Primer Extension Reactions
Primer Extension Reactions - Related Videos
15:28
Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
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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
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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
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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.
13:27
PCR: The Polymerase Chain Reaction
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2023
The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling – cyles of temperature changes at fixed time intervals. Using a thermostable DNA polymerase, PCR can create numerous copies of DNA from DNA building blocks called dNTPs. There are three steps in PCR: denaturation, annealing, and elongation. Denaturation is the first step in the cycle and causes the DNA to melt by disrupting hydrogen bonds between the bases resulting in single-stranded DNA. Annealing...
10:17
Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers
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Cited by 7 •
2017
Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.
FAQs
The extending enzyme adds nucleotides from the primer’s available 3′ end, so the primer must anneal in an orientation that permits copying toward the relevant template region. Each added nucleotide is complementary to the template. Consequently, the resulting product provides two interpretable signals: its sequence reports template identity, while its length indicates the distance to the feature being examined.
Primer annealing determines which nucleic acid region is interrogated. Because the oligonucleotide is sequence-specific, complementarity between primer and template positions the extension reaction at a defined site rather than across an unspecified region. This positioning is especially important when researchers want to distinguish nearby transcript or sequence features, since the final product reflects the primer’s placement relative to the target.
Enzyme choice follows the chemical nature of the template. A DNA polymerase extends a primer on a DNA strand, whereas a reverse transcriptase extends it using RNA information as the template. This distinction connects the reaction chemistry to the biological question: the selected enzyme determines whether the product reports a DNA region directly or records information present in RNA.
Product length is not merely a reaction yield measurement; it can locate a nucleic acid feature. If the primer position is known, the distance represented by the extension product can identify a transcription start site or another mapped position. Sequence analysis adds a second layer by showing which nucleotides were copied, allowing length-based localization to be evaluated alongside sequence information.
A basic workflow begins by selecting a short oligonucleotide complementary to the region adjacent to the feature of interest. The primer is allowed to anneal to the DNA or RNA target, and the appropriate extending enzyme adds complementary nucleotides from its 3′ end. Researchers then separate or sequence the product, using its length or sequence to interpret the target.
For transcription-start-site mapping, the primer is positioned on a known portion of the transcript, and extension proceeds toward the previously uncharacterized end. The length of the resulting product therefore reports the distance between the primer and the start site. This makes the technique useful for examining gene regulation, where the precise initiation position provides information about transcript organization.
Primer extension can address different questions by changing what is read from the product. In RNA studies, extension results can contribute to RNA-structure analysis; in sequence studies, copied products can reveal variants or support targeted sequencing. These applications share the same interpretive basis: a defined primer creates a localized readout of template position and sequence.