Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 8

LINKING CLONES

255

well or sample plug. In this way the linking clones are selectively captured. They can be

religated at low concentration to avoid the production of chimeras, and then re-introduced

into a bacterial host.

The alternative approach, after the rare-site-containing clones have been linearized, is

to ligate a selectable marker into the rare-cutting site. Both suppressor tRNA genes and

kanamycin resistance genes have been successfully used for this purpose. The library is

then used to transform a bacterial host in the presence of the

selection. The only clones

that should survive are the linking clones. Both of the methods described work reasonably

well. However, the procedures are not perfect, and it is important, after a linking library

has been made, to screen out artifactual clones. These may include such annoyances as

chimeras of rare cutter fragments, clones that contain a rare cutter site that is not actually

cut in the parental genome because it is methylated, and clones containing small individ-

ual rare cutter fragments. One way to screen for useful clones is to use them as probes in

Southern hybridizations with genomic DNA singly and doubly digested with the restric-

tion enzyme used for constructing the library such as

Eco

R I or Hin

d III, and the rare-cut-

ting enzyme. Proper linking clones will show single bands or no bands in the absence of

rare cutter cleavage, and double bands once the rare site has been cut.

Once linking clones are available, they have many other useful applications besides the

ordering of macrorestriction fragments. Linking clones serve as effective bridges between

two different kinds of restriction nuclease digests (or other

types of DNA samples) be-

cause they come from a known position, the ends, of large DNA fragments. As a second

example, one can cut linking clones at their internal rare cutter site and isolate the two

fragments. The resulting samples are called

half-linking clones.

They are useful for the

analysis of the internal structure of large DNA fragments, as shown by the schematic ex-

periment illustrated in Figure 8.18. Here a

Not

I half-linking clone is used as an indirect

end label by hybridizating it to a blot of a PFG-fractionated partial

Eco R I digest of a

Not

I fragment. The sizes of the DNA pieces seen in the partial digest provide the location of

the internal

Eco R I sites. This type of analysis was originally developed for smaller DNA

pieces by Smith and Birnstiel. It is a very powerful approach. Half-linking clones are also

very useful for the analysis of partial digests with enzymes

that cut

infrequently,

as we

will demonstrate later.

JUMPING LIBRARIES

257

Figure 8.20

Basic notion of a jumping clone: Two discontinuous pieces

of the genome (

a ) and ( b ), but related by some map or fragment informa-

are then digested with a restriction enzyme

that

has much more frequent sites in

the

target

DNA but no sites in the vector. This can

be

arranged by suitable design of

the

vector.

The result is a mixture of relatively small DNA pieces;

only those near the junction of the

two ends of the original large DNA fragments contain vector sequences. A second ligation is

now carried out, again at very low concentration. This circularizes all of the DNAs in the

sample. When these are reintroduced into

E. coli, under conditions where selectable markers

on the vector are required for growth, a jumping library results.

There are potentially powerful uses for jumping libraries that span adjacent sites of in-

frequently cutting restriction enzymes. These are illustrated in Figure 8.22. Note that each

of the jumping clones will overlap two linking clones.

If one systematically examines

which linking clones share DNA sequences with jumping clones, and vice versa, the re-

sult will be to order all the linking clones and all the jumping clones. This could be done

by hybridization, PCR, or by direct DNA sequencing near the rare-cutting sites. In the lat-

ter case DNA sequence comparisons will reveal clone overlaps. Such an approach, called

sequence-tagged rare restriction sites

(STARs), becomes increasingly attractive as the

ease and throughput of automated DNA sequencing improves.

A more general form of jumping library is shown schematically in Figure 8.23. Here a

genome is partially digested by a relatively frequent cutting restriction nuclease. The re-

sulting, very complex, mixture is fractionated by PFG, and a very narrow size range of

material is selected. If this is centered about 500 kb, then the sample contains all contigu-

ous blocks of 500-kb DNA in the genome. This material is used to make a jumping li-

brary in the same general manner as described above, by

circularizing it around a vector

at low concentration, excising internal material, and recircularizing. The resulting library

is a size-selected jumping library, consisting, in

principle, of all discontinuous sets of

short DNA sequences spaced 500 kb apart in the genome. The major disadvantage of this

library is that it is very complex. However, it is

also very useful, as shown in Figure

8.24 a. Suppose that one has a marker in a region of interest, and one would like another

marker spaced approximately 500 kb away. The original

marker is used to screen a 500-

Figure 8.23

DNA preparation (

a ) used to generate a more general jumping library (

b ) consisting

PARTIAL DIGESTION

259

Figure 8.24

Example of the use of a general

jumping library to move rapidly from one site in a

genome to distantly spaced sites. (

a )

Half-jumping clones provide new probes, at known distances

from the starting probe. (

b ) Information about the direction of the jump can be preserved if the map

tion site (Fig. 8.24

b ). Selection of jumping clones by using portions

of the original

marker will let information about its orientation be preserved after the jump. The major

limitation in the use of

this otherwise very powerful approach is that it is relatively

hard

to make long jumps because it is difficult to ligate such large DNA circles efficiently.

