BAC-based physical mapping of the pine genome will be initiated as part of APG. Specifically, molecular markers will be mapped onto corresponding BACs using macroarray hybridization techniques. Physical mapping is slated to begin in mid 2006.
Below are some definitions (from Peterson et al. 2000) that may help in understanding the process of BAC-based physical mapping.
Ordered libraries: In an ordered BAC library, bacteria from positive colonies (i.e., colonies presumably containing insert DNA) are picked from agar trays and placed into freezing medium in individual wells of microtiter plates (one clone per well). Letters along the Y-axis and numbers along the X-axis of each plate provide each well with a specific alphanumerical designation (e.g., well G13). Additionally, the microtiter plates in a library are numbered consecutively. Consequently, any particular clone in the library possesses its own unique address (e.g., plate 131, well G13).
Segregation of individual clones into separate wells coupled with automation allows complete libraries to be gridded onto filters in a highly specific manner, i.e., each clone is gridded onto a filter based on its address in the library. If a probe hybridizes to a specific spot(s) on a grid, the relative location of the spot can be used to determine the exact location of the clone within the library.
Ordered libraries save valuable time and resources by increasing the efficiency and speed of library screening (see Choi and Wing 1999 for review).
Physical mapping: Physical mapping is the grouping of clones into contigs using physical mapping techniques (see below). The goal of most physical mapping projects is to assemble contigs that encompass entire chromosomes/genomes as a prelude to genome sequencing (e.g., Mozo et al. 1999).
Contig: A contig is a set of clones containing partially overlapping pieces of insert DNA that collectively contain an uninterrupted stretch of genomic DNA. Contigs are constructed using physical mapping techniques.
Physical mapping techniques are any techniques used in contig construction. The most common BAC physical mapping techniques include chromosome walking, BAC-end sequencing, STS-based mapping, map-based cloning, and DNA fingerprinting.
Chromosome walking: In chromosome walking, the end sequences of a “starter” clone(s), typically associated with an EST or RFLP marker, are used to probe colony blots/grids. DNA fingerprints of positive clones are compared to the fingerprint of the starter clone, and those exhibiting a minimal amount of overlap with the starter are grouped into a contig with that clone.
DNA fingerprinting is a means of analyzing the similarity between several DNA samples based upon the presence or absence of specific restriction sites within their sequences. In DNA fingerprinting, two or more DNA samples (e.g., BAC clones) are digested with the same set of restriction enzymes. The digested DNA samples are run on a gel and blotted onto nitrocellulose (Southern blotting). Blots are hybridized with labeled probe sequences, and similarities/differences between the hybridization patterns for the DNA samples are noted.
In BAC-based physical mapping, DNA fingerprints of BAC clones can be compared. Those clones that have considerable overlap in their fingerprint patterns can be grouped together into contigs (Marek and Shoemaker 1997; Marra et al. 1997).
BAC-end sequencing is a powerful technique used in association with chromosome walking to construct contigs. It is rooted in the principles of Sanger dideoxy DNA sequencing and DNA amplification (Rosenblum et al., 1997). In brief, (a) BAC DNA is isolated from clones using a miniprep procedure, (b) vector-based primers, Taq polymerase, and a nucleotide cocktail containing dye-labeled ddNTPs are added to each miniprep, (c) the mixtures are heat-denatured, cooled to allow primer hybridization, and warmed to a temperature that allows DNA polymerization, (d) each reaction is electrophoretically resolved on an acrylamide sequencing gel, and (e) the gel is analyzed using an automated gel analysis system (see Boysen et al. 1997; Rosenblum et al. 1997; Kelly et al. 1999 for reviews). If the BAC DNA concentration in the minipreps is particularly low, several thermocycle runs can be used to produce more sequencing substrates (Liu and Whittier 1995).
The sequences generated by BAC-end sequencing represent regions of insert DNA adjacent to insert sites (“BAC ends”). Once BAC end sequences for a particular clone are obtained, probes based on that clone’s end sequences can be used to screen the BAC library and find clones that overlap the starting clone (i.e., chromosome walking). When applied on a large scale, probing libraries with BAC end sequences can lead to relatively rapid construction of physical maps.
Multiplex screening is a colony hybridization strategy for efficiently screening ordered libraries with multiple radioisotope- or fluorochrome-labeled probes. Briefly, probes of interest are labeled and arranged in a series of rows and columns in a microtiter plate. Probes from an entire row are pooled and used to screen a set of library grids. Likewise, probes from one column are pooled and used to probe an identical set of grids. Hybridization patterns on both sets of grids are recorded and compared. If a particular clone is recognized by both the pooled row and pooled column probes, that clone most likely contains a DNA sequence complementary to the probe found at the intersection of the row and column on the microtiter plate. Computer analysis of the hybridization patterns of all pooled column and row combinations allows clones to be assigned to probes using a minimum number of hybridizations (Cai et al. 1998).
STS-based mapping is a physical mapping technique rooted in the principles of PCR. In general, primer pairs are designed from cDNAs and/or genomic regions known to be single-copy in nature. In the presence of labeled nucleotides, PCR is performed using a particular primer pair(s) and a set of BAC templates (e.g., minipreps from a BAC library). Those BACs that function as templates for a particular primer pair are grouped into a contig. If different primer pairs produce amplification products from the same BAC template, those primers represent loci that are physically close to one another. A primer that results in isolation of clones containing a region of single-copy DNA is called an STS marker. Those primers derived from cDNAs often are called ESTs (expressed sequence tags) because they presumably correspond to expressed genes.
*This material is based upon work supported by the National Science Foundation under Grant No. DBI-0421717. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. MGEL © 2006. Web design by Daniel G. Peterson. Last updated 08-Jun-2006.
