Name: array comparative genomic hybridization (array cgh) for detection of genomic copy number variants
Uploaded: 2015-02-21T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants at JoVE.com

The authors have no acknowledgements.

1. Etiketleme Reaksiyon <ol><li> Kullanıma hazır -20 ° C&#39;de plakalar ve mağaza 96 oyuklu içine üretici tarafından tavsiye edilen derişimlerde, ön kısım siyanin 3 ve 5 etiketli dUTPs, etiketlenmemiş nükleotidleri ve rastgele primerler Kullanımdan önce. Bu protokol, kısım uygun etiketli dUTP&#39;nin 10.5 ul ve 10.5 ul etiketsiz nükleotidler ve her kuyuya rastgele primerler amaçları için. </li><li> Işıksız bir ortamda, yaklaşık 1 saat süreyle 4 ° C &#39;de nükleotid primerler hazır kullanımlı 96 gözlü levha, çözülme. </li><li> Çözülmüş sonra, ışıksız bir ortamda, en az 30 dakika, oda sıcaklığında, bu plaka dengeye getirin. </li><li> En az 15 dakika boyunca 60 ° C &#39;de DNA örnekleri dengelenmesi. </li><li> Bir sıvı kotarma robotu kullanılarak, 96 oyuklu bir plakanın her bir oyuğuna, nükleaz içermeyen su akıtın. Not: su hacmi, her giriş bir DNA numunesi konsantrasyonuna bağlı olacaktır ve 50 ng / ul nihai konsantrasyona neden olmak için yeterli olmalıdır. </li><li> Bir sıvı han kullanılmasıdling robotu, 96-çukurlu plaka içinde bir kuyu içine pipet DNA 1 ug (1.5), 20 ul toplam hacim getirmek. NOT: Veri Elde niteliği tehlikeye bununla birlikte DNA&#39;nın küçük miktarları kullanılabilir (değerli örnekler için, 400 ng kadar giriş DNA miktarları kullanılabilir). </li><li> Bir sıvı kotarma robotu kullanılarak seyreltilmiş DNA plakanın her oyuğuna dengelenmiş nükleotid primerler 20 ul transfer. </li><li> Şerit kapaklar (sıkı mühür önemlidir) ile 96-plaka Seal. </li><li> Bir ısıtılmış kapak ile, bir PCR makinesi içinde, 10 dakika boyunca 99 ° C&#39;de DNA&#39;nın denatüre edilmesi ve daha sonra tavlama primerler, 5 dakika için buz üzerinde soğuk ek. </li><li> Bir sıvı kotarma robotu kullanarak, iyice her DNA&#39;lar ve pipet karışımı elde etmek için Klenow DNA polimeraz enzimi Exo 10 ul ekle. NOT: toplam hacim 50 ul olmalıdır. </li><li> 96 oyuklu plaka kapatın ve 16 saat 37 ° C&#39;de inkübe edin. Not: 4 st daha kısa bir kuluçkalama, ancak bir O / N yeterlidircubation daha uygun olabilir. </li><li> Bir sıvı kotarma robotu kullanılarak, her bir durdurma tamponu 5 ul ilave edin. Not: etiketleme reaksiyonu için kullanılan reaktif bir özeti Tablo 1&#39;de gösterilmiştir. </li></ol> Adi Nükleotidlerinin 2. Kaldırma <ol><li> Silika-membran esaslı spin sütunları ve özel spin kolon işleme robot (spin sütunları elle de işlenebilir) kullanarak nükleotidleri çıkarın. Üreticinin talimatlarını izleyin. <ol><li> Iyi bir 2 ml tüp her içeriğini aktarmak; kuyu kimlikleri ile ön-etiket bu. NOT: Bu adım, bir sıvı kotarma robotu kullanılarak elle veya tercihen gerçekleştirilebilir. </li><li> Bir spin kolon işleme robot kullanıyorsanız, o zaman 2 ml tüpler, DNA saflaştırma kolonlanyla ve ilişkili tamponlar ile robot yükleyin. </li><li> Silika etiketli DNA bağlamak için etiketleme reaksiyonuna 250 ul yüksek tuz, DNA bağlama tamponu (tampon PB) EkleMembran. </li><li> 500 ul yıkama tamponu (PE tampon A) ile iki kez membran yıkama ile yabancı maddelerin çıkarın. </li><li> ~ 12 ul bir hacim içinde, saflaştırılmış etiketli DNA kurtarmak için zara 15 ul düşük tuz elüsyon tamponu (EB tampon A) ekleyin. </li></ol></li></ol> 3. Hyb Çiftleri birleştirin <ol><li> Cyanine 3 ve 5-etiketli DNA uygun çiftleri birleştirin, COT-1 DNA üretici tarafından sağlanan engelleme karışımı ve sıvı taşıma robotu kullanarak üretici tarafından sağlanan hibridizasyon tamponu. NOT: Paylaşılan CNVs bir sorun yoksa bu hyb çiftleri (örneğin, rutin çok olduğu gibi, böyle bir kraniyosinostozlar hasta vs renal