An Effcient Method to Clone Chicken Microsatellite Repeat Sequences.

Abstract

An efficient method, that is easy to use, for cloning of microsatellite sequences was developed for chickens. Chicken genomic DNA fragments, digested with restriction enzymes, were ligated into pCR-Script SK(+) vector and the ligation mixture was transformed into XLl-Blue MRF' competent cells. Without the plating step, the cells were infected with helper phage and single stranded DNA (ssDNA) was prepared by standard procedures except with a modification as follows. After the phage precipitation step, DNaseI and RNaseA were used to remove contaminating Escherichia coli DNA and RNA from the resulting ssDNA in the phage mixture. The ssDNA was used as a template for selective second-strand DNA synthesis, primed with (CA)10 oligonucleotide, by thermostable DNA polymerase. Subsequently, ssDNA remaining in the mixture was digested by mung bean nuclease, and the mixture was transformed into XLl-Blue MRF' cells. About 70% of the clones in a library enriched for (CA)n microsatellites with this approach were positive in hybridization analysis using (CA)10 as a probe. Sequence analysis revealed that positive clones contained CA repeats and the length of CA repeat units varied between 6 and 28 units with a mean value of 13.1. This method would make the search for new (CA)n repeat length polymorphisms more efficient and contribute to genetic mapping of chicken.