Primer Extension Assay
Primer Extension Assay - Related Videos
15:28
Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
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Cited by 41 •
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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.
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Proboscis Extension Response (PER) Assay in Drosophila
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Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.
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 •
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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 •
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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.
05:52
Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay
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Cited by 5 •
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This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.
FAQs
The primer’s binding position determines which RNA region is copied and provides the reference point for interpreting the extension product. Because reverse transcriptase proceeds along the complementary template until the RNA 5′ terminus, the product length reflects the distance between the primer and that endpoint. This makes primer placement central to accurate transcription-start-site mapping.
A sequencing ladder or size standard converts product length into positional information. Researchers compare the extended DNA product with known reference fragments, then use the matching size to infer where the RNA ends at its 5′ terminus. Without that comparison, the assay would show that extension occurred but would not precisely locate the transcript endpoint.
Distinct extension-product lengths can reveal more than one transcription initiation site. Each length represents a different distance from the same primer-binding region to an RNA 5′ endpoint, allowing start sites to be distinguished within the analyzed transcript population. This is useful when investigating how gene-regulatory activity produces alternative transcript beginnings.
Changes in the assay can be interpreted in relation to RNA processing or transcript abundance, not only transcription initiation. A shifted product length suggests a different mapped endpoint, whereas an altered extension pattern can reflect a change in the detected transcript population. Comparing samples therefore helps connect RNA-level changes with gene-regulation questions.
A basic workflow starts with an RNA sample and a labeled DNA primer designed to anneal to the complementary transcript. After primer binding, reverse transcriptase extends the primer, and the resulting DNA product is assessed by size against a sequencing ladder or standard. Keeping these stages conceptually separate helps distinguish hybridization, extension, and endpoint measurement.
Primer Extension Assay is useful when the research question concerns the precise beginning of an RNA transcript rather than RNA presence alone. It can support promoter studies by showing which transcription start site is associated with a gene and whether that pattern changes between samples. The resulting map contributes to analysis of gene expression and regulation.
In biology, the method links sequence-level information to functional questions about gene expression. Mapping a transcript’s 5′ end can help researchers examine transcription initiation, promoter activity, RNA processing, and RNA structure within molecular genetics studies. Its value comes from translating the length of a defined extension product into an interpretable feature of the RNA being analyzed.