PCR-based strategies to speed up cloning after tagging with Mutator
--Guri Johal and John Gray

The Mutator (Mu) transposable element system is the most efficient system for tagging genes in maize. This is largely due to the transposition of hundreds of Mu elements that are present in active Mutator lines. Although the high copy number increases the chances of getting an insertional mutation, it also is the root cause of a significant problem, i.e., to determine which of hundreds of Mu elements in the mutant has caused the mutation. The limited resolution of ordinary agarose gels makes it impossible to identify a Mu element that cosegregates with the mutant allele, unless the number of background Mu elements is decreased. This is usually done by outcrossing the mutant a few times with an inbred that lacks Mutator activity. During these crosses, Mu elements unlinked with the mutation are segregated out. This however, takes a couple of seasons, and sometimes it also leads to the suppression of the mutant phenotype making the cosegregation analysis impossible. Another problem with the Mutator system is that although nine different types of Mu elements have been identified, the mutation of interest may be caused by a Mu element that has not been identified yet.

A couple of PCR-based approaches have been conceived to simplify and speed up the cloning process once a mutant or even a mutant sector is obtained during a Mutator tagging project. These strategies are based on employing a Mu-TIR primer (preferably from the end sequence) as an anchor primer. The other primer can be used randomly or ligated onto the other end by a method devised for the ligation mediated PCR (Mueller and Wold, Science, 246:780, 1989). Following PCR, a polymorphism will be sought between the products derived from DNA extracted from tissue (sector or pooled plants) that harbors the mutation and tissue (sector or pooled plants) that lacks the mutation. One problem that can affect the success of this approach is that maize plants, including Mu inactive stocks or inbreds, contain hundreds of TIR's. Thus, hundreds of products may be amplified, confounding their resolution even on a sequencing gel. To alleviate this problem, 4-16 different types of the anchor primers can be made by introducing one to two random nucleotides on the 3' end of the TIR sequence, and different anchor primers may be used singly or in combinations in different reactions to impose selectivity on the PCR products that can be amplified. Although it will increase the number of reactions, it can dramatically reduce the number of PCR bands in a given reaction, making them resolvable, and thereby increasing the chances of identifying a polymorphic product.

This procedure can conceivably also be used at the transcript level. When performed on RNA, the procedure may not even require multiple species of the Mu-TIR anchor primer, and RNA ligase can be used to ligate a specific primer at the other end of the cDNA (as in the 5' RACE technique). The instability of the mutant transcript can be a problem, however. In Mu-induced mutations that show suppressible properties, a modification of this procedure, in which two anchor primers, one coming from the Mu-TIR and the other containing poly T, may be even more specific, and therefore may be more successful, in the identification and cloning of a tagged gene. 

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