Sequencing By Ligation
Sequencing By Ligation - Related Videos
07:34
DNA Ligation Reactions
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2023
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
08:57
Amplicon Sequencing using the Long-Read Sequencing Technologies
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2025
This protocol was optimized for targeted deep sequencing of 18 drug-resistance regions in Mycobacterium tuberculosis using a long-read sequencing platform, followed by analysis with a tuberculosis-specific bioinformatics pipeline designed for long-read data.
14:15
Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
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Cited by 6 •
2014
MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.
11:53
Cecal Ligation Puncture Procedure
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Cited by 200 •
2011
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
05:47
Modified Technique for Coronary Artery Ligation in Mice
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Cited by 8 •
2013
The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.
FAQs
DNA ligase evaluates whether a hybridized oligonucleotide probe is correctly matched to the template beside the sequencing primer. When the match is correct, ligase joins the probe, creating a connection that can be detected through its fluorescent label. This makes base complementarity the decision point for recording sequence information, rather than relying on one-at-a-time strand extension.
Fluorescent labeling converts each successful ligation event into an optical readout. After imaging records the probe's color, that probe is removed so another probe can test the next cycle. Repeating this hybridization, ligation, imaging, and removal sequence produces a series of color signals, which researchers use to infer nucleotide order across the analyzed DNA fragment.
Sequencing by ligation differs from sequencing by synthesis in the chemical event used to identify bases. It relies on ligase joining a correctly matched probe, whereas synthesis-based approaches extend a strand one nucleotide at a time. The ligation strategy is especially relevant when a project requires massively parallel analysis of many DNA fragments and high-throughput sequence information.
The sequencing primer provides the reference point beside which oligonucleotide probes hybridize. That placement lets the ligation reaction test complementarity at a defined location on the template, while repeated cycles build the sequence information gathered from the same analysis. In practice, the primer helps organize how probe colors are interpreted during successive rounds of detection.
Imaging is the measurement step that preserves the identity of each successful ligation event. The fluorescent color of the joined probe is recorded before the probe is removed, linking the observed signal to that cycle. Repeating this record-and-remove process creates the color data needed to determine nucleotide order rather than merely confirming that a reaction occurred.
The method is useful when researchers need sequence information from many DNA fragments at once. Identified applications include variant detection, genome profiling, and other high-throughput genomics research. These uses connect the ligation signal series to questions about genetic differences and broader patterns across analyzed DNA, rather than restricting the approach to a single fragment.