Ligation-mediated Primer Extension
Ligation-mediated Primer Extension - Related Videos
15:28
Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
0 Views •
Cited by 41 •
2009
We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.
10:51
Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo
0 Views •
Cited by 17 •
2014
We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.
11:14
Primer-Free Aptamer Selection Using A Random DNA Library
0 Views •
Cited by 20 •
2010
SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.
07:34
DNA Ligation Reactions
0 Views •
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...
11:53
Cecal Ligation Puncture Procedure
0 Views •
Cited by 200 •
2011
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
FAQs
The two stages contribute different recognition functions. Hybridization positions the primer on a complementary template, while polymerase activity creates an extension product from its 3′ end. Ligase then joins that product to an adjacent nucleic acid or adapter. This sequence of events produces a defined molecule whose structure reflects both target recognition and successful joining.
The 3′ end is the starting point for polymerase-driven nucleotide addition, so its placement depends on correct primer-template hybridization. Extension therefore records the position at which the primer recognizes the complementary sequence. In assays designed for precise sequence recognition, this creates a molecular product that can be distinguished from products formed without the intended alignment.
Ligation provides a second molecular requirement after extension. The newly produced strand must be joined to an adjacent nucleic acid or adapter, rather than merely existing as an extension product. Because detection can focus on the defined joined molecule, the combined design supports precise recognition and can help connect a sequence-specific event to a downstream assay signal.
Primer extension alone produces a polymerase-generated strand from a hybridized primer. Ligation-mediated Primer Extension adds a subsequent structural constraint: the extension product must be joined to an adjacent nucleic acid or adapter. That extra step creates a defined molecular configuration, making the method useful when analysis depends on both sequence recognition and formation of a specific product.
First, the primer is hybridized to its complementary DNA or RNA template. Polymerase then extends the primer by adding nucleotides at its 3′ end. The resulting product is subjected to ligase activity so it joins an adjacent nucleic acid or adapter. The defined joined molecule can then be used for downstream detection or analysis.
It is suited to assays that require precise recognition of a nucleic acid sequence and a defined product for subsequent analysis. Supported uses include mutation analysis, transcript characterization, targeted sequencing, and related detection assays. The selected application determines whether the joined molecule is interpreted as information about a sequence variant, a transcript, or a targeted nucleic acid region.
Sequence-specific primer hybridization establishes recognition of the intended template region, while extension copies information from that site. Ligation then creates a defined molecule that can be examined downstream. In mutation analysis, this design supports focused assessment of sequence differences; in targeted sequencing, it provides a structured product associated with the selected nucleic acid target for further analysis.