A rapid capture technique was developed to efficiently isolate specific DNA targets from a variety of genomes. 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. These targets include but are not limited to transposable elements, microsatellites, repetitive sequences, and possibly unique sequences. 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. Further, this method allows sequences flanking known internal regions to be co-isolated, facilitating the development of flanking primers for downstream applications. Considerable reduction in the frequency of nonspecific binding between key components (background) obviates the need for subsequent screening steps. Rapid capture of DNA targets quickly provides information about target and flanking sequences.
The isolation of specific DNA targets within genomes is critical for a variety of research. Standard approaches, however, have been cumbersome and time consuming, with most requiring some type of library screening. The high background associated with such screens often requires the addition or repetition of several steps to reduce that background.
Recently, considerable effort has been expended isolating microsatellites and their flanking sequences. Microsatellites are sequences containing a low level of sequence complexity comprised of short tandem repeats of two to five base pairs (bp). They are often the marker of choice for population genetics studies mainly due to their higher mutation rate (1). 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 (1,2,3). The efficiency of these enrichment protocols varies greatly among species. Avian taxa have one of the lowest recovery efficiencies (1), probably because they contain low levels of microsatellites (4).
A 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). This background likely stems from the high level of nonspecific binding of streptavidin magnetic beads to DNA. Consequently, many protocols require an additional screening step after enrichment. Linker design may also contribute to the inefficiencies of these methods. To date, these protocols have typically been used for microsatellite isolation.
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). Unfortunately, these methods fail to address problems associated with the isolation of targets that may be in multiple copies in the genome. 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.
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.Materials And Methods DNA Sources
DNA was obtained from a variety of taxa ((Table 1)) with extraction methods dependent upon tissue type. Only high molecular weight DNA was used, such as that extracted from blood or fresh tissue.Table 1. Species and Targets Tested with the Rapid Capture Method
aTarget was transposable element loci.
bTarget was microsatellite loci.
cSuccess rate represents the percent clones containing the target DNA. Siblings or duplicate clones were discarded.
dRepresents combined results of several hybridizations using both linker-ligated DNA directly and hybridizations performed using the product from post-linker ligation PCR.
eThe first percentage is from hybridizations using the linker-ligated DNA directly while the second percentage is from hybridizations performed using the product from post-linker ligation PCR.
fHybridizations were performed using the product from post-linker ligation PCR.
gPCR product from an initial hybridization was directly used in a second identical hybridization.
hHybridization with four probes simultaneously: AAC, GCA, CAT, and GATA.