{"id":17071,"date":"2022-09-26T18:22:27","date_gmt":"2022-09-26T18:22:27","guid":{"rendered":"https:\/\/admin.acceleratingscience.com\/behindthebench\/?p=17071"},"modified":"2022-09-26T18:22:27","modified_gmt":"2022-09-26T18:22:27","slug":"double-stranded-dna-quality-control-using-capillary-electrophoresis","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/double-stranded-dna-quality-control-using-capillary-electrophoresis\/","title":{"rendered":"Double-Stranded DNA Quality Control Using Capillary Electrophoresis"},"content":{"rendered":"<h2><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter wp-image-17104 size-large\" title=\"Double-Stranded DNA Quality Control - Start With DNA Isolation\" src=\"http:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/dna-quality-control-1065x607-1-1024x584.jpg\" alt=\"scientists viewing dna quality control data on computer screens\" width=\"760\" height=\"433\" srcset=\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/dna-quality-control-1065x607-1-1024x584.jpg 1024w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/dna-quality-control-1065x607-1-300x171.jpg 300w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/dna-quality-control-1065x607-1-768x438.jpg 768w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/dna-quality-control-1065x607-1.jpg 1065w\" sizes=\"auto, (max-width: 760px) 100vw, 760px\" \/>Start With Isolating DNA<\/h2>\n<p>All genomic analyses start with isolating DNA. This DNA could be genomic DNA from intact organisms, frozen tissues, or from fixed tissues embedded in paraffin (FFPE). It may include PCR amplicons that are used as intermediate steps in a cloning or sequencing reaction. Finally, the construction of next generation sequencing libraries involves the manipulation of pools of DNA fragments.<\/p>\n<h3>Analyze the Amount and Integrity of the Isolated DNA<\/h3>\n<p>In all of these procedures, investigators need to analyze the amount and integrity of the DNA isolated before moving on to more complicated or expensive steps. This ensures efficient use of budget, unbiased NGS results, and general data reliability and reproducibility for all downstream applications.<\/p>\n<p>Examples include:<\/p>\n<ul>\n<li>DNA extracted from FFPE-preserved tissue to examine the level of DNA fragmentation that limit the usefulness of information from subsequent assays;<\/li>\n<li>NGS library QC to determine the size distribution and abundance of library fragments to predict NGS success;<\/li>\n<li>Success of PCR amplification prior further manipulation.<\/li>\n<\/ul>\n<h3>Agarose Gels for Analyzing DNA Fragments<\/h3>\n<p><strong>Agarose gels<\/strong> have long been a standard for analyzing DNA fragments, but while they are inexpensive, they are <strong>no<\/strong><strong>t very sensitive, precise or high throughput<\/strong>. Other platforms have been developed that provide better sensitivity and higher throughput. However, these often rely on expensive specialty chips or reagents. In addition, each of these solutions require a dedicated instrumentation, taking up more space in the lab and requiring additional training and maintenance costs.<\/p>\n<h2><\/h2>\n<h2>The Need for Reliable, Cost-effective dsDNA Integrity Testing<\/h2>\n<h3>Putting Your CE-based Genetic Analyzer to Work<\/h3>\n<p>Scientists at Thermo Fisher Scientific developed a <strong>method for double-stranded DNA (dsDNA) fragment QC<\/strong> that <strong>runs on Applied Biosystems\u2122 existing capillary electrophoresis (CE) genetic analyzers<\/strong>. <strong>Fragment analysis by CE<\/strong> yields quantitative information, while providing further library information such as the size of DNA fragments. This method does not require the purchase of specialty polymers or capillaries, therefore providing additional flexibility and utility for the existing CE systems including the 4-cap <span style=\"text-decoration: underline\"><a title=\"Simple, fast, low-throughput Sanger sequencing and fragment analysis system\" href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing\/sanger-sequencing-technology-accessories\/seqstudio-genetic-analyzer.html\" target=\"_blank\" rel=\"noopener\">SeqStudio Genetic Analyzer<\/a><\/span> and the latest mid-throughput <span style=\"text-decoration: underline\"><a title=\"Flexibility, usability, connectivity, and serviceability in a mid-throughput genetic analyzer\" href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/life-science\/sequencing\/sanger-sequencing\/genetic-analyzers\/models\/seqstudio-flex.html\" target=\"_blank\" rel=\"noopener\">SeqStudio Flex Series Genetic Analyzers<\/a><\/span>.<\/p>\n<h2>A New Twist on Fragment Analysis<\/h2>\n<h3>Turning Dyes and DNA Digests into Data<\/h3>\n<p>The new protocol relies on incubating DNA with a double-stranded DNA intercalating dye, followed by electrophoresis using existing polymers and buffers. One key difference between this dsDNA QC protocol and standard fragment analysis is performing CE separation at low temperature. This maintains the DNA in a partial duplex with the intercalating dye that can be detected as a positive signal. The resulting electropherogram can be examined for relative size(s) of a sample and provide a semi-quantitative measurement of the amount of DNA in the sample.<\/p>\n<p>Details of the method are provided in protocol entitled Analysis of double-stranded DNA fragments using capillary electrophoresis.