Stem-looped Primers
Stem-looped Primers - Related Videos
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.
11:27
Studying DNA Looping by Single-Molecule FRET
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Cited by 13 •
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This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.
02:48
Loop-Mediated Isothermal Amplification for Bacterial Gene Detection
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2025
Source: Domesle, K. J., et al. Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation. J. Vis. Exp. (2020).This video demonstrates the loop-mediated isothermal amplification (LAMP) technique for detecting a target bacterial gene by amplifying DNA at a constant temperature. The reaction mixture is prepared by adding an isothermal master mix, specific primers for the target gene, and samples containing DNA templates. The...
17:15
Catheterization of Intestinal Loops in Ruminants
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Cited by 4 •
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We describe a novel surgical method for catheterizing 'intestinal loops' within the ileum of sheep. Once animals have recovered from surgery and have cleared antibiotics and analgesics, multiple treatments can be deposited directly in loops via the catheters.
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.
FAQs
The 3′ end provides the sequence-specific contact with the target RNA, including short molecules such as microRNAs. Because recognition is concentrated at this end, binding can distinguish related sequences that differ only in their nucleotide composition. This selective interaction initiates reverse transcription and links accurate target recognition to the production of a measurable amplification product.
The folded stem-loop gives a short target-associated primer a longer structural context during reverse transcription. That added configuration supports formation of a reverse-transcription template while preserving sequence-specific recognition at the 3′ end. As a result, the assay can analyze short RNA molecules more selectively than approaches using conventional primer designs alone.
Discrimination depends on the primer’s engineered sequence and its requirement for correct pairing at the target-binding end. Closely related molecules may therefore produce different reverse-transcription outcomes when their sequences do not match the primer sufficiently. This property is particularly important for microRNA analysis, where short targets can be difficult to distinguish with less selective primers.
Reverse transcription converts recognition of the target RNA into a complementary nucleic acid template that can enter a later amplification step. The stem-looped primer both contacts the RNA through its 3′ end and provides the longer configuration needed for this conversion. PCR or quantitative PCR can then amplify the resulting product for detection or measurement.
A typical workflow begins with a stem-looped primer designed to recognize the selected microRNA at its 3′ end. Reverse transcription then generates a longer nucleic acid template from that short RNA target. Researchers subsequently use PCR or quantitative PCR to amplify the product, allowing the presence or relative abundance of the microRNA to be assessed.
They are especially useful when investigators need selective analysis of short RNA molecules or closely related sequences. In biology, reported applications include microRNA studies connected with gene regulation, developmental processes, and disease-associated biomarkers. Quantitative PCR can extend these applications by providing measurements of target abundance rather than detection alone.