T-dna Primers
T-dna Primers - Related Videos
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.
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.
10:17
Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers
0 Views •
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.
02:40
DNA Replication
0 Views •
2020
DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied. After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells. Replication in Prokaryotes DNA replication uses a large number of...
13:16
Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA
0 Views •
Cited by 26 •
2018
Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.
FAQs
Primer placement determines which portion of the insertion becomes detectable. Primers designed near the left or right border can amplify internal T-DNA regions, while pairing a T-DNA primer with a plant-specific flanking primer can amplify the junction between the transferred DNA and neighboring plant sequence. This distinction helps characterize the insertion rather than merely detect construct-derived DNA.
The two primer types provide complementary sequence information. A T-DNA primer recognizes the known transferred construct, whereas a plant-specific flanking primer recognizes DNA adjacent to the integration site. Amplification with both types of primers supports identification of a T-DNA–plant junction, helping researchers determine where the inserted region connects with the plant genome.
Different constructs contain different T-DNA sequences, so primer sets can be selected to target the relevant inserted regions. Comparing which primer combinations produce amplification allows researchers to screen lines for particular constructs or insertion events. This approach supports characterization of transformed plants when several genetic lines must be evaluated or compared.
A typical workflow begins with plant genomic DNA and primer selection based on the known T-DNA sequence, often near a border. Researchers perform PCR to amplify the targeted region, examine the resulting amplification, and use plant-specific flanking primers when junction analysis is needed. DNA sequencing can then help validate the amplified insertion or integration site.
PCR indicates whether a targeted T-DNA region or junction can be amplified, but sequencing provides additional characterization of that amplified region. When T-DNA primers are combined with plant-specific flanking primers, sequencing can support identification of the genomic integration site and confirm the relationship between the transferred DNA and adjacent plant sequence.
They are useful during screening and validation of plants produced through Agrobacterium-mediated transformation. Researchers can use them to confirm insertion events, assess lines carrying different constructs, and support genetic studies involving integrated T-DNA. Their value increases when PCR results are combined with sequencing, because the analysis can address both construct presence and genomic integration.