The Sca forward oligonucleotide (5′-CAGTGCTCTAGACGTGCTAGT-3′) and the Sca reverse oligonucleotide (5′-ACTAGCACGTCTAGAGCACTGAAAA-3′) were annealed by heating an equal volume of 10 µM oligonucleotides for 5 min at 94☌ followed by a room temperature incubation for 10 min resulting in 5 µM Sca linkers. Sca linkers specifically designed for this method were ligated to the blunt-ended dephosphorylated fragments. Here we report a method that integrates several aspects of methods previously developed for microsatellite isolation, but that ( i) significantly decreases the isolation of untargeted (background) DNA sequences, ( ii) extends the method to any DNA target including those much more complex and varied sequences such as dispersed transposable elements, and ( iii) improves upon the design of linkers and restriction endonucleases used in the isolation process so that flanking regions suitable for the development of PCR primers are more frequently co-isolated with the DNA target. Rapid capture of DNA targets isolates 5′ and 3′ flanking sequences concurrently with the target sequence and does so simultaneously for targets occurring multiple times in a genome. Unfortunately, these methods fail to address problems associated with the isolation of targets that may be in multiple copies in the genome. Methods that allow for the efficient isolation of flanking sequences from specific unique sequences exist (e.g., GenomeWalker from Clontech Laboratories, Mountain View, CA, USA). To date, these protocols have typically been used for microsatellite isolation. Linker design may also contribute to the inefficiencies of these methods. Consequently, many protocols require an additional screening step after enrichment. This background likely stems from the high level of nonspecific binding of streptavidin magnetic beads to DNA. Avian taxa have one of the lowest recovery efficiencies ( 1), probably because they contain low levels of microsatellites ( 4).Ī significant issue remaining with these improved methods is that isolated pools of targeted DNA often include high proportions of DNA fragments lacking the microsatellite targets (background). The efficiency of these enrichment protocols varies greatly among species. Many of the techniques recently developed for microsatellite isolation involve enzymatic fragmentation of the genomic DNA, hybridization of fragments to a biotinylated oligonucleotide containing the repeat of interest, and subsequent capture of the hybridized fragments with streptavidin magnetic beads ( 1–3). They are often the marker of choice for population genetics studies mainly due to their higher mutation rate ( 1). Microsatellites are sequences containing a low level of sequence complexity comprised of short tandem repeats of two to five base pairs (bp). Recently, considerable effort has been expended isolating microsatellites and their flanking sequences. The high background associated with such screens often requires the addition or repetition of several steps to reduce that background. Standard approaches, however, have been cumbersome and time consuming, with most requiring some type of library screening. The isolation of specific DNA targets within genomes is critical for a variety of research. Rapid capture of DNA targets quickly provides information about target and flanking sequences.

Considerable reduction in the frequency of nonspecific binding between key components (background) obviates the need for subsequent screening steps. Further, this method allows sequences flanking known internal regions to be co-isolated, facilitating the development of flanking primers for downstream applications.

Additionally, because the thermodynamics of nucleic acid hybridizations differ from processes such as PCR, a wider variety of targets with a range of mismatches to any customized probe can be isolated. These targets include but are not limited to transposable elements, microsatellites, repetitive sequences, and possibly unique sequences. The specificity can be easily adapted to any target for which partial sequence is known, allowing for the isolation of a wide set of target molecules from either characterized or uncharacterized genomes. A rapid capture technique was developed to efficiently isolate specific DNA targets from a variety of genomes.

Rapidlink dna