displazi hasta olarak fenotip-uyumsuz örnekleri, eşleştirmek numune vs örnek ve referans veya örnek olabilir ) aynı klinik olarak anlamlı KNV taşımak için olası. Bir referans DNA kullanıyorsanız, ticari DNA kaynağı önerilir, ve numune ile birlikte işleme tabi tutulmalıdır. <ol><li> Bir sıvı taşıma ro kullanılmasıBot, COT-1 DNA (3333 ng / ul) ekleyin üretici tarafından sağlanan her bir çukura 1.1 ul COT-1 DNA, 4.95 ul bloke karışım ve 24,75 ihtiva ettiği şekilde, 96 oyuklu plakanın her oyuğuna karışım ve hibridizasyon tamponu bloke ul hibridizasyon tamponu. </li><li> Her bir oyuğa, uygun siyanin 3-etiketli DNA 9.35 ul ekleyin. Ön-ıslak her ipucu doğruluğunu pipetle geliştirmek için. </li><li> Her bir oyuğa, uygun siyanin 5-etiketli DNA 9.35 ul ekleyin. Ön-ıslak her ipucu doğruluğunu pipetle geliştirmek için. </li><li> 1 dakika boyunca 96 gözlü plaka ve vorteks kapatın. Her bir alt içeriğini toplamak için 96 oyuklu plaka dönerler. </li></ol></li></ol> 4. Melezleme <ol><li> 65 ° C bir melezleşme firinda bir destek slayt ve her dizi slayt için bir melezleme odasına prewarm. <ol><li> Diziler için geçerli olmadan önce bir ön-hibridizasyon inkübasyon etiketli DNA denatüre. 24 saat için bir döner fırın içinde hibridizasyon yapın. </li><li> 30 dakika boyunca 70 ° C&#39;de 96-çukurlu plaka inkübe ve ardından en az 5 dakika boyunca 37 ° C&#39;de bırakın. </li><li> 42 ° C&#39;de ısıtılmış bir platform üzerinde çalışarak, kullanımdan önce bir hibridizasyon odasına her sırt slayt yükleyin. Conta slayt şeffaf kısmı melezleme odasının &#39;pencereli&#39; kısmı ile aynı hizada olduğundan emin olun. </li><li> Pipet önceden hazırlanmış melezleme karışımı 42 ul slayt destek dizi üzerindeki uygun pozisyonda üzerine (bölüm 3). Ön-ıslak her ipucu doğruluğunu pipetle geliştirmek için. Pipet yavaş yavaş emin her pozisyon merkezinde, üzerine sıvı kauçuk halka sınır dokunmaz. </li><li> Destek slayt tüm pozisyonları doldurulur sonra, dikkatle üzerinde dizi slayt düşürmek ve melezleme odasına monte. Üzerinde yazılı olan dizinin yan destek slayt baktığından emin olun. Tam melezleme odası vidasını sıkın emin olun. </li><li> Monte hibridizasyon odasına kontrol edin. EOrada kauçuk halka sınırları dışında melezleme karışımı kaçak ve melezleme odası dikey sonuna dinlenmiş olduğunda her hava kabarcığı yüksekliği ~ 4 mm olduğunu nsure. Hibridizasyon odasına döndürün ve konumda ve hareket etmiyor sıkışmış hava kabarcıkları olmadığını kontrol edin. Bunlardan herhangi varsa, odayı bankta bir keskin musluk verin. Tüm hava kabarcıkları hareket olduğunu kontrol edin. </li><li> 24 saat boyunca 65 ° C&#39;de döner fırına melezleştirme odasına yerleştirin. Fırın rotor dengeli olduğundan emin olun; Gerekirse diğer boş odaları kullanın. </li></ol></li></ol> 5. Yıkama ve Tarama Slaytlar <ol><li> Fazla etiketli DNA kaldırmak ve daha sonra her sonda floresan ölçmek için tarama diziler yıkayın. <ol><li> Fırın ve demonte dışarı hibridizasyon odaları atın. dizi ve conta slaytlar araya sıkışmış olacak; yıkama tamponu 1 bu batığın ve onları ayrı gözetlemek için plastik, düz kenarlı forseps bir çift kullanın. </li><li> Diconta slayt scard ve tampon 1 batık bir rafa dizi slayt yerleştirin. </li><li> Kuvvetli bir şekilde karıştırma 5 dakika için taze yıkama tamponu 1 ~ 700 ml bir dizi slayt yıkayın (pire ve manyetik bir karıştırıcı kullanılır). </li><li> ~ 700 ml yıkama tamponu 2 dizi slayt aktarın ve kuvvetli bir şekilde karıştırıldı, 90 sn için yıkayın. Yavaşça yıkama tamponu 2 dışarı dizi slaytlar asansör, onlar kuru ortaya çıkmalıdır. </li><li> Bir slayt koruyucusu kullanarak, tarayıcı bir slayt tutucu içine dizi slayt yükleyin. Dizi tarayıcıya slayt yükleyin ve dizi üreticisinin talimatları izleyerek tarayın. </li></ol></li></ol>

