He cites the low success rates so far in other, more commonly used species such as cows and mice, and the even greater difficulty of cloning monkeys, man's nearest neighbour.
Pure subtractive methodologies are of limited use due to the need for a large quantity of mRNA to drive hybridization to completion as well as the difficulty in cloning the tiny amount of cDNA remaining after hybridization.
The difficulty in cloning ω-gliadin genes has been reported previously, i.e., where recombination-deficient host bacteria and a vector that is less-susceptible to insert deletions were necessary to clone two genomic fragments each containing an ω-gliadin gene (Hsia and Anderson 2001).
Among the other advantages of the new TAR cloning system is its ability to isolate centromeric and telomeric regions, which have been generally excluded as potential targets by the Human Genome Project because of the difficulty of cloning large blocks of repetitive DNA.
The wheat ω-gliadin genes are an elusive subject for detailed research, and anyone studying these genes needs to be aware of major complicating factors among which are technical difficulties in cloning these highly repetitive DNA sequences, chimeric ESTs, the high frequency of pseudogenes, and the difficulty in estimating both the total and active numbers of ω-gliadin genes.
Indeed, many immature or partially spliced mRNAs have been found in cDNA libraries, in particular of G protein-coupled receptors [36], partly due to difficulties in cloning long transcripts.
These disparities are due to variations of the density of restriction sites in certain genome regions or difficulties in cloning too large or too small fragments [ 40].
Scientists have been working on such techniques (see box) for some time but their work has been hampered by the difficulties involved in cloning human cells in the laboratory.
Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method.
Due to cloning difficulties, SOAT cDNA (OriGene) was cloned into the Interplay Mammalian TAP vector, pCTAP (Stratagene) which contains a dual streptavidin binding peptide tag and a calmodulin binding peptide tag.
