Name: Sanger sequencing
Uploaded: 2022-01-10T00:00:00.0000000
Duration: T00H03M00S
Description: Sanger sequencing

Name: Sanger sequencing

Description: Sanger sequencing

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Upload Date: 2022-01-10T00:00:00.0000000

Transcript: Language: EN.
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SPEAKER 1: The DNA sequencing technique Sanger sequencing was initially invented in 1977 by Frederick Sanger, a creation for which he won his second Nobel Prize in Chemistry in 1980. This technique soon became the primary method through which researchers sequenced DNA, and although numerous updates to the technologies involved have enabled this technique to be completed in a faster, more automated manner, the core working principles of this technique remain the same.
SPEAKER 1: Although more contemporary next-generation sequencing technologies offer higher-throughput parallel sequencing of multiple samples and gene targets, Sanger sequencing remains valued due to its fast, cost-effective sequencing capabilities when dealing with fewer than 20 DNA targets. Its low error rate makes it a common feature in clinical sequencing. It can also be trusted to validate findings made through next-generation sequencing.
SPEAKER 1: Automated Sanger sequencing operates via the following key steps. First, target template DNA must be purified and quantitated, which takes approximately 30 minutes. Then the template strands of DNA are subjected to several rounds of thermal cycling. These reactions contain a mixture of regular deoxynucleotides and fluorescently labeled dideoxyribonucleotides or ddNTPs.
SPEAKER 1: These ddNTPs lack the three prime OH group required for further phosphodiester bonds to form and therefore prevent the continued extension of the DNA template in the reaction when incorporated into a strand. There are more regular deoxynucleotides than ddNTPs in the mixture so target DNA strands of different lengths are created. This step takes up to two hours and 30 minutes.
SPEAKER 1: Next, the reaction needs to be cleaned up to remove any excess ddNTPs. This step is important to avoid dye blobs caused by unincorporated dye terminators remaining in solution. That can obscure the sequencing data and can take up to one hour and 30 minutes. Once cleanup has occurred, the DNA strands created by the reaction are separated by capillary electrophoresis in a sequencing instrument.
SPEAKER 1: Once separated by size, with smaller DNA fragments moving through the polymer gel in the capillary faster, a laser excites the dye-labeled DNA fragments, and the 'basecaller' technology within the instrument detects the fluorescent signals to identify the type of ddNTP that terminated each fragment. This can take anywhere from 30 minutes to three hours depending on target sequence length, instrument setup, and other factors.
SPEAKER 1: To find out more about Sanger sequencing, check out our In Focus on the topic in association with Promega over on www.BioTechniques.com.

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