<ol>
	<li>Miller, D. T., 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=Consensus+statement:+chromosomal+microarray+is+a+first-tier+clinical+diagnostic+test+for+individuals+with+developmental+disabilities+or+congenital+anomalies.">Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.</a> Am. J. Hum. Genet. 86, 749-764 (2010).</li><li>Ahn, J. W., 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=Array+CGH+as+a+first+line+diagnostic+test+in+place+of+karyotyping+for+postnatal+referrals+-+results+from+four+years'+clinical+application+for+over+8,700+patients.">Array CGH as a first line diagnostic test in place of karyotyping for postnatal referrals - results from four years' clinical application for over 8,700 patients.</a> Mol. Cytogenet. 6 (16), 1755-8166 (2013).</li><li>Ahn, J. W., 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=Validation+and+implementation+of+array+comparative+genomic+hybridisation+as+a+first+line+test+in+place+of+postnatal+karyotyping+for+genome+imbalance.">Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance.</a> Mol. Cytogenet. 3 (9), 1755-8166 (2010).</li><li>Beaudet, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+chromosomal+microarray+analysis+in+developmental+and+behavioral+pediatrics.">The utility of chromosomal microarray analysis in developmental and behavioral pediatrics.</a> Child Dev. 84, 121-132 (2013).</li><li>Park, S. J., 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=Clinical+implementation+of+whole-genome+array+CGH+as+a+first-tier+test+in+5080+pre+and+postnatal+cases.">Clinical implementation of whole-genome array CGH as a first-tier test in 5080 pre and postnatal cases.</a> Mol. Cytogenet. 4, 12 (2011).</li><li>Iafrate, A. J., 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=Detection+of+large-scale+variation+in+the+human+genome.">Detection of large-scale variation in the human genome.</a> Nat. Genet. 36, 949-951 (2004).</li><li>Redon, R., 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=Global+variation+in+copy+number+in+the+human+genome.">Global variation in copy number in the human genome.</a> Nature. 444, 444-454 (2006).</li><li>Cafferkey, M., Ahn, J. W., Flinter, F., Ogilvie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+features+in+patients+with+15q11.2(BP1-BP2)+deletion:+Further+delineation+of+an+emerging+syndrome.">Phenotypic features in patients with 15q11.2(BP1-BP2) deletion: Further delineation of an emerging syndrome.</a> Am. J. Med. Genet. A. 164, 1916-1922 (2014).</li><li>Molin, A. M., 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+novel+microdeletion+syndrome+at+3q13.31+characterised+by+developmental+delay,+postnatal+overgrowth,+hypoplastic+male+genitals,+and+characteristic+facial+features.">A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.</a> J. Med. Genet. 49, 104-109 (2012).</li><li>Tropeano, M., 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=Male-biased+autosomal+effect+of+16p13.11+copy+number+variation+in+neurodevelopmental+disorders.">Male-biased autosomal effect of 16p13.11 copy number variation in neurodevelopmental disorders.</a> PLoS One. 8, e61365 (2013).</li><li>Bon, B. W., 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=Further+delineation+of+the+15q13+microdeletion+and+duplication+syndromes:+a+clinical+spectrum+varying+from+non-pathogenic+to+a+severe+outcome.">Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome.</a> J. Med. Genet. 46, 511-523 (2009).</li></ol>

