Construction of small-insert libraries enriched for short tandem repeat sequences by marker selection.
These protocols construct a representative small-insert genomic DNA library in a phagemid vector. First, size-selected DNA fragments are ligated into a phagemid vector. In the second protocol, the resulting small-insert phagemid library is propagated in a bacterial strain combining mutations at the dut and ung loci, which permit incorporation of uracil in place of thymidine during DNA replication. Infection of the phagemid library with M13 helper phage permits recovery of this library as single-stranded DNA (ssDNA). Finally, this uracil-substituted ssDNA is used as a template for primer extension using an oligonucleotide whose sequence corresponds to the STR class of interest [e.g., (GATA)10] as primer. The products of this primer-extension reaction are transformed into an E. coli strain maintaining wild-type genes at the dut and ung loci. Under these conditions, uracil-substituted ssDNA will be restricted from growing by the host-encoded uracil-N-glycosylase, while the primer-extended products are capable of replicating.
Pulido
J C
JC
Millenium Pharmaceuticals, Cambridge, Massachusetts, USA.
Duyk
G M
GM
D003090:Coliphages / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005819:Genetic Markers; D005821:Genetic Techniques; D005822:Genetic Vectors; D005826:Genetics, Medical; D015698:Genomic Library; D006801:Humans; D018895:Microsatellite Repeats
D005819:Genetic Markers; D004247:DNA