, Volume 20, Issue 4, pp 509-520

The Use of Competitive PCR Mimic to Evaluate a Limulus Lambda Phage Genomic DNA Library

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Abstract

1. A λ phage genomic DNA library for Limulus (L.) polyphemus brain was constructed using the λGEM-12 vector and the host strain KW251.

2. The primary library contained approximately 1.275 × 106 independent clones, increasing upon amplfication to 6.66 × 109 pfu/ml in a total volume of 58 ml.

3. A total of 28 clones was randomly chosen for a determination of the average size of inserts in the library. All clones contained inserts and the average size was 14.9 kb, ranging from 11.7 to 28.0 kb. The library provides a 10-fold equivalent of the L. polyphemus genome.

4. A new approach for evaluating a genomic DNA library was developed, in which competitive PCR MIMIC was employed to determine the target gene copy number in both constructed library and brain genomic DNA. The putative protein kinase C ε (PKCε) was selected as the target gene because its partial sequence of cDNA was recently cloned from L. polyphemus brain in our laboratory (Cao et al., 1998). A 419-bp fragment of nonhomologous sequence derived from putative PKCε and a 306-bp fragment from plasmid pUC 18 were generated for use as target and competitor in PCR MIMIC, respectively.

5. Within the genomic library DNA, a 0.8 value was obtained for the copy number of the putative PKCε gene that was detected in 0.1 amol of one equivalent L. polyphemus genome in terms of the average recombinant molecular weight. In the genomic DNA, a single copy of putative PKCε was found in 0.1 amol of one coverage for the L. polyphemus genome. Thus, it was implied that nearly 80% genetic resource was incorporated into the library. This percentage was termed the incorporation rate.

6. Based on these findings, we suggest that the incorporation rate is an essential factor for evaluating genomic libraries, particularly, when using partial digestion with restriction enzymes for library construction.