The authors have nothing to disclose.

Dizi CGH gibi triploidi gibi dengeli düzenlenmeleri veya ploidisi anormallikleri tespit olmayacaktır. Ayrıca, düşük seviyeli mozaik dengesizlikler tespit edilemeyebilir. Ancak dizi CGH birçok sitogenetik laboratuvarda yerini aldı G-bantlı kromozom analizi daha KNV tespiti için daha yüksek bir çözünürlüğe sahip. Bu nedenle genom KNV tespiti için geçerli altın standarttır. Gelecekte yeni nesil dizileme teknolojileri ile değiştirilebilir, ancak şu anda, kendi maliyet ve teknik karmaşıklığı bu h...

Bu silmeler veya kromozomal bölümlerinin ekstra kopyaları zihinsel engeli, dismorfik ve congentical malformasyonlar neden olduğu uzun yıllardır bilinmektedir ve bazı durumlarda genetik sendromlara neden olmuştur 1. Ancak, bu değişikliklerin genom tespiti için 2000&#39;li yılların ortalarında kadar mevcut tek teknoloji olamaz dengesizlik bölge ve doğasına bağlı olarak, 5-10Mb etrafında bir çözünürlüğe sahip kromozom analizi,-bantlı, G, ve hangi Malzeme, aynı bant modeli farklı bir kromozom bir bölge ile ikame edilmiş anormal kromozom algılar. Böyle situ hibridizasyon ve multipleks ligasyon-spesifik prob amplifikasyon floresan gibi yardımcı sitogenetik teknikler şüpheli spesifik sendromik dengesizlik durumlarında, belirli loci sorgulama için kullanılabilir olmuştur, ama bu dizi karşılaştırmalı genomik hibridizasyon getirilmesi kadar değildi (dizi CGH) Rutin klinik tanı s içinekopya sayısının genom algılama (CNVs) varyantları ervice 2-5 (genellikle 120KB civarında) bir ölçüde artmış çözünürlükte mümkün oldu. Bazı bölgelerde normal popülasyona 6-7 yaygındır için araştırma çalışmaları yanında, klinik hizmet çalışmaları diğer CNVs ederken, daha önce saptanamayan, otizm ve epilepsi 8-11 gibi neurodisabilities ile ilişkili, CNVs olduğunu göstermiştir. Bu yazıda anlatılan protokol bizim İngiltere Ulusal Sağlık Servisi (NHS) klinik tanı laboratuvarında kullanılan; Bu devlet destekli hizmet maliyetini en aza indirmek için yeni melezleme stratejileri, toplu test ve robotik kullanın. Aşağıda detayları verilen protokol önce, yüksek kaliteli DNA, uygun başlangıç ​​malzemesi, yaygın olarak kan, kültürlenmiş hücreler ya da doku numunelerinin elde edilmesi gerekmektedir. 280 emme oranı (b gerekir: Spektrofotometri konsantrasyonunun ölçülmesi için kullanılabilir ve 260 kontrol (&gt; 50 ng / ul olması gerekir)e 1,8-2,0). Jel elektroforezi, DNA önemli bir bozulma olmadan yüksek moleküler ağırlığa sahip olup olmadığını kontrol etmek için kullanılabilir. Bu protokol otomatik sıvı taşıma robotik kullanarak çalıştırmak başına 96 numune etiketleme yüksek verim laboratuvarlar için tasarlanmıştır. Bununla birlikte, otomasyon olmaksızın daha düşük verim laboratuarlar için adapte edilmiş olabilir.