<sup>1<\/sup>\u00a0 Briefly, TOTO\u2122-1, an inexpensive fluorescent DNA intercalating dye, is incubated with a DNA sample for approximately 10 minutes in water. The sample is then run through POP-7\u2122 polymer on 3500 or SeqStudio Flex systems, or POP-1\u2122 on SeqStudio, with the capillary heater turned off or minimized. Concurrently, a single or set of test-defined fragments, for example phage lambda DNA digested with HindIII or phage phiX174 digested with BsuRI are run in a separate capillary. The peaks generated during electrophoresis are detected using the E5 (DS-02) filter set.<\/p>\n<div id=\"attachment_17074\" style=\"width: 770px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" aria-describedby=\"caption-attachment-17074\" class=\"wp-image-17074 size-large\" title=\"A New Twist on Fragment Analysis - Turning Dyes and DNA Digests into Data\" src=\"http:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/figure1-lambda-hind_iii-digest-3276x946-1-1024x296.png\" alt=\"graph lambda hind iii digest\" width=\"760\" height=\"220\" srcset=\"https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/figure1-lambda-hind_iii-digest-3276x946-1-1024x296.png 1024w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/figure1-lambda-hind_iii-digest-3276x946-1-300x87.png 300w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/figure1-lambda-hind_iii-digest-3276x946-1-768x222.png 768w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/figure1-lambda-hind_iii-digest-3276x946-1-1536x444.png 1536w, https:\/\/admin.acceleratingscience.com\/behindthebench\/wp-content\/uploads\/sites\/9\/2022\/09\/figure1-lambda-hind_iii-digest-3276x946-1-2048x591.png 2048w\" sizes=\"auto, (max-width: 760px) 100vw, 760px\" \/><p id=\"caption-attachment-17074\" class=\"wp-caption-text\"><strong>Migration of well-defined dsDNA fragments on genetic analyzer<\/strong>. The fragment pattern of phage lambda DNA digested with HindIII seen by agarose gel electrophoresis is recapitulated through CE-based fragment analysis using the low temperature protocol.1 LIZ\u21221200 single-stranded standards shown in yellow.<\/p><\/div>\n<p>&nbsp;<\/p>\n<h2>Confidently Perform NGS<\/h2>\n<h3>Confirming Library Quality to Ensure NGS Data Quality<\/h3>\n<p>Once the electropherograms from samples are obtained, the size of the resulting fragment(s) can be estimated by comparing peaks to the fragment standard with known sizes. Small PCR artifacts, intact gDNA, and the fragment sizes of interest are quickly and easily visualized.<\/p>\n<h2>Let LIZ Standards Show You the Way<\/h2>\n<h3>Internal Standard for Increased Precision<\/h3>\n<p>For additional precision, each sample can be run with a LIZ\u21221200 single-stranded standard. By calibrating the known double-stranded fragments to the LIZ1200 peaks, a precise determination of the size of the unknown peak can be determined. Finally, the peak height will be correlated with the amount of DNA in that peak, leading to a semi-quantitative estimate of the amount of DNA present. Of course, there are many variables that determine how much DNA is ultimately injected into the capillary. Therefore, precise quantification requires more extensive calibration and normalization using defined amounts of a similar-sized fragment.<\/p>\n<p>In summary, this method provides a path to fast and simple QC of dsDNA samples using existing Applied Biosystems genetic analyzers.<\/p>\n<p>&nbsp;<\/p>\n<h2>Download Protocol<\/h2>\n<h3><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/global\/forms\/life-science\/dsdna-capillary-electrophoresis-app-note.html\">\u00bb Analysis of Double-Stranded DNA Fragments Using Capillary Electrophoresis\u00a0<\/a><\/h3>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p>\u00ad&#8211;<\/p>\n<h2>Reference<\/h2>\n<ol>\n<li><a href=\"https:\/\/www.thermofisher.com\/us\/en\/home\/global\/forms\/life-science\/dsdna-capillary-electrophoresis-app-note.html\" target=\"_blank\" rel=\"noopener\">Analysis of double-stranded DNA fragments using capillary electrophoresis<\/a>. Thermo Fisher Scientific Application note.<\/li>\n<\/ol>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Start With Isolating DNA All genomic analyses start with isolating DNA. This DNA could be genomic DNA from intact organisms, frozen tissues, or from fixed tissues embedded in paraffin (FFPE). It may include PCR amplicons that are used as intermediate steps in a cloning or sequencing reaction. Finally, the construction of next generation sequencing libraries<\/p>\n","protected":false},"author":120,"featured_media":17104,"comment_status":"open","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[172],"tags":[36,2303,2304,2305,2306,2294,206],"division":[],"class_list":{"0":"post-17071","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-general","8":"tag-capillary-electrophoresis","9":"tag-dna","10":"tag-dna-fragments","11":"tag-double-stranded-dna","12":"tag-dsdna","13":"tag-genetic-analysis","14":"tag-genetic-analyzer","15":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.8 (Yoast SEO v27.8) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Capillary Electrophoresis for Double-Stranded DNA Quality Control<\/title>\n<meta name=\"description\" content=\"The need for reliable, cost-effective dsDNA integrity testing is key to quality control. 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