Biz tavsiye ve teknik yardım için Blaschke, Grunwald ve Wittwer laboratuarları üyelerine teşekkür ederim. Bu çalışma JLB için PCMC Vakfı, NIH R01 MH092256 ve DP2&#39;deki MH100008 ve Dimes Vakfı araştırma bursu # 1-FY13-425, Mart, tarafından desteklenmektedir.

1.. DNA hazırlanması <ol><li> 50 mM KCI, 10 mM Tris-HCl pH 8.3,% 0.3 Tween 20,% 0.3 NP-40: DNA lizis tamponu hazırlayın. Kullanım 12 günde 1 mg / ml &#39;lik bir nihai konsantrasyona kadar proteinaz K ilave taze. </li><li> Doku toplama: <ol><li> Yetişkin balık yüzgeç klibi için: <ol><li> Balık anestezisi: 0.004% MS-222 (tricaine) çözümü de balık yerleştirin. Solungaç hareketi yavaşlatır kadar bekleyin. </li><li> 5-10 Kimwipes bir yığın balık koymak ve steril bir jilet ile kuyruk yüzgecinin, yaklaşık 2-3 mm, küçük bir parça kesti. </li><li> Hızla geri kazanımı için temiz su ile etiketlenmiş bir tankta balık yer, özenle fin klip içerecek tankı ve ilgili tüpü hem etiket. Su değiştirin ve her gün balık yemi. </li><li> Steril bir pipet ucu ile kanat klibi almak ve 100 ul DNA liziz tamponu ile dolu bir tüp içine aktarın. </li><li> Pipet atmak ve bir kesici kabın içine jilet atmak. </li></ol></li><li> Embriyo kuyruk klibi için: <ol><li> % 0.004 MS-222 (tricaine) çözelti içine embriyolar yerleştirin. 2 dk bekleyin. </li><li> Bir tahlil mikroskobu altında forseps ile kuyruk bir parça kesin. </li><li> 30 ul DNA liziz tamponu ile dolu bir etiketli tüp içine kuyruk koyun. </li><li> Hızla 100 ul% 4 paraformaldehid içeren bir tüp içine embriyo koydu. </li><li> Embriyo ve bunlara karşılık gelen kuyruk tüpleri ile Etiket tüpler. </li><li> Tespit için 4 ° C sıcaklıkta gece boyunca embriyolar ile tüpleri saklayın. </li><li> Kirlenmeyi en aza indirmek için her bir embriyonun arasında Milli-Q su ve Kimwipes kullanarak forseps temizleyin. </li></ol></li><li> Bütün embriyo için: <ol><li> % 0.004 MS-222 (tricaine) çözelti içine embriyolar yerleştirin. 2 dk bekleyin. </li><li> DNA, 100 ul lizis tamponu ile doldurulmuş bir tüp içinde 1-5 embriyolar koyun. Dechorionation gerekli değildir. </li></ol></li></ol></li><li> DNA sindirim 55 ° C&#39;de doku (fin veya kuyruk klipleri ya da embriyolar) ile inkübegecede 12 4 saat kadar için. </li><li> Proteinase K inaktive 15 dakika 12 boyunca 95 ° C&#39;ye kadar ısı boruları. DNA PCR için hemen kullanılabilir veya en fazla 3 ay boyunca -20 ° C&#39;de saklanmalıdır. </li></ol> 2. PCR <ol><li> Tasarım Primerler elle veya primer tasarım yazılımı (örneğin Lightscanner Primer Tasarım Yazılımı-Biofire) tarafından. HRMA için PCR ürünleri genellikle 50-200 bp, SNP tespiti için daha küçük PCR ürünleri, tipik haliyle, 50-80 bp olması gerekir. </li><li> PCR <ol><li> 96 kuyucuklu veya 384 oyuklu bir plaka içerisinde PCR reaksiyonu yapın. </li><li> 10 ul toplam hacim kullanın: 4 ul LightScanner ana karışımı (0.1 U sıcak başlangıç ​​Taq-polimeraz AB, tampon, 0.2 mM dNTPler, 2 mM magnezyum klorür ve 1x floresan çift bükümlü DNA-bağlama boya); 5 pmol her bir primer, ve yetişkin kuyruk fin ya da embriyonik kuyruk 13 3 ul genomik DNA ekstre 1 ul genomik DNA şablonu. </li><li> 30 ul mineral yağ ile kapak örnekleri. </li&gt; <li> Optik-şeffaf yapışkan conta ile kapak plakası. </li><li> PCR çevrim koşulları tipik şartlar 95 ° C&#39;de 