<table><tbody><tr><td>CGH microarray 8 x 60K</td><td>Agilent</td><td>G4126</td><td></td></tr><tr><td>Array CGH wash buffers</td><td>Agilent</td><td>5188-5226</td><td></td></tr><tr><td>Array CGH hybridisation buffer</td><td>Agilent</td><td>5188-5380</td><td></td></tr><tr><td>Minelute purification kit</td><td>Qiagen</td><td>28006</td><td></td></tr><tr><td>Array CGH labelling kit</td><td>Enzo</td><td>ENZ-42672-0000</td><td></td></tr></tbody></table>

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.

Bir hibritlenmiş dizi Her prob kırmızı ve yeşil fluorochromes bir karışımı olarak görüntülenmiştir (Şekil 1). her bir prob için yeşil floresan sinyalinin kırmızı oranı tarayıcı tarafından ölçülür ve ilgili yazılım genomik pozisyonuna göre log2 oranları gibi, bu çizer ve dış önceden belirlenmiş sınırları düşen bölgeleri tanımlar. Elde edilen dizi izleri genomik dengesiz olarak tanımlanan bölgeler yoruma imkan vermek. Örneğin, Williams sendromu, kromozom 7&#3...

Watch this Scientific Journal Video about Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants at JoVE.com

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

Genomik kopya sayısı varyantları tespiti için Dizi CGH G-bantlı karyotip analizi yerini aldı. Bu kağıt teknolojisi ve teşhis hizmet laboratuvarında uygulanmasını açıklar.

Genomik Kopya Sayısı Variantların Algılama için dizi Karşılaştırmalı Genomik Hibridizasyon (CGH Array)

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

array-comparative-genomic-hybridization-array-cgh-for-detection

Research

JoVE Journal

Biology

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

19.5K Görüntüleme.  Guy's & St Thomas' NHS Foundation Trust.  Genomik kopya sayısı varyantları tespiti için Dizi CGH G-bantlı karyotip analizi yerini aldı. Bu kağıt teknolojisi ve teşhis hizmet laboratuvarında uygulanmasını açıklar. 

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

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Tek Hücre sıralama kullanarak Kopya sayısı Değişmeler Algılama

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genetik

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantasyon Genetik Tanı için FISH

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Tıp

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum ve plazma kopya sayı algılama gerçek zamanlı PCR kullanarak

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Kanser Araştırmaları

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

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

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

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

Dizi tabanlı Karşılaştırmalı genomik hibridizasyon için bir Platform verimli algılama varyasyonları kopya numarası hızlı nötron kaynaklı Medicago truncatula mutantlar içinde

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

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

Bir CDH1 nadir transkript değişken mide kanseri taze donmuş dokularda çip tabanlı dijital PCR tarafından algılanması

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Klinik kohort çalışmaları çoğaltılmış Genotipleme ve kütle spektrometresi ile test aday gen

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

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

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

Tüm Genom Dizisi Karşılaştırmalı Genomik Hibridizasyon Teknik gösteri

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 ...

Biyoloji

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

Yarı İletken Tabanlı Yeni Nesil Dizileme Platformunda Anöploidi için İmplantasyon Öncesi Genetik Test

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Hedefe Yönelik Sıralama Yaklaşımı ile Karşılaştırmalı Lezyonlar Analizi

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

Davranış

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Dijital Nükleik Asit Kantifikasyon Tahliller Basit Toplu Saati

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...