Table 3.

Strategies to screen for PPIs in the yeast two-hybrid system

Library type	Features, with advantages (+) and disadvantages (−)
Genomic DNA	For organisms with low intron occupancy and small intergenic regions; genomic DNA is cut with ClaI-compatible restriction enzymes (95, 299); +, cheap, incomplete fragments may facilitate positive outcome (e.g., with membrane proteins or incorrectly annotated protein-encoding genes); −, small fraction of in-frame protein coding fragments, introns are present
cDNA	For organisms with high intron occupancy and large intergenic regions (614); +, cheap, exclusion of noncoding fragments and introns, correct orientation; −, only partial fraction of in-frame protein coding fragments, strong difference in abundance between different cDNA fragments
Normalized cDNA	Normalizes the amount of cDNA fragments for each gene (683); +, exclusion of noncoding fragments and introns, correct orientation, better representation of each cDNA fragment; −, relatively expensive, only partial fraction of in-frame protein coding fragments
Full-length cDNA	Full-length cDNA fragments created with gene-specific forward primers (627); +, exclusion of noncoding fragments and introns, correct frame and orientation, balanced representation of each gene; −, expensive, complete cDNA fragments reduce the positive outcome rate for specific types of interactions (e.g., with membrane proteins)
Open reading frame DNA	Each open reading frame is individually cloned into the prey library by in vivo (gap repair) or in vitro (Gateway from Invitrogen) recombinational cloning (294, 650); +, exclusion of noncoding fragments and introns, correct frame and orientation, balanced representation of each gene; −, expensive, introns are present, complete ORF fragments reduce the positive outcome rate for specific types of interactions (e.g., with membrane proteins)