MATS: a rapid and efficient method for the development of microsatellite markers from YACs.
In this report, we describe the successful application of a rapid and efficient procedure, based on subtractive hybridization and PCR amplification, for generating microsatellite-based markers directly from yeast artificial chromosomes (YACs). This strategy, termed MATS (marker addition through subtraction), exploits the fact that the only difference between a yeast host strain harboring a YAC and the host strain alone is the artificial chromosome. Given the low complexity of the yeast genome and relatively large target size presented by a YAC, only a single round of subtraction is required before amplification of the target sequences (YAC) and cloning into a plasmid vector for further analysis. Several key steps have been designed to achieve optimal subtraction and to obtain preferential amplification and recovery of the target sequences. Methods for efficient construction of small insert libraries and rapid, nonradioactive screening have also been integrated into the protocol. Using a 750-kb YAC as a target, we identified a minimum of 14 unique microsatellite containing clones, leading to the development of 12 polymorphic STSs (sequence-tagged sites). These new markers will facilitate the genetic localization of targeted locus and allow the accurate ordering by STS content mapping of a cloned contig spanning the interval. In addition to the utility of this approach in positional cloning, this strategy may provide an approach for filling gaps in the emerging genetic maps.
Chen
H
H
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Pulido
J C
JC
Duyk
G M
GM
D001483:Base Sequence; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics; D004276:DNA, Satellite / Q000032:analysis* / Q000235:genetics; D015723:Gene Library; D005819:Genetic Markers; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016324:Sequence Tagged Sites
D017931:DNA Primers; D004271:DNA, Fungal; D004276:DNA, Satellite; D005819:Genetic Markers; D015345:Oligonucleotide Probes