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

tions. Thus they can be eliminated.

The

use

of multiple configurations of the array is

rather foolish and inefficient for

small arrays and small numbers of false positives. However, this process becomes very

efficient for large arrays with large numbers of false positives. Procedures exist called

transformation

matrices

or

Latin squares

that each show how to reconfigure an original

two-dimensional array into informative alternates. It is not obvious if efficient procedures

are known for arrays in higher dimensions. It is also not clear that any general reconfigu-

ration procedure is optimal, since the best scheme at a

given point may depend on the

prior results. Suffice it to say that the use of several alternate configurations appears to be

a very powerful tool. This is illustrated by the example shown in Figure 9.25. Here five

positive clones are correctly identified using row and column pools from three configura-

tions of a 7

7 array. This requires testing a total of 42 pools, which is only slightly less

than the 49 tests needed if the clones were examined individually. Again, however, the ef-

ficiency of the approach grows tremendously as the array size increases. Additional dis-

cussion

of quantitative aspects of

pooling

strategies can be found in Bruno et al., (1995).

INNER PRODUCT MAPPING

319

RH. An array is made of the YAC PCR products, and this is probed successively by hy-

bridization with each RH PCR product. This assigns the YACs to RHs.

In previous STS mapping strategies, PCR with STS primers had to be used directly to

analyze YACs in YAC pools. This is relatively inefficient for reasons that have been de-

scribed earlier in this chapter. Instead, in IPM, the STS-YAC correspondences are built

mathematically as shown in Figure 9.27. The inner product

C

of two matrices

A

and B is

computed as

C ij

A ik B kj

k

The YAC versus RH hybridization results can be scored as positive (

1) or negative (

1)

as shown for matrix

A. The RH

versus STS PCR results can also be

scored as

positive

( 1) or negative (

1) as shown in

matrix

B. The inner product

matrix

C

is computed

el-

ement by element in a very simple fashion. It is a matrix that describes comparisons be-

tween STS’s and YACs that reflects the separate RH results with each. The best estimate

for each YAC-STS

direct correspondence is

the largest (most positive) element in

each

row of matrix

C. This is shown as

or * in the simplified matrix

C which indicates only

320

ENHANCED METHODS FOR PHYSICAL MAPPING

the largest

elements of C. The

symbols in

C

indicate a YAC (column) that actually

contains

the

indicated STS (row). The * symbols in

C indicate a YAC does not actually

contain

the

STS (i.e., it would

be scored negative in a direct

PCR

test) but must be lo-

toxic to clone in available vector systems. Cloning a region, even if

it is stable, may also

be too time-consuming or costly if one needs to examine the region in a large number of

different samples. This would be the case where, for example, a region

expanded in a set

of different tumor samples is to be characterized. One way to circumvent the problem of

subcloning a region is to find one or more slices of a PFG-fractionated restriction digest

that contains the region. With enzymes like

Not

I that have rare recognition sites, gener-

ally 1 to 2 Mb regions will reside on at most a few fragments. Only a few probes from the

region will suffice to identify these fragments. PFG separation conditions can then be op-

timized to produce these fragments in separation domains where size

resolution

is

opti-

mum. The resulting slice of separation gel will then contain, typically, about 2% of the to-

tal genome. For a 600-kb human DNA fragment, this slice will consist of 100 fragments,

only one of which is the fragment of interest. This is probably too

dilute to

permit

any

kind of direct isolation or purification. But the slice can serve as an efficient sample for

PCR amplifications that try to assign additional STSs or ESTs to the region. If the slice is

examined from a digest of a single chromosome hybrid, it will contain only 1 or 2 human

DNA fragments. Then, as shown in Chapter 14, PCR amplification based

on

human-

specific repeating sequences

can

be

used to produce numerous

human-specific

DNA

probes from the region of interest.

RESTRICTION LANDMARK

GENOME

SCANNING

An alternative method for systematically generating a dense array

of

samples

from

a

genome has been developed. This method is called

restriction landmark genome scanning

(RGLS). It was originally conceived as a way of facilitating the construction of genetic

maps by finding large numbers of useful polymorphic sequences. Thus it

is a

method set

up to reveal differences between two genomes, and as such it fits the spirit of the kind of

differential analysis that needs to

be developed. RLGS, as

currently

practiced, is

based,

however, on genomic DNA rather than

on cloned DNA. The basic idea behind RLGS is

illustrated in Figure 9.28. A genome is digested with a rare-cutting restriction nuclease

like

Not

I, and the ends

of the

fragments

are labeled. This generates about 6000 labeled

sites

because

there are about

3000

Not

I sites in the genome, and each fragment will be

RESTRICTION LANDMARK GENOME SCANNING

321

labeled on both ends. The sample is then digested with a second restriction enzyme, one

that cuts more frequently, say at a six base site. The resulting fragments are then fraction-

ated by agarose gel electrophoresis in the size range of 1 to 20 kb. The agarose lane is ex-

cