Name: an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants
Uploaded: 2017-11-08T15:00:00
Description: Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

Financiering van dit werk wordt geleverd ten dele door een subsidie van NSF plantenonderzoek genoom (IOS-1127155).

Opmerking: afbeelding 1 ziet u de vijf stappen voor de array CGH protocol. Ze zijn: 1) voorbereiding van plantaardig materiaal; 2) isolatie van DNA-monsters van hoge kwaliteit; 3) labelen en zuivering van DNA-monsters; 4) hybridisatie, wassen, en scannen van gehele genoom arrays; en 5) CGH data-analyse. M. truncatula hele genoom betegeling matrices bevatten een totaal 971,041 unieke oligo sondes gericht op meer dan 50.000 genen of modellen van het gen in het genoom (Zie ta...

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We hebben een arraygebaseerd CGH platform ontwikkeld voor de detectie en karakterisatie van snelle neutronen bombardement (FNB)-geïnduceerde mutanten in M. truncatula cv. Jemalong A17. Om aan te tonen het gebruik van de array CGH methode voor het opsporen van genmutaties, we aCGH analyse van de mutant FN6191, die tentoongesteld van een hyper-nodulatie fenotype in tegenstelling tot wild type planten, wanneer geënt met S. meliloti Sm1021uitgevoerd. Voor segmentatie analyse, werd een segment beschouwd al...

Peulvruchten (Fabaceae) is de derde grootste familie van tweezaadlobbige planten, met vele economisch belangrijke soorten zoals sojabonen (Glycine max) en luzerne (Medicago sativa). Peulvruchten planten kunnen communiceren met stikstof-vaststelling bodem bacteriën, Rhizobium om root knobbeltjes waarin de atmosferische dinitrogen is gereduceerd tot ammoniak voor gebruik door de waardplant genoemd. Als zodanig, teelt van peulvruchten gewassen vereist weinig inbreng van stikstof meststoffen en draagt aldus bij aan duurzame landbouw. Peulvruchten gewassen produceren bladeren en zaden met een hoog eiwitgehalte, bijeenkomen uitstekend ruwvoer en graan gewassen. Gecultiveerde peulvrucht soorten hebben echter over het algemeen complex genoom structuren, functionele studies van genen die spelen een sleutelrol in peulvruchten-specifieke processen omslachtig maken. Medicago truncatula is wijd goedgekeurd als een soort model voor peulvruchten studies vooral omdat (1) het heeft een diploïde genoom met een relatief kleine haploïde genoom grootte (~ 550 Mbp); (2) planten kunnen stabiel worden omgezet voor gene functionele studies; en (3) het is nauw verwant aan alfalfa (M. sativa), de koningin van voedergewassen en vele andere economisch belangrijke gewassen voor translationeel onderzoek. Onlangs, het genoom van M. truncatula cv Jemalong A17 geweest vrijgegeven1,2. Annotatie van het genoom laat zien dat er meer dan 50.000 voorspelde genen of modellen van het gen in het genoom. Om te bepalen van de functie van de meeste van de genen in de M. truncatula is genoom een uitdagende taak. Ter vergemakkelijking van functionele studies van genen, een uitgebreide verzameling van mutanten in het bereik van meer dan 150.000 M1 lijnen is gegenereerd met behulp van snelle neutronen bombardement (FNB) mutagenese in M. truncatula cv Jemalong A173 ,4. Snelle neutronen, een energierijke ionisatie mutageen, is gebruikt bij het genereren van mutanten in vele plantensoorten met inbegrip van Arabidopsis5,6, rijst (Oryza sativa)7, tomaat (Solanum lycopersicum), sojabonen (Glycine soja; G. max)8,9, gerst (Hordeum vulgare), en Lotus japonicus10. Een groot deel van mutaties afgeleid van FNB mutagenese zijn als gevolg van DNA deleties die variëren in grootte van een paar basenparen op mega basenpaar9,11. Vele fenotype-geassocieerde genen met succes zijn geïdentificeerd en gekarakteriseerd4,12,13,14,15,16, 17 , 18 , 19. eerder, moleculaire klonen van de onderliggende genen van FNB mutanten vertrouwd op een kaart gebaseerde benadering, die is tijdrovend en beperkt het aantal mutanten te worden gekenmerkt op moleculair niveau. Onlangs, verscheidene gratis benaderingen, met inbegrip van transcriptie gebaseerde methoden, genoom arraygebaseerd vergelijkende genomic hybridisatie (CGH) voor DNA kopie nummer variatie detectie, en hele genoom sequencing, plavuizen zijn tewerkgesteld te vergemakkelijken de karakterisering van schrapping mutanten in verschillende organismen, met inbegrip van dieren en planten20,21,22,23,24,25, 26,27,28,29,30,31.
Om te vergemakkelijken de karakterisatie van FNB mutanten in M. truncatula, een geheel-genoom arraygebaseerd vergelijkende genomic hybridisatie (CGH) is platform ontwikkeld en gevalideerd. Zoals gemeld in dierlijke systemen, maakt het platform CGH arraygebaseerd de ontdekking van kopie aantal variaties (CNVs) op het niveau van de hele genoom in M. truncatula FNB mutanten. Bovendien, letsels kunnen worden bevestigd door PCR en schrapping grenzen kunnen worden geïdentificeerd door sequentiebepaling. Over het geheel genomen is de matrix CGH platform een efficiënte en effectieve hulpmiddel bij het identificeren van de laesies in M. truncatula FNB mutanten. Hier, worden de matrix CGH procedure en PCR karakterisering van schrapping grenzen in een M. truncatula FNB mutant geïllustreerd.
Het volgende protocol voorziet experimentele stappen en informatie over reagentia, apparatuur en analysetools voor onderzoekers die geïnteresseerd zijn in het uitvoeren van volledige genoomanalyse arraygebaseerd vergelijkende genomic hybridisatie (CGH) van exemplaaraantal variaties in planten. Als voorbeeld, werd Medicago truncatula FN6191 mutant gebruikt om te identificeren schrapping regio's en de kandidaat-genen mutant fenotypen is gekoppeld. M. truncatula FN6191 mutant, oorspronkelijk geïsoleerd van een snelle neutronen schrapping bombardement-geïnduceerde gemuteerde collectie32 (Zie Tabel of Materials), een hyper-nodulatie fenotype tentoongesteld na inoculatie met de bodem bacterie, Sihorhizobium meliloti heeft Sm1021, in tegenstelling tot wild type planten.

<table>
	<tbody>
		<tr>
			<td>Medicago truncatula genome array, 1 x 1 M</td>
			<td>Agilent</td>
			<td>G4123A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula FN6191 (mutant)</td>
			<td>In house</td>
			<td>FN6191</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula Jemalong A17 (reference)</td>
			<td>In house</td>
			<td>A17</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>1000D</td>
			<td></td>
		</tr>
		<tr>
			<td>SureTag DNA Labeling Kit</td>
			<td>Agilent</td>
			<td>5190-3400</td>
			<td></td>
		</tr>
		<tr>
			<td>Random primer</td>
			<td>Agilent</td>
			<td>5190-3399</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>MJ research</td>
			<td>PTC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Labnet international Inc</td>
			<td>Spectrafuge 24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and Drying Solution</td>
			<td>Agilent</td>
			<td>5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Hybridization Kit</td>
			<td>Agilent</td>
			<td>5188-5380</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber gasket slides</td>
			<td>Agilent</td>
			<td>G2505</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Agilent</td>
			<td>5190-3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Wash Buffer 1 and 2</td>
			<td>Agilent</td>
			<td>5188-5221</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber, stainless</td>
			<td>Agilent</td>
			<td>G2534A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Agilent</td>
			<td>G2545A</td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Columns</td>
			<td>Agilent</td>
			<td>5190-3391</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanner</td>
			<td>Roche</td>
			<td>MS200</td>
			<td></td>
		</tr>
		<tr>
			<td>NimbleScan 2.6</td>
			<td>Roche Nimblegen</td>
			<td>5225035001</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal Map 1.9</td>
			<td>Roche Nimblegen</td>
			<td>Signalmap1.9</td>
			<td></td>
		</tr>
	</tbody>
</table>

