BAC-by-BAC Sequencing


Also see: EST Sequencing | Shotgun Sequencing

A third approach has been to sequence overlapping BAC clones, also referred to as a minimum tiling path (MTP). Like the previous case, the BAC clones are anchored to the genetic map. A great aid to anchoring the clones is a process called DNA fingerprinting. If a BAC library contains genomic fragments that covers the genome several times over, then many BAC clones share restriction patterns in common. Based on common restriction patterns tiles of overlapping BAC clones can be assembled into fingerprinted contigs (FPCs).

A graphical description of this process is at the BOTTOM of this page.

A map of this type was instrumental in directing and organizing the Human Genome Project.  A similar map was also assembled for the 400-Mb rice genome. The rice map was constructed from a 23x BAC library and represents the rice genome in 438 contigs. Recently, the larger (2.8Gb) mouse genome of was constructed. Despite the fact that the mouse genome is much larger than that of rice, it is constructed from 305,716 BAC clones (~16X coverage) which have been assembled into 296 contigs. The significantly fewer gaps in the mouse map relative to that of rice have resulted from leveraging the human map information during the mouse map construction. Specifically, 453,962 mouse STCs were compared with the human genome sequence. About 11.3% of these STCs established co linearity between mouse and human, thereby facilitating contig ordering. The advantage of these long tiles of BAC clones is that it becomes easier to anchor them to the genetic map because they span a distance of many centiMorgans (cMs). Although these tiles are reasonably correct, their limited resolution makes them somewhat unsuitable for determining a MTP because they are based on a restriction pattern with six-base-pair cutters. Therefore, it has been necessary to use STCs in combination with FPCs to identify the minimum overlap between two neighboring BAC clones. The disadvantage of this strategy is that sequencing a whole genome can take a great deal of time and resources.  The advantage is that the  genome sequence is well organized, and can be finished to an accuracy of one base error in 10,000.

A Selection of BAC-by-BAC Genome Sequencing Projects:
A Graphic Description of BAC-by-BAC:


1. BAC clones are grown in 96-well format, digested to completion with the appropriate restiction enzymes, and fragments are separated by agarose gel electrophoresis. Gel images are converted to "bands files" where every clone is represented by a list of the band sizes making up the digest.

2. Using finger printing, overlapping clones with common restriction fragments are identified. Groups of clones are ordered into contigs.

3. Markers are addressed to BAC clones by using overlapping radio-labeled oligonucleotides. Probes are pooled and hybridized to nylon membranes supporting high-density arrays of clones. Alternatively, markers can be assigned to BAC clones by PCR. PCR primers made from marker sequences can be amplified across pools.
GO BACK HOME