17 Genomic Libraries
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clonable will depend on the enzyme and vector selected. Plasmid libraries
generally lack fragments greater than 5- 10 kb in length and preferentially
contain small inserts. Libraries constructed using either lambda bacteriophage or cosmids have both upper and lower limits of insert size since
recombinant vectors >50 kb or <38 kb cannot be packaged.
Partial digest libraries
One can avoid many of the aforementioned limitations by using partial digest libraries. Consider the genomic segment in Figure 2. There are 14 restriction sites for enzyme A in this example. Complete restriction with enzyme A yields 15 fragments, most ofwhich are below the minimum insert
size of the vector. Reducing the amount of enzyme or decreasing the duration of the reaction results in incomplete digestion and yields a ladder of
fragments. Segments that were previously below the cloning limit of the
vector are now clonable, and the total fraction of the original source material that is represented in the library is greatly improved. The ratio of clonable/unclonable segments is optimized by careful selection of the reaction
conditions. The cloning efficiency is highest when the size of the most abundant fragments equals the vector capacity. This is achieved by using reduced
quantities of dilute enzyme, brief restriction times, and an enzyme that cuts
DNA frequently and without bias in selection of its restriction sites (Seed,
Parkerand Davidson 1982). The enzyme Sau3A or Mbol is usually selected
because it recognizes a 4-bp sequence and yields fragments that can be ligated to vectors cleaved with BamHI. One empirically determines the optimal conditions in a test digest prior to scaling up for a preparative digest
(see Procedure). Aliquots of the test digest are removed at various times
after addition of the enzyme and evaluated by gel electrophoresis. The
time point and enzyme concentration that yield the most clonable fragments are selected for the preparative reaction.
There is a second major advantage of partial digest libraries. A well-made
library typically is comprised of clones containing overlapping inserts (Figure 2). A "deep" or representative library (see Comments, Library Complexity) often includes between 5 and 10 copies of a locus, each clonedas a unique, overlapping fragment. It is this feature that makes such libraries especially suitable for chromosome walking.
One disadvantage of partial digest libraries is that they are technically
more challenging to construct. The depth of a library correlates directly
with the quality of the DNA used for its construction. For optimal results,
the average size of the source DNA before partial digestion must be at least
267
clonable will depend on the enzyme and vector selected. Plasmid libraries
generally lack fragments greater than 5- 10 kb in length and preferentially
contain small inserts. Libraries constructed using either lambda bacteriophage or cosmids have both upper and lower limits of insert size since
recombinant vectors >50 kb or <38 kb cannot be packaged.
Partial digest libraries
One can avoid many of the aforementioned limitations by using partial digest libraries. Consider the genomic segment in Figure 2. There are 14 restriction sites for enzyme A in this example. Complete restriction with enzyme A yields 15 fragments, most ofwhich are below the minimum insert
size of the vector. Reducing the amount of enzyme or decreasing the duration of the reaction results in incomplete digestion and yields a ladder of
fragments. Segments that were previously below the cloning limit of the
vector are now clonable, and the total fraction of the original source material that is represented in the library is greatly improved. The ratio of clonable/unclonable segments is optimized by careful selection of the reaction
conditions. The cloning efficiency is highest when the size of the most abundant fragments equals the vector capacity. This is achieved by using reduced
quantities of dilute enzyme, brief restriction times, and an enzyme that cuts
DNA frequently and without bias in selection of its restriction sites (Seed,
Parkerand Davidson 1982). The enzyme Sau3A or Mbol is usually selected
because it recognizes a 4-bp sequence and yields fragments that can be ligated to vectors cleaved with BamHI. One empirically determines the optimal conditions in a test digest prior to scaling up for a preparative digest
(see Procedure). Aliquots of the test digest are removed at various times
after addition of the enzyme and evaluated by gel electrophoresis. The
time point and enzyme concentration that yield the most clonable fragments are selected for the preparative reaction.
There is a second major advantage of partial digest libraries. A well-made
library typically is comprised of clones containing overlapping inserts (Figure 2). A "deep" or representative library (see Comments, Library Complexity) often includes between 5 and 10 copies of a locus, each clonedas a unique, overlapping fragment. It is this feature that makes such libraries especially suitable for chromosome walking.
One disadvantage of partial digest libraries is that they are technically
more challenging to construct. The depth of a library correlates directly
with the quality of the DNA used for its construction. For optimal results,
the average size of the source DNA before partial digestion must be at least