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- W2025124372 abstract "▼Partial cDNA clones from the 3′-ends of mRNAs can often be obtained using a degenerate primer to a conserved region of a protein in an anchored PCR reaction (Ref. 1). However, further sequencing requires some form of 5′RACE. The methods are PCR based and require the addition of a synthetic priming sequence to the 3′-end of the cDNA. This is usually done either using the enzyme, terminal deoxynucleotidyl transferase (Ref. 2, 3, 4), or by ligation of a singlestranded oligonucleotide to the 3′-end of the cDNA using RNA ligase (Ref. 5). However, we have found the terminal deoxynucleotidyl transferase enzyme very difficult to use and the RNA ligase procedure long and complicated and also unreliable. In this report we describe a simple technique to obtain 5′ends of mRNA by adding an artificial priming site to the 3′end of newly synthesized cDNA using T4 DNA ligase (Figure 1). The procedure relies on the fact that DNA–RNA hybrids can act as substrates for T4 DNA ligase (in fact such hybrid molecules can be ligated directly into a plasmid vector to create a library of cDNA clones, although the efficiency is quite low). In this procedure, newly synthesized cDNA is retained as a cDNA–RNA hybrid. This hybrid is then ligated, using T4 DNA ligase, to plasmid DNA digested with a restriction enzyme that leaves blunt ends. The plasmid DNA then supplies the external priming sequence for either a T7 primer or M13 sequencing primer. The main advantages of" @default.
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- W2025124372 date "1998-01-01" @default.
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- W2025124372 title "A simple method to obtain the 5′ ends of mRNA sequences by direct ligation of cDNA–RNA hybrids to a plasmid vector" @default.
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- W2025124372 doi "https://doi.org/10.1016/s1366-2120(08)70120-x" @default.
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