Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics1-10 / 9

286

ENHANCED METHODS FOR PHYSICAL MAPPING

quite

susceptible to shear breakage, which increases as

the square

of the length of the

DNA. Any DNA that is fragmented by shear breakage or

nuclease contamination during

the ligation procedure, and also any contaminating vector arms, will be eliminated by the

second

size-fractionation. This is important because, otherwise, large

numbers of vector

average, for unknown reasons

as the team gained more experience. All of these libraries

are made from PFG-fractionated

Eco

R I partial digests of genomic DNA. Usually DNA is

transformed into yeast by electroporation.

The major problem with megaYACs (and most YAC libraries for that matter) are re-

arranged clones. These include chimeric clones, clones with deletions, and clones with in-

sertions of yeast DNA. These are illustrated in Figure 9.1. Deletions and yeast insertions

make it difficult to use the YACs directly

as DNA sources for finer mapping or sequenc-

ing. However, such clones are still useful for the kinds of mapping strategies we will de-

scribe later in this chapter. Chimeric clones

are more of a problem because they can lead

to serious errors in mapping if they are not detected. The chimeric clones appear to con-

tain two or more disconnected genomic regions. In some YAC libraries more than 50% of

the clones are chimeras.

There are two potential origins for

the

occurrence

of chimeras. Some

may

arise

during ligation, especially

if the

insert

DNA

is

at

too

high a concentration relative to

the amount of YAC arms present. Co-ligation can be reduced substantially by more com-

plex cloning strategies than

used in

early YAC

library

construction (Wada et

al.,

1994).

Figure 9.1

Some common artifacts in YAC cloning: chimeras, deletions, and insertion of yeast se-

quences.

LARGER YEAST ARTIFICIAL CHROMOSOMES (YACs)

287

For example, suppose that genomic DNA is

partially digested with

Mbo

I in agarose,

which produces fragments ending in

5 ———

3 ———CTAG

The single-stranded ends of the resulting sample are then partially filled in treatment with

Klenow fragment DNA polymerase and dpppA and dpppG only. The resulting genomic

DNA fragments will then have ends like

5 ————GA

3 ————CTAG

and thus they cannot be ligated to each other. In parallel, the YAC cloning vector is di-

gested to completion with

Bam

H I to generate telomeres (see Box 8.1) and then digested

with

Sal I to yield fragments that end in

5 ————G

3 ————CAGCT

The

ends of these are then partially

filled in

by treatment with Klenow DNA polymerase

ligating to the genomic DNA fragments prepared as described above.

A second source

of chimeras will arise from recombination.

In preparing

yeast for

DNA transformation,

usually a

small fraction of

the cells

is rendered competent

to

pick

up DNA, and it is not at all

uncommon for these

cells to

pick up

several YACs.

Usually

daughters are produced. If each of these retains a centromere,

they will usually segregate

to separate daughter cells, each of which will now maintain

a different single chimeric

from

observations on libraries made from hybrid cell lines. In most

of these cases, the

amount of chimerism is very low. This presumably arises because the

human DNA is

much

more dilute in these samples, and human-rodent recombination is

much less effi-

cient because most repeated sequences are not well conserved between the two species.

When YACs are prepared directly from DNA obtained from flow-sorted chromosomes,

HOW FAR CAN YACs GO?

Larger insert clones facilitate mapping projects

in

several ways. The number of clones

one needs to order to fill the

minimum tiling path

is

reduced. This greatly simplifies the

tier is determined by the largest stable and reliable clones that are

available.

The

chromosomes of

S. cerevisiae

range

in size

from

250

kb to 2.4 Mb. Thus the

largest

current YACs

are in the midsize range

of yeast chromosomes.

It is

not

clear

290

ENHANCED METHODS FOR PHYSICAL MAPPING

In contrast, while the irreducible minimum centromere of

S. pombe

is unknown, past experi-

ence suggests that it could well be in excess of 100 kb. This would make the construction of

cloning vectors for use in

S. pombe

very cumbersome.

An alternative cloning system for large DNA where considerable recent progress has

been made uses bacterial artificial chromosomes or BACs (see Box 8.2). This system em-

ploys single-copy

E. coli

F-plasmids as vectors for DNA inserts. Natural F-factors can be

a megabase in size. Thus BACs ought to have capacities in this size range if the resulting

DNAs can be transfected into

E. coli

efficiently. The first BACs were rather small with in-

serts mostly in the 100to 200-kb size range. However, recently larger BACs, with sizes

from greater than 300 kb, have been reported. It remains to be seen how much this range

can be enhanced by further modification and protocol optimization. BACs have the intrin-

sic advantage that the background

E. coli

DNA

is only a

third that of background yeast

DNA. It is also relatively easy

to purify the BAC DNA away from the host genomic

DNA.

