{"id":11128,"date":"2018-04-05T18:15:05","date_gmt":"2018-04-05T18:15:05","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/?p=11128"},"modified":"2020-05-28T19:15:23","modified_gmt":"2020-05-28T19:15:23","slug":"how-to-evaluate-forensic-dna-quality-with-quantifiler-trio-dna-quantification-kit","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/how-to-evaluate-forensic-dna-quality-with-quantifiler-trio-dna-quantification-kit\/","title":{"rendered":"How To Evaluate Forensic DNA Quality With Quantifiler Trio DNA Quantification Kit"},"content":{"rendered":"

Wouldn\u2019t it be wonderful if there was a tool that forensic scientists could use\u00a0to assess the quality of an unknown DNA sample<\/a>\u00a0before attempting to generate an STR profile?\u00a0DNA samples collected from crime scenes range from DNA-rich fluids, like blood and saliva, to a few skin cells left behind from a casual touch.\u00a0\u00a0For example, there is generally much more DNA in a bloodstain than on, say, a knife handle. Getting an accurate estimate of DNA concentration is crucial to generating a robust DNA profile.\u00a0\u00a0But what if the process of DNA quantification could provide even more information?\u00a0\u00a0What if it could provide information about the quality of a DNA sample?<\/p>\n

Well, luckily there is the\u00a0Quantifiler Trio Quantification Kit<\/a>. The Quant Trio kit provides DNA concentration, in addition to quality assessments for degradation and inhibition of the sample. And it can help you make workflow decisions based on quantity of autosomal vs male DNA present.<\/p>\n

DNA can be degraded by environmental influences like sunlight, extreme heat, and humidity; degradation may manifest as a ski-slope pattern in the STR electropherogram.\u00a0\u00a0You see this pattern with degraded DNA because small fragments remain intact and amplify well, but the large fragments are damaged and don\u2019t amplify well.<\/p>\n

<\/p>\n

To evaluate degradation in forensic samples, a Degradation Index, or DI, has been added to the Quantifiler Trio Kit.\u00a0\u00a0The DI is the ratio of the smaller to larger DNA fragments in a sample.\u00a0\u00a0It is\u00a0automatically calculated in the software<\/a>.<\/p>\n

\"\"<\/p>\n

The DI for intact DNA will be \u22641, as the concentration of the small and large fragments are approximately equal. Any DI over 1 could indicate degradation. To overcome degradation, you could\u00a0target more DNA during STR amplification<\/a>\u00a0in an effort to increase the signal of the large DNA fragments.<\/p>\n

Impurities in a DNA extract can also suppress amplification of DNA \u2013 we call this inhibition of the reaction.\u00a0Inhibition can occur at the quantification or amplification stages and can affect the interpretability of your DNA profile. Inhibition could appear similar to degradation, because large DNA fragments don\u2019t amplify as well as small DNA fragments in the presence of the inhibitor.<\/p>\n

<\/p>\n

Although the STR profiles from degraded and inhibited samples may appear similar, don\u2019t be fooled.\u00a0Unlike with degraded DNA, increasing your target adds even more inhibitor to the reaction, making the inhibition even worse.<\/p>\n

The Internal PCR Control (IPC) is synthetic DNA that is amplified along with each sample.\u00a0\u00a0It just confirms that the assay worked as expected.\u00a0\u00a0Inhibitors can affect the IPC amplification; an increase in the threshold cycle value for the IPC indicates that it took the synthetic DNA longer than expected to reach a defined threshold; therefore something was impeding the reaction.<\/p>\n

There is one tricky thing about interpreting elevated IPC CT<\/sub>values.\u00a0\u00a0High concentrations of DNA in an extract (above 5ng\/uL) can elevate the IPC Ct slightly because the entire reaction becomes saturated.\u00a0Because of this, it is very important to evaluate the IPC\u00a0CT<\/sub>value in conjunction with the DNA concentration. For example, if you were to question whether a sample that is at a concentration of 50ng\/ul is inhibited, you would have to compare it to other samples or standards with a similarly high concentration.\u00a0\u00a0The same is true for a sample with a low concentration \u2013 you should only compare like to like.<\/p>\n

<\/p>\n

Because degradation and inhibition both affect large DNA targets more than small, it is necessary to assess the quality flags for degradation and inhibition together.\u00a0\u00a0For example, if the DI is elevated, and the IPC is as expected then the sample is degraded and not inhibited.\u00a0\u00a0However, if the\u00a0IPC and DI are both elevated, you may not be able to determine if the sample is simply inhibited, or both degraded and inhibited.\u00a0In a severely inhibited sample, the inhibition should be addressed by dilution or clean up and if necessary, the treated sample can be re-quantified to assess whether degradation is present.<\/p>\n

<\/p>\n

You can learn more from forensic scientists,\u00a0who work with bone, and use real-time PCR analysis<\/a>\u00a0to make decisions that deliver improved recovery of alleles.<\/p>\n

For licensing and limited use restrictions visit\u00a0thermofisher.com\/HIDlicensing<\/a>.<\/p>\n

For Research Forensic or Paternity Use Only. Not for use in diagnostic procedures.\u00a0<\/em><\/p>\n

<\/p>\n

<\/p>\n

<\/p>\n","protected":false},"excerpt":{"rendered":"

Wouldn\u2019t it be wonderful if there was a tool that forensic scientists could use\u00a0to assess the quality of an unknown DNA sample\u00a0before attempting to generate an STR profile?\u00a0DNA samples collected from crime scenes range from DNA-rich fluids, like blood and saliva, to a few skin cells left behind from a casual touch.\u00a0\u00a0For example, there is<\/p>\n","protected":false},"author":115,"featured_media":10951,"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":[71],"tags":[204,43],"division":[],"class_list":{"0":"post-11128","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-forensics","8":"tag-forensic-focus","9":"tag-pcr","10":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"\nHow 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