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One-sided polymerase chain reaction: the amplification of cDNA.

ABSTRACT: We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.

SUBMITTER: Ohara O

PROVIDER: S-EPMC297692 | biostudies-other | 1989 Aug

REPOSITORIES: biostudies-other

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One-sided polymerase chain reaction: the amplification of cDNA.

Ohara O O Dorit R L RL Gilbert W W

Proceedings of the National Academy of Sciences of the United States of America 19890801 15

We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted eithe ...[more]

PMID: 2788276

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Project description:Background: Precise determination of amplification efficiency is critical for reliable conversion of within-sample changes in fluorescence occurring on a logarithmic scale to between-sample differences in DNA content occurring on a linear scale. This endeavor is especially challenging for the telomere length (TL) quantitative-PCR (qPCR) assay, where amplification efficiency can vary between reactions targeting telomeric repeats (T) and those targeting a single-copy gene (S) to calculate TL as the T/S ratio. Methods: We compared seven different approaches toward estimating amplification efficiency, including the standard-curve method utilized by the qPCR instrument software, and alternative approaches which estimate efficiency on a reaction-by-reaction basis using the stand-alone program LinRegPCR. After calculating T/S ratios using efficiency estimates from each approach (N = 363), we tested their relative performance on metrics of assay precision and correlates of external validity including chronological age (age range = 1-72 years), across tissues within-person (leukocyte-buccal), and between parents and offspring. Results: Estimated amplification efficiency for telomere reactions was significantly lower than estimates for single-copy gene reactions. Efficiency estimates for both reaction sets were significantly higher when estimated with the standard-curve method utilized by the qPCR instrument relative to estimates reconstructed during the log-linear phase with LinRegPCR. While estimates of single-copy gene efficiency reconstructed using LinRegPCR measured within 90% of perfect exponential doubling (E = 1.92), estimates generated using the standard-curve method were inflated beyond 100% (E = 2.10-2.12), indicating poor fidelity. Despite differences in raw value, TL measurements calculated with LinRegPCR efficiency estimates exhibited similar relationships with external validity correlates to measurements generated using the qPCR instrument software. Conclusion: Since methods to estimate amplification efficiency can vary across qPCR instruments, we suggest that future analyses empirically consider external methods of efficiency calculations such as LinRegPCR, and that already generated data be re-analyzed to glean possible improvements.

Dataset Information

One-sided polymerase chain reaction: the amplification of cDNA.

Publications

One-sided polymerase chain reaction: the amplification of cDNA.

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