Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription - PubMed (original) (raw)

Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription

Douglas Kyung Nam et al. Proc Natl Acad Sci U S A. 2002.

Abstract

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.

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Figures

Figure 1

Figure 1

Schematics of internal poly(A) priming by oligo(dT) primer. Besides priming the poly(A) sequence at the 3′ end of mRNA, the oligo(dT) primer can also prime the poly(A) sequences present internally within mRNA. The initiation of cDNA synthesis from the oligo(dT) primed at both locations results in the generation of two truncated cDNAs from a single mRNA template.

Figure 2

Figure 2

Comparison of internal poly(A) priming and 3′ poly(A) priming between oligo(dT) primer and anchored oligo(dT) primers. An in vitro transcript containing an internal A20 and 3′ A50 was used for reverse transcription with either oligo(dT) primer or anchored oligo(dT) primers. (A) Sequencing image of cDNA clones primed by oligo(dT) primer and anchored oligo(dT) primers. (B) Summary of the results of the analyses.

Figure 3

Figure 3

Internal poly(A) priming prevents the extension of 3′ end-primed cDNA along the mRNA template. A group of mRNAs with internal poly(A) sequences was primed with oligo(dT) or anchored oligo(dT) primers for reverse transcription. Quantitative PCR was used to determine cDNA levels originating from internal poly(A) priming, and 3′ end poly(A) priming extending beyond the internal poly(A) tract. (A) Demonstration of the levels of cDNA from internal poly(A) priming and 3′ end poly(A) priming in proteoglycan mRNA. It shows the diminishing of cDNA originating from 3′ end priming when oligo(dT) primer was used, whereas the level was high between internal and 3′ end-primed cDNA when anchored oligo(dT) primers were used. (B) Summary of the results from the analyses. The value from internal priming is set as 1 for comparing the relative levels between the cDNA from internal priming and cDNA from external priming.

Figure 4

Figure 4

Effects of high-dose oligo(dT) primers on the internal poly(A) priming and 3′ end poly(A) priming. mRNA from proteoglycan with 10 internal As was used in this analysis. Increased doses of oligo(dT) primer were used for the first-strand cDNA synthesis. Quantitative PCR was used to determine cDNA levels originating from internal poly(A) priming and 3′ end poly(A) priming extending beyond the internal poly(A) tract. The same amount of control templates was used in the quantitative PCR for each set of reactions.

Figure 5

Figure 5

Estimated prevalence of ESTs in dbEST originating from internal poly(A) priming. (A) Summary of the analyses. See the text for detailed description. (B) Distance between the sequence matched by EST to the internal poly(A) sequence in the matched reference sequence.

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