Powerful bacterial

genetic

procedures can be used to manipulate the BAC se-

quences in vivo, and it is fair to say that comparable

procedures can be used to manipu-

late YACs within their host cells. A key feature for both systems is that we now know the

entire DNA sequence of both

E. coli

and

S. cerevisiae.

Procedures are likely to be devel-

oped and in place soon for direct DNA sequencing of large insert clones in one or both of

these organisms. By knowing all of the host DNA sequence ahead of time, it will be pos-

sible to design sequencing or PCR primers in an intelligent, directed way. Any accidental

host sequence that results from primer errors or homology will be immediately apparent

once the putative clone sequence is compared with that of its host genome. Another bac-

terial large DNA cloning system that is in widespread use is the P1 artificial chromosome

(PACs), described in Box 2.3.

VECTOR OBSOLESCENCE

Based on past experience with ordinary recombinant

DNA

procedures

over

the

past

decade, the highly desirable vector

of today is an inefficient, undesirable vector tomor-

row. There is no way we can predict what the optimal vectors will be like five years from

now; what bells and whistles they must have to facilitate the rapid mapping and sequenc-

ing procedures that will then be in use. To demonstrate how cloudy our crystal ball is in

this respect, within five years the development of rapid methods to screen clones for pos-

sible functions is very likely. Just what form these

screens will take, and what require-

ments they will impose on cloning vectors, are entirely unknown.

Imagine that tomorrow a vast improvement in some cloning vector has been achieved.

All of the current map data and samples do not use this vector. How will we transfer the

enormous number of samples used in

genomic mapping from yesterday’s obsolete vec-

tors to the new ones? Certainly it will not be efficient to do this clone by clone. New

strategies are badly needed that allow flexibility in the handling of samples to allow mass

recloning or rescreening of entire ordered or partially ordered libraries to retain useful or-

der information but equip the clones with the newly desired features. It is fair to say that

today, while the problem is recognized, creative solutions to it are still lacking. We will

either need clever selection, very effective automation for large numbers of separate sam-

ples,

or very effective multiplexing

that will

allow many samples to be

handled

together

HYBRID MAPPING STRATEGIES: CROSS-CONNECTIONS BETWEEN LIBRARIES

291

One

consequence of

the

virtual certainty of

vector obsolescence is that it

is desirable

to minimize the numbers of samples archived for storage and subsequent redistribution.

Instead, it seems more efficient to develop procedures that will allow desired clones to be

pulled easily from whatever new libraries are made. The advantage of PCR-based ap-

proaches is obvious in this regard. These approaches require storing only DNA sequence

information that allows primers to be made whenever they are needed to assay for a given

sequence in a sensitive

way. Whatever library a

desired DNA sequence is in,

it should

then be possible to find

it in a relatively quick and inexpensive way, by PCR assays on

pools of clones or hybridization assays on arrays of clones from that library, as we will

describe later in this chapter. In this way no large numbers of samples need be stored for

long time

periods nor

for

mass distribution.

Only pools of samples will

have to

be

archived.

different types of samples. These include cell lines, radiation hybrids,

and large restric-

tion fragments for regional assignments and gaining an overview, as well as various clone

libraries such as megaYACs, YACs, P1, and cosmid clones that actually form the eventual

basis for DNA sequencing. Past projects have tended to concentrate on ordering at most a

few of these samples across

the chromosome, and then they resorted to using

other types

of samples in selected regions where these were needed to address particular problems.

Based on these

experiences,

it now seems evident that much of the labor in handling all

of these types of samples is preparing a dense set of labeled DNA probes or PCR primers

(e.g., STSs

vide infra

) that are needed for most fingerprinting or mapping activities.

Once one has such probes or primers, an attractive scheme for ordering them is shown

in Figure 9.4. The labeled probes or primers are used to interrogate, simultaneously, all

samples that are of potential interest for the chromosome of question. If

a dense enough

set of probes

or primers exists, and if the clone libraries are highly

redundant, we will

show that the result of the interrogation should be to order all the samples of interest in

parallel. Ordering by any of the methods currently at our disposal involves finding over-

lap information. The larger the number of different samples used in the

overlap proce-

dure, the more likely one or more will cover the key region needed to form an informative

overlap. This approach is neither top down or bottom up; in most respects it combines the

best features of the two extreme views of map construction. They key issue is how to im-

plement such a

strategy in

an efficient way. There are three basic issues: how to handle

the probes, how to handle the samples, and how to do the interrogation.

Tens of thousands of DNA samples are involved in most large-scale mapping efforts.

These cannot be handled routinely as individual liquid DNA preparations. One viable ap-

shown schematically in Figure 9.5. It consists of 2

104 individual cosmids. This must

be prepared by an automated arraying device (Box 9.1). The key fact is that once the sam-

ples are prepared, the device can make as many copies of the filter as needed with rela-

tively little additional effort. If each cosmid has an average of 4

104 base pairs of DNA,

the array represents a total of 8

108 base pairs. This is

a fivefold redundant coverage of

some screening procedures like PCR. It also

has the advantage

that a

relatively small

number of pools can replace a very large number of

individual samples

or spots

on an ar-

mine which elements of particular pools

were responsible

for

positive signals generated

by the probe.

Hundreds to thousands of probes or

primers are used

in

a large-scale mapping effort.