an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants

Mutanten zijn onschatbare waarde genetische hulpbronnen voor gene functie studies. Voor het genereren van mutant collecties, kunnen drie soorten mutagene agentia worden gebruikt, met inbegrip van biologische zoals T-DNA of transposon, zoals ethyl methanesulfonate (EMS) chemische of fysische zoals ionisatie straling. Het soort mutatie waargenomen varieert afhankelijk van de mutagene stof gebruikt. Mutaties omvatten ionisatie straling geïnduceerde mutanten, verwijdering, duplicatie of omlegging. Terwijl T-DNA of op basis van transposon mutagenese beperkt tot de soorten die vatbaar voor transformatie is zijn, kan chemische of fysische mutagenese worden toegepast op een breed scala van soorten. De karakterisering van mutaties afgeleid van chemische of fysische mutagenese traditioneel is echter afhankelijk van een kaart-gebaseerde klonen benadering die arbeid intensief en tijdrovend is. Hier, laten we zien dat een high-density genoom arraygebaseerd vergelijkende genomic hybridisatie (aCGH) platform kan worden toegepast om efficiënt detecteren en karakteriseren van kopie aantal variaties (CNVs) in mutanten afgeleid van snelle neutronen bombardement (FNB) mutagenese in Medicago truncatula, een peulvrucht soorten. Volledige genoomanalyse van de reeks toont aan dat er meer dan 50.000 genen of gene modellen in M. truncatula. Bij de huidige, FNB-geïnduceerde mutanten in M. truncatula zijn afgeleid van meer dan 150.000 M1 lijnen, die van onschatbare waarde plantgenetische hulpbronnen voor functionele studies van genen in de genoom. Het platform van de aCGH hier beschreven is een doeltreffend instrument voor het karakteriseren van FNB-geïnduceerde mutanten in M. truncatula.

Figuur 2 geeft de distributie van genormaliseerde log2 verhoudingen van mutant versus WT signalen over het hele genoom. Analyse van CGH gegevens bleek benaderend 22 kb schrapping op chromosoom 4, die de gehele SUNN gene33 en diverse andere omvat geannoteerde genen in de mutant FN6191 (Figuur 2, Figuur 3). De kandidaat-verwijderde regio werd gecoverd door 73...

Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Dit protocol biedt experimentele stappen en informatie over reagentia, apparatuur en analysetools voor onderzoekers die geïnteresseerd zijn in het hele genoom arraygebaseerd vergelijkende genomic hybridisatie (CGH) analyse van kopie nummer variaties in uit te voeren planten.

Een Array gebaseerde vergelijkende Genomic hybridisatie Platform voor de efficiënte opsporing van kopie-nummer variaties in snelle neutronen geïnduceerde Medicago truncatula mutanten

