB326F </td><td> 50 ग्राम / μl </td></tr><tr><td> निक अनुवाद किट </td><td> Abbott आण्विक (Vysis) </td><td> 07J00-0001 </td><td></td></tr><tr><td> स्पेक्ट्रम ऑरेंज dUTP </td><td> Abbott आण्विक (Vysis) </td><td> 02N33-050 </td><td></td></tr><tr><td> सीईपी </td><td> Abbott आण्विक (Vysis) </td><td> बदलता रहता है </td><td></td></tr><tr><td> / LSI WCP संकरण बफर </td><td> Abbott आण्विक (Vysis) </td><td> 06J67-011 </td><td></td></tr><tr><td> सीईपी संकरण बफर </td><td> Abbott आण्विक (Vysis) </td><td> 07J36-001 </td><td></td></tr><tr><td> क्रोमोजोम पैपड़ोसी देशों की यात्रा योजना </td><td> MetaSystems समूह </td><td> D-14NN-050-टी.आर. </td><td></td></tr><tr><td> ओलिंप BX61 फ्लोरोसेंट माइक्रोस्कोप </td><td> ओलिंप </td><td> BX61TRF-1-5 </td><td></td></tr><tr><td> माइक्रोस्कोप इमेजिंग सॉफ्टवेयर प्रणाली </td><td> एप्लाइड इमेजिंग </td><td> 3.93.1 Cytovision </td><td></td></tr><tr><td> डिजिटल कैमरा </td><td> ओलिंप </td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> स्वस्थानी संकरण व्यंजनों में Formamide समाधान 70% Formamide/2x एसएससी 35 मिलीलीटर * Formamide (सिग्मा) 10 मिलीलीटर एसएससी 10x 5 मिलीलीटर घ 2 2 0 एच एचसीएल के साथ 7.0 पीएच (सिग्मा) * यह महत्वपूर्ण है Formamide उपयोग करें कि -20 डिग्री सेल्सियस पर संग्रहीत किया गया है लंबे समय तक कमरे के तापमान का भंडारण formic एसिड और पीएच w उत्पन्न होगाबीमार भी कम हो सकता है. 50% Formamide/2x एसएससी 25 मिलीलीटर Formamide (सिग्मा) 10 मिलीलीटर एसएससी 10x 15 मिलीलीटर DH 2 हे एचसीएल के साथ 7.0 पीएच (सिग्मा) 20x एसएससी, 4 एल 702 छ NaCl (सिग्मा) 358 छ ना साइट्रेट (सिग्मा) DH 2 मात्रा हे पी.एन. बफर [4 0.1 NaP0 एम 0.1% NP_40 (सिग्मा)] एक 0.1 एम समाधान सोडियम फास्फेट का प्रत्येक (फ़िल्टर बाँझ बनाना और 500 मिलीलीटर aliquots में दुकान). 0.1 नाह एम 2 P0 4, 1 एल 13.8 छ 2 नाह 4 P0 (सिग्मा): DH 2 मात्रा हे 0.1 नः 2 4 1 P0 एल एम </strong&gt; 14.2 P0 2 नाह 4 g (सिग्मा) DH 2 मात्रा के लिए हे. पी.एन.: 2 HP0 4 8.0 पीएच ना .1 नाह एम 2 4 P0 के साथ 0.1 एम के पीएच को समायोजित करें. फ़िल्टर बाँझ बनाना और NP-40 के 1 मिलीलीटर जोड़ें. PNM 50 मिलीलीटर 1.25 छ गैर वसा सूखा दूध (सिग्मा) 25 मिलीलीटर पी.एन. बफर (ऊपर पकाने की विधि) मिक्स लगातार सरगर्मी के साथ 15-20 मिनट के लिए. 10 मिनट के लिए 400 XG पर 2 बार स्पिन. तैरनेवाला का प्रयोग करें, और उपजी दूध प्रोटीन परेशान नहीं करने के लिए सुनिश्चित करें. </td></tr></tbody></table>

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.

प्रतिकृति मानव गुणसूत्र 6 के लिए समय विश्लेषण का एक उदाहरण चित्रा 2 में दिखाया गया है. युक्त कोशिकाओं का एक विलोपन ASAR6 4 जीन, 6q16.1 में स्थित BrdU 5 घंटे के लिए खुल गए थे, mitotic कोशिकाओं के लिए काटा और एक गुण?...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

गुणसूत्र प्रतिकृति समय के विश्लेषण के लिए एक मात्रात्मक विधि का वर्णन है. विधि फ्लोरोसेंट के साथ संयोजन में BrdU निगमन का इस्तेमाल<em&gt; स्वस्थानी में</em&gt; संकरण (मछली) स्तनधारी गुणसूत्रों की प्रतिकृति समय का आकलन. इस तकनीक rearranged और संयुक्त राष्ट्र rearranged क्रोमोसोम के एक ही कोशिका के भीतर प्रत्यक्ष तुलना के लिए अनुमति देता है.

क्रोमोजोम नकल फ्लोरोसेंट के साथ संयुक्त समय<em&gt; सीटू में</em&gt; संकरण

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

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Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

प्रारंभिक चिकी भ्रूण के स्वस्थानी संकरण में डबल पूरा पर्वत

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish पूरा पर्वत उच्च संकल्प डबल प्रतिदीप्त स्वस्थानी में संकरण

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Fluorescence in situ hybridization FISH Protocol in Human Sperm

प्रतिदीप्ति बगल में</emमानव शुक्राणु में> संकरण (मछली) प्रोटोकॉल

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

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

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

पूरे माउंट<em&gt; स्वस्थानी में</em&gt; E8.5 E11.5 माउस भ्रूण संकरण

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

In Situ Hybridization for the Precise Localization of Transcripts in Plants

In Situ Hybridization for the Precise Localization of Transcripts in Plants

संयंत्रों में देखिए का सटीक स्थानीयकरण के लिए स्वस्थानी संकरण में

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

वायरल शाही सेना के प्रतिदीप्ति द्वारा जांच<em&gt; बगल में</em&gt; संकरण (मछली)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

उच्च throughput स्वचालित का उपयोग गुणसूत्रों की शारीरिक मानचित्रण<em&gt; बगल में</em&gt; संकरण

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

एकल कोशिका के जीन प्रतिलेखन की शाही सेना प्रतिदीप्त द्वारा विश्लेषण<em&gt; स्वस्थानी</em&gt; संकरण (मछली)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

पूरे माउंट आरएनए प्रतिदीप्त<em&gt; स्वस्थानी में</em&gt; संकरण<em&gt; ड्रोसोफिला</em&gt; भ्रूण

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

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

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प्रतिदीप्ति का प्रयोग mRNA अणुओं के दृश्य और विश्लेषण<em&gt; स्वस्थानी</emमें&gt; संकरण<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...