Which technique is most suited for identifying the 5′ end of an RNA transcript?
Determination of the authentic 5 prime end of a transcript requires extension of a labeled primer annealed downstream. In primer extension, reverse transcriptase synthesizes complementary DNA until reaching the extreme 5 prime terminus of messenger RNA, generating product whose length indicates transcriptional start site. Northern blotting estimates size and abundance but lacks nucleotide resolution at ends. S1 mapping provides approximate termini, while 3 prime RACE specifically targets the polyadenylated tail. Therefore, primer extension remains a classical method of choice for high-resoluti
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.