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

The overall goal of this array-based comparative genomic hybridization protocol is to rapidly identify copy number variations in fast neutron bombardment induced mutants of the legume plant Medicago truncatula. This method helps to answer key questions in the field of legume biology. Such as, facilitating identification of empathogens on low site involved in regulating nodule development in legumes.The main advantage of this technique is that it is of highest repute and sensitive in detecting copy number variations such as deletion mutations in the neutron bombardment mutants of the legume plant Medicago truncatula. Demonstrating this procedure will be myself, and Hongcheng Wang, Host Doctor Fellows from Genetic Lab. Quickly freeze one gram of young leaf tissue collected from single plants in liquid nitrogen.Then, use a mortar and pestle and grind the frozen leaf tissue to fine powder within the liquid nitrogen. Extract the wild type and mutant genomic DNA samples from the fine powder using a DNA isolation kit. Use 500 microliter centrifuge tubes to dilute one microgram of each wild type and mutant genomic DNAs with double distilled water to a final volume of 20 microliters.Then, add five microliters of random primer to the centrifuge tubes. Quickly vortex the tubes, and after a quick spin down place them in a thermocycler for 10 minutes to denature the DNA samples at 98 degrees Celsius. After 10 minutes remove the tubes from the thermocycler, and instantly place them on ice water for five minutes.Then, prepare two labeling mixes and add 25 microliters of labeling mix as one and two to the wild type and mutant DNA tubes respectively. Pipette the DNA and the labeling mix three times, and then briefly spin the tubes. After spinning, incubate the tubes in the thermocycler at 37 degrees Celsius for two hours, and then at 65 degrees Celsius for 20 minutes to inactivate the exo Klenow enzyme.Then, add 430 microliters of One X TE Buffer, and mix the contents in each tube. Next, briefly spin the tubes and transfer the solution in the tubes to purification columns with two milliliter collection tubes. Centrifuge the purification columns at 14, 000 times G for 10 minutes and discard the flow through.Add 480 microliters of One X TE Buffer to each column, and again centrifuge at 14, 000 times G for 10 minutes. Discard the flow through. Transfer each of the wild type and mutant labeled DNAs from the bottom of the column to new centrifuge tubes.After measuring the volume of each of the labeled DNAs with the pipette adjust the final volume to 80 microliters with One X TE Buffer, then measure the concentration of the labeled DNAs in a spectrophotometer. Next, mix equivalent volumes of mutant and wild type DNAs to hybridize probes on a micro array chip for comparative hybridization. Bring the final volume to 160 microliters with One X TE Buffer.Next, prepare the hybridization solution and pipette it three times to mix three agents well with mixed DNA. After briefly spinning the tubes incubate them in a thermocycler at 98 degrees Celsius for 10 minutes, and at 37 degrees Celsius for 20 minutes. Remember to set the temperature to 67 degrees Celsius to preheat the hybridization oven four hours before hybridization.Then, place a gasket slide on the base of the hybridization chamber and load 490 microliters of the hybridization solution onto it after 20 minutes of incubation at 37 degrees Celsius in the thermocycler. To form the hybridization chamber cover the gasket slide with Medicago truncatula genome micro array chip, then cover the hybridization chamber and securely tighten it with the clamps. Incubate the assembled hybridization chamber in the hybridization oven at 67 degrees Celsius for 40 to 48 hours.Meanwhile, prepare two slide washing dishes with 250 milliliters of Washing Buffer One kept at room temperature. Then, make one slide washing dish with Washing Buffer Two kept at 37 degrees Celsius. Then, prepare one slide washing jar with 70 milliliters of acetonitrile, and another slide washing jar with 70 milliliters of stabilization and drawing solution.Place both the slide washing jars in a fume hood at room temperature. After incubation remove the hybridization chamber from the hybridization oven and loosen the chamber clamps to open the cover. Then, remove the micro array chip in the gasket slide and place them on slide washing dish with Wash Buffer One.Isolate the micro array chip from the cover slide. Next, transfer the micro array chip to the second slide washing dish with Wash Buffer One. Using a stir bar, gently stir the solution on a magnetic stir plate at room temperature for five minutes.Place the micro array chip on the slide washing dish with pre-warmed Washing Buffer Two, then stir with the stir bar to wash the solution at 37 degrees Celsius for one minute. Remove the micro array chip and place it in the acetonitrile containing slide washing jar in a fume hood to wash for 30 seconds. Transfer the micro array chip to the slide washing jar with stabilization and drying solution and wash again for 30 seconds.Carefully remove the chip and dry for one minute inside the fume hood. Scan the micro array chip using a scanner under two micrometer resolutions. A signal mapping software was used as an interface to analyze the distribution of normalized log two ratios of mutant versus wild type signals across eight chromosomes.For putative deletions value of log two ratio equal to or less than minus two point five standard deviation is considered. Segmentation analysis of the software demonstrates an estimated 22 kilobase deletion region on chromosome four shown in the graph. Analysis of the deletion borders flanked by the micro array probes show a reduced mean normalized log two ratio.The graph also shows six other annotated genes including the son gene encompassing the deleted region. Son gene is responsible for controlling nodulation in Metecago truncatula. Next, PCR amplification done to confirm the deletion borders shows a one point five kilobase product amplified from FN6191 mutant, but not in the wild type.DNA sequencing was done to show the deletion junction in FN6191 mutant shown in the black arrow. Sequencing was also done that confirmed the location of the deletion borders. Once mastered, this technique can be completed in 72 hours if it is performed properly.To obtain high quality data, remember to keep the ozone concentration low in the room where performing the procedure. Following this procedure, other measures such as polymerase chain reactions of all genome sequencing can be performed to answer additional questions like the size and the copy number variations in the genome. Event and validation of this technique has paved the way for researchers to explore copy number variation they induce to mutants and the natural variants in plant species that are not amenable to insertional mutagenesis such as Garden P.Don't forget that working with acetonitrile, stabilization and drying solution can be extremely hazardous.Precautions, such as wearing eye proctection, avoiding direct contact with the agents, and working careful is absolutely essential.

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An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

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