7.25G: Primer Extension Analysis
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Primer extension is used to map the 5′ ends of DNA or RNA fragments.
Outline primer extension analysis
- Primer extension assay is done by annealing a specific oligonucleotide primer to a position downstream of that 5′ end.
- The primer is radiolabelled, usually at its 5′ end. This is extended with reverse transcriptase, which can copy either an RNA or a DNA template, making a fragment that ends at the 5′ end of the template molecule.
- Primer extension analysis includes selection and preparation of a labeled primer complementary to the RNA transcript of interest; hybridization of the primer to a region of the RNA under study; extension from the primer by an RNA-dependent DNA polymerase to synthesize a cDNA strand.
- Analysis of primer extension of the extended cDNA products is done on denaturing polyacrylaminde gels and autoradiography.
- radiolabelled: Tagged with a radiotracer.
- polyacrylamide: Any of a range of cross-linked polymers of acrylamide; used to form soft gels.
Primer Extension Analysis
Primer extension is a technique whereby the 5′ ends of RNA or DNA can be mapped. Primer extension can be used to determine the start site of RNA transcription for a known gene. This technique requires a radiolabelled primer (usually 20 to 50 nucleotides in length) which is complementary to a region near the 3′ end of the gene. The primer is allowed to anneal to the RNA and reverse transcriptase is used to synthesize cDNA from the RNA until it reaches the 5′ end of the RNA. By running the product on a polyacrylamide gel, it is possible to determine the transcriptional start site, as the length of the sequence on the gel represents the distance from the start site to the radiolabelled primer.
Applications of Primer Extension Analysis
Primer extension analysis has three main applications. First, it is used for mapping the 5′ end of transcripts. This allows one to determine the transcription initiation site (assuming the mRNA isn’t further processed), which helps localize promoters or TATA boxes. Second, it can be used to quantify the amount of transcript in an in vitro transcription system.
Third, it can be used to determine the locations of breaks or modified bases in a mixed population of RNA or DNA samples. This is useful in applications like footprinting. Two different methods are used. In one, the modified nucleotide cannot be recognized by the polymerase or reverse transcriptase; in such cases, the chain ends at the site of modification. In the other, the modification is converted in a later step of the analysis to a strand break by chemical treatment. For instance, the sites of modifications by dimethyl sulfate (DMS) can be identified by treating DNA with DMS, exposing the sample to conditions that break the backbone at the site of modification, followed by primer extension.