5 dakika ve 10 saniye 30 döngü için 95 ° C olan optimize, 60 ° C&#39;de 25 saniye, 72 ° C&#39;de 30 saniye, 30 saniye boyunca 95 ° C ile biten ve soğutuldu 15 ° C. </li><li> Mağaza PCR 4 ° C&#39;de plakalar veya HRMA doğrudan devam etmektedir. </li><li> PCR&#39;nin ilk duruma getirilmesi sırasında, agaroz jel elektroforez kullanılarak boyutu analizi ile teyit eden PCR ürünü ve PCR ürününün dizilimi yolu ile değerlendirildiler. </li></ol></li></ol> 3. HRMA <ol><li> Isı plakaları <ol><li> Bir eriyik analiz sisteminde plaka koyun. </li><li> (LightScanner Yazılım Çağrı-IT 2.0) yazılımını açın. </li><li> 60-95 ° C arasında ısı plakaları </li><li> Yeni bir veri depolama dosyası oluşturun. </li></ol></li><li> HRMA verileri analiz (Şekil 2). Analiz pek çok aşamadan sonra mümkündür. Bu veri m en yaygın olarak kullanılan bir dizi göstermektediranipulations. <ol><li> Alt Küme ayarı Altkümesini sekmesini tıklatın. Örnekleri ile kuyu seçin. </li><li> Normalleştirme <ol><li> Sol paneldeki Normale sekmesini seçin. Floresan varyans ortadan kaldırmak için, elle öncesi (başlangıç) olarak paralel çift çizgi konumlandırmak ve sonrası (son)-eriyik bölgeleri (Şekil 2C, ok uçları) gösterildiği gibi ve 1 ve 0 tüm örneklerin eritmek ve post-eriyik floresan sinyalleri normalleştirmek sırasıyla 14. </li><li> Erime bölgesi öncesi ve sonrası eriyik hatları arasındaki bölgede, ama seçilmemiş böylece hatlarının konumlandırma ayarlayın. Genel olarak, Aşağı Min ve Aşağı Max (ve Yukarı Min ve Üst Max) sıcaklık hatları birbirinden yaklaşık 1 ° C konulmalıdır. </li><li> Normalleştirilmesi için sıcaklık pozisyonlar seçin tüm örnekler (eritmek bölge ve en az ya da post-eritme bölgesi için bir (% 0) floresan için maksimum veya tam (% 100) sahip olacak şekilde floresan kutuplu olarak en iyi olarakmak). Normalizasyon erime noktası ve 0 en az floresan de erime noktasından daha sonra yakın bir yatay çizgi kadar uzanır 1 en yüksek floresan da yakın bir yatay bir çizgi ile sonuçlanacaktır, 1 üzerinde veya 0 ° C&#39;nin altındaki floresan seviyeleri olmamalıdır . Örneğin, Şekil 2C&#39;de, 79-80 ° C&#39;lik sıcaklık aralıkları kullanılarak önceden eritilmesi eğrileri normalize </li></ol></li><li> Gruplama / Kümelenme Erime sıcaklığı (Şekil 2C, yeşil kutu) dayalı genotipleri ayırt. Gruplamasını seçin. <ol><li> Gruplama bölümünde Standartları seçim listesinden Autogroup seçin. </li><li> Gruplama bölümünde Hassasiyet seçim listesinden sırasıyla tek geçişler veya çoklu geçişler ile profiller eritmek için Normal veya Yüksek seçeneğini seçin. </li><li> Seç ComputeGruplama bölümünde gruplar. </li><li> Dosya menüsüne gidin ve sonuçları kaydetmek için Kaydet&#39;i tıklatın. </li></ol></li></ol></li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

HRMA ile birlikte PCR Zebra balığı genotiplendirebilmek güçlü bir teknolojidir. Bu yaklaşımın avantajı, hız, sağlamlık, ve hatta nokta mutasyonları tespit etmek için hassasiyet vardır. Tüm protokol, fin-klip için eritilerek eğri analizi, tek bir kişi tarafından daha az sekiz saat içinde gerçekleştirilebilir. Buna ek olarak, teknik, yüksek miktarda madde taraması için müsait olan, etidyum bromid kullanımını gerektirmeyen ve kirlenme sorunları en aza indirir ve her PCR analizi adımları i?...

