Abstract
Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is a widely used enzyme for cDNA synthesis. Here we show that MMLV-RT has a strong template-independent polymerase activity using blunt DNA ends as substrate that generates 3′ overhangs of A, C, G, or T. Nucleotides were appended efficiently in the order A > G > T > C, and tail lengths varied from 4 to 5, 2 to 7, 2 to 4, and 2 to 3 for A, C, G, and T, respectively. The activity was so strong that nearly all our test DNA ends were appended with at least one A, C, G, or T. The N-tailing activity of MMLV-RT was enhanced in the presence of Mn2+, and the G-, C-, and T-tailing activities were further enhanced by dCMP, dGMP, and dAMP, respectively. This is the first report of an enzymatic activity that almost thoroughly appends two or more As, or one or more Cs, Gs, or Ts to the 3′ end of double-stranded DNA, which would enable exhaustive analysis of DNA samples. The N-tailing activity of MMLV-RT is potentially useful in many biotechnological applications.
Highlights
Ligation of two DNA molecules with one containing a single 3′A-overhang and the other a single 3′T-overhang is universally used in a wide range of applications, including TA-cloning[1,2], GC-cloning[3], and library preparations for next-generation sequencing[4,5]
In the presence of a single-stranded DNA often called template-switching oligonucleotide (TSO), which contains a stretch of ribonucleotide Gs at its 3′end, Moloney murine leukemia virus reverse transcriptase (MMLV-reverse transcriptase (RT)) switches template from RNA to TSO, and synthesizes DNA that is complementary to TSO and covalently bound to the cDNA
Heating of MMLV-RT at 42 °C for 15 min resulted in loss of 50% of activity[16], and our data showed that G-tailing activity at 30 °C corresponded to 93% of that at 40 °C
Summary
Ligation of two DNA molecules with one containing a single 3′A-overhang and the other a single 3′T-overhang is universally used in a wide range of applications, including TA-cloning[1,2], GC-cloning[3], and library preparations for next-generation sequencing[4,5]. MMLV-RT shows such a strong activity to add a few As, Cs, Gs, and Ts to the 3′end of blunt-ended DNA that most molecules are tailed at the end of the reaction. To evaluate the tailing efficiency of MMLV-RT, FAM70 DNA was incubated with the enzyme in the presence of dATP, dCTP, dGTP, or dTTP and analyzed under denaturing conditions in a capillary sequencer.
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