maximum entropy arguments to select the most likely fragment orderings consistent

with

a given digestion pattern. The difficulty is that many restriction enzymes appear to have

preferred cutting sites. In the case of

Sfi

I these are generated by the peculiarities of the

interrupted recognition sequence. In the case of

Not

I and many other enzymes, hot spots

for cutting appear to occur in regions where a

number of sites

are clustered very

closely

in the genome (Fig. 8.26). Since the possibility of a hot spot in a particular region is hard

to rule out, a priori, one plausible candidate for a restriction map that fits the partial diges-

tion data is a null map in which all partially

cleaved sites lie to one side of the probe and

a fully cut hot spot lies to the other side (Fig. 8.27). While such a map is statistically im-

plausible in a random genome, it becomes quite reasonable

once the possibility of hot

spots is allowed.

There are three special cases of partial digest patterns that can be analyzed without the

complications we have just raised above. The first of these

occurs when the region is

flanked by a site that is cut in every molecule under examination. There are two ways to

generate this site (which is, in effect, a known hot spot). Suppose that the probe used to ana-

lyze the digest is very close to the end of a

chromosome (Fig.

8.28). Then the null map is

curs if

we can

integrate

into a

chromosome

the

recognition

sequence

of

a very

rare

cutting

enzyme,

such that

this

will be the

only

site of its

kind in

the

region

of interest.

PARTIAL DIGESTION

261

Then we cleave at the very rare site completely

and partially digest with an enzyme that

has somewhat more frequent cleavage sites. The power of this approach as a rapid map-

ping method is considerable. However, it is dependent on the ability to drive the very rare

cleavage to completion. As we have indicated before, in many of the existing schemes for

very rare cleavage, total digestion cannot be guaranteed. Thierry and Dujon in Paris have

recently shown that sites for the nuclease I-

Sce

I (Table 8.1) can be inserted at convenient

densities in the yeast genome and that the enzyme cuts completely enough to make the

strategy we have just described very effective.

A second case occurs that allows analysis of partial digestion data if the region of in-

terest is next to a very large DNA fragment. Then, as shown in Figure 8.29, all DNA frag-

ments seen in the partial digest that have a size less than the very large fragment can be

ordered so long as the digest is probed by hybridization with a probe that is located on the

first small piece next to the very large fragment. Again, as in the case of telomeric probes,

relaxing this constraint a bit still enables

considerable map

information

to be

inferred

from the digest.

The third case where partial digests can be analyzed occurs when one has two probes

known, a priori, to lie on adjacent DNA fragments. This is precisely the case in hand, for

example, when two half-linking clones are used as probes of a partial digest made by us-

ing the same rare site present on the clones. In this case, as shown in Figure 8.30, those

fragment sizes seen with one probe and not the other must extend in the direction of that

probe. Bands seen with both probes are generally not that

informative.

Thus

linking

The power of the approaches that employ partial digests is that a probe in one location

can be used to reach out and obtain map data across considerable distances, where no other

probes may yet exist. In order to carry out these experiments, however, two requirements

must be met. First, reliable length standards and good

high-resolution PFG fractionations

are essential. The data afforded by a partial digest is all inherent in the lengths of the DNA

bands seen. For example, if one used a probe known to reside on a 400-kb band, and found

in a partial 1000-kb band, this is evidence that a 600-kb band neighbors the 400-kb band.

One could then try to isolate specific probes from the 600-kb region to try to prove this as-

signment. However, if the 1000-kb band was mis-sized, and it was really only 900 kb, when

one went to find probes in the 600-kb region of a

gel, this would be the wrong region.

The second constraint for effective partial digest analysis is very sensitive hybridization pro-

tocols. The yields of DNA pieces in partials can easily be only 1 to 10%. Detecting these re-

quires hybridizations that are 10 to 100 times as sensitive as those needed for ordinary sin-

gle-copy DNA targets. Autoradiographic exposures of one to two weeks are not uncommon

in the analysis of partial digests with infrequently cutting enzymes.

EXPLOITING DNA POLYMORPHISMS TO ASSIST MAPPING

Suppose that two different DNA probes detect a

Not I fragment 800 kb long. How can we

tell if they lie on the same fragment, or if it just a coincidence and they derive from two

different fragments with sizes too similar to resolve? One approach is to cut the

Not I di-

gest with a second relatively rare-cutting enzyme. If the band seen by one probe shortens,

and the band seen by the other does not, we know two different fragments are involved. If

both bands shorten after the second digestion, the result is ambiguous, unless two differ-

ent size bands are seen by the two probes and the sum

of their sizes is greater than the

size of the band originally seen in the

Not

I digest.

A more reliable approach is to try the two different probes on DNA isolated from a se-

ries of different cell lines. In practice, eight cell lines with very different characteristics

usually suffices. When this is done with a single DNA probe, usually one or more of the

lines shows a significant size difference or cutting difference from the others (Fig. 8.31).

There are many potential origins for this polymorphism. Mammalian genomes are rampant

with tandem repeats, and these differ in size substantially

from individual to individual.