Zebrafish (Br.rerio) yaygın gelişimi ve hastalığın modelleme çalışmaları için kullanılan bir omurgalı bir model sistem. Son zamanlarda çok sayıda transgenik ve mutasyon zebrabalıkları teknolojileri geliştirilmiştir. Genellikle Tol2 transpozon sistemi 1 göre hızlı transgenesis teknikleri, birden fazla DNA fragmanı düzeneği 2 için gelişmiş klonlama seçenekleri ile kombine edilmiştir. Çinko-Parmak nükleazlar (ZFNs) ve transkripsiyon aktivatörü gibi efektör nükleazlar (TALENS) zebra balığı 3,4 hem somatik hem de germlin hücrelerinde mahalleri hedeflemek üzere kullanılmıştır. Bu teknikler, etkili bir şekilde yüksek frekanslı mutasyon oluşturulması ve germ-hattı iletim 3,4 olan, genetik olarak modifiye edilmiş hayvanlar oluşturabilir. Bu ilerlemelere rağmen, zebrabalıkları geleneksel genotip teknikleri mütajeni ve Transgenesis araçlarının tam gücünü sınırlamak. PCR bazen KISITLAMAL ile birlikte, jel elektroforezi, ardındann enzim sindirimi, yaygın genom değişiklik tespit etmek için kullanılır, ancak zaman alıcı ve küçük eklemeleri veya silmeleri tanımlamak için daha az hassas olmasıdır. TaqMan probu tahliller yüksek başlangıç ​​maliyetleri ve dikkatli optimizasyon gerektirir. PCR ürünlerinin dizileme birkaç gün sürebilir ve büyük ölçekli tarama için pratik değildir yapabilirsiniz. RFLP (RFLP) analizi sadece kısıtlama enzimi tanıma siteleri, sınırlı etkileyen SNP ayırt edebilir. Yüksek çözünürlüklü erime analizi (HRMA), kapalı-tüp post-PCR analiz yöntemi, hızlı, hassas, ucuz ve örneklerin çok sayıda tarama için müsait bir yeni geliştirilen bir yöntemdir. HRMA SNP, mutasyonlar ve transgenlerin 5-7 tespit etmek için kullanılabilir. HRMA çift sarmallı DNA &#39;ların termik denatürasyona göre ve her bir PCR amplikonu karakteristik benzersiz bir ayrışma (erime) 5 vardır. Numuneler nedeniyle t ayrımcılık olabiliro Farklı nükleotid bileşim olup, GC içeriği veya uzunluğu, tipik olarak, bir floresan boya ile kombinasyon halinde, sadece çift sarmallı DNA 8 bağlar. Böylece, HRMA farklı erime eğrisi özelliklerine göre farklı genotipleri ayırt edebilir. HRMA düşük maliyetli reaktifler kullanır ve bir tek aşamalı post-PCR işlemi olduğu için, yüksek verimli stratejileri için kullanılabilir. HRMA böylece analiz aşağıdaki PCR amplikonları diğer uygulamalar için de kullanılabilir, yıkıcı olmayan bir. HRMA hücre çizgileri, fare ve insan da dahil olmak üzere pek çok, 9-11 organizmalar ve sistemleri, uygulanmıştır. Kullanımı son çinko parmak nükleazlara (ZFNs) ve TALENS 6,12,13 neden mutasyonları tespit etmek için zebrabalıkları tarif edilmiştir. Bu yazıda, (Şekil 1), embriyonik ve yetişkin zebrabalıkları PCR tabanlı HRMA gerçekleştirmek için nasıl açıklar. Bu protokol si de dahil olmak üzere, SNP, transgenlerin ve mutasyonları tespit etmek için uygundurngle temel-çift değişiklikler, eklemeler veya silmeler.

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		<tr height="42">
			<td height="42" style="height:42px;width:232px;">100 Reaction LightScanner Master Mix</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">HRLS-ASY-0002</td>
			<td style="width:157px;">www.biofiredx.com</td>
			<td style="width:167px;">Store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Hard-Shell PCR 96-well BLK/WHT Plates</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">HSP9665</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microseal &#39;B&#39; Adhesive Seals</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">MSB1001</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">96-Well LightScanner Instrument</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">LSCN-ASY-0040</td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Software with Call-IT 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">High-Resolution Melting Analysis 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Primer Design Software</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vector NTI Software</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:141px;"></td>
			<td>www.invitrogen.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tricaine</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:232px;">Paraformaldehyde</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
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rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis

Zebra balığı, geliştirme eğitimi hastalık modelleme ve uyuşturucu taraması gerçekleştirmek için güçlü bir omurgalı model sistem. En son genetik araçlar çeşitli mutasyonlar neden olan ve transgenik çizgiler üretmek için çeşitli stratejiler de dahil olmak üzere, getirilmiştir. Bununla birlikte, büyük ölçekli tarama zaman alıcı ve emek yoğun olan geleneksel genotipleme yöntemleri ile sınırlıdır. Burada yüksek çözünürlüklü erime analizi (HRMA) ile birlikte PCR ile Zebrafish genotipleri analiz etmek için bir tekniği açıklar. Bu yaklaşım, kirlenme eserler düşük riski, hızlı, hassas ve ucuzdur. HRMA ile PCR ile genotipleme yüksek verimli tarama protokolleri de dahil olmak üzere, embriyo veya yetişkin balıklar için kullanılabilir.

Protokol (iş akış diyagramı Şekil 1 &#39;de gösterilmiştir), birkaç gün boyunca tek bir gün boyunca gerçekleştirilen ya da kademeleri içinde ayrılabilir. DNA ekstraksiyon PCR amplikonların eriyik ve analiz takip eder. Amplikonun eriyik için sıcaklıklar büyüklüğü ve GC-içeriğine bağlıdır, ancak genellikle başlangıç ​​ve 50 ˚ C ve 95 ° C ˚ uç sıcaklıkları (Şekil 2A ve 2B) uygundur. Eriyik gerçekleştirildiğinde, floresan erime ...

Watch this Scientific Journal Video about Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis at JoVE.com

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Yüksek çözünürlüklü erime analizi (HRMA) ile bir araya PCR zebrafish genotip için hızlı ve etkili bir yöntem olarak gösterilmiştir.

Yüksek çözünürlüklü Melt Analizi ile PCR kullanarak hızlı ve verimli Zebra Genotiplemesi

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

rapid-efficient-zebrafish-genotyping-using-pcr-with-high-resolution

Research

JoVE Journal

Biology

22.1K Görüntüleme.  University of Utah School of Medicine.  Yüksek çözünürlüklü erime analizi (HRMA) ile bir araya PCR zebrafish genotip için hızlı ve etkili bir yöntem olarak gösterilmiştir. 

Video: Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Sigma&#39;nın Özü-N-Amp Doku PCR Kit kullanarak Fare Doku hızlı Genotiplendirme

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Biyoloji

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.

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

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

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

Mikro Termal Isı Yayımı kullanarak Rapid PCR Thermocycling

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-tip ve Süs Çeşitler arasında ayırt A PCR tabanlı Genotiplendirme Yöntemi Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Bitki damarları ve Üç Boyutlu Yapı ve Fonksiyon Visualize&#39;a Yüksek Çözünürlüklü Bilgisayarlı Tomografi Kullanımı

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Erken evre Embriyolar verimli ve hızlı İzolasyon<em&gt; Arabidopsis thaliana</em&gt; Tohumlar

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Yüksek Çözünürlüklü Tüm Dağı<em&gt; Yerinde</emGen İfade ve Fonksiyon Eğitim için Zebra balığı Embriyolar içinde&gt; Hibridizasyon

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag Analizi - A Yüksek Verimli, Sc2.0 Genotiplendirme için Real Time PCR Testi

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Yüksek çözünürlüklü görüntüleme ve Bireysel Astral Mikrotubul Dynamics Analizi Budding Maya

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...