Genome-Scale Cloning and Expression of Individual Open Reading Frames Using Topoisomerase I-Mediated Ligation

Table 1.

Cloning and Expression Results for Yeast Genome and Human Kinase Studies

Yeast first-pass process No. successful No. attempted Success rate (%)
pYES2 pcDNA3.1 pYES2 pcDNA3.1 pYES2 pcDNA3.1
ORF amplification 5632 6035 93 93
Topoisomerase I-mediated  cloning + orientation 3749 3762 5632 5632 67 67
Expression QC positive 659 558 3749 3762 18 15
Start-to-finish summary 659 558 6035 6035 11 9
Yeast second-pass process
ORF amplification 5000 5093 5376 5477 93 93
Topoisomerase I-mediated  cloning + orientation 3750 3266 5000 5093 75 64
Expression QC positive 1553 1197 3750 3266 41 37
Start-to-finish summary 1553 1197 5376 5477 29 22
Human kinase process No. successful (pcDNA3.1) No. attempted (pcDNA3.1) Success rate (%) (pcDNA3.1)
RT–PCR 179 288 62
Topoisomerase I-mediated  cloning + orientation 140 179 78
Expression QC positive 108 140 77
Start-to-finish summary 108 288 38

  • Cloning and expression results for the yeast genome and human kinase studies. Success rates are expressed as percentages of successful attempts over the total number of attempts for each step of the cloning and expression process.

This Article

  1. Genome Res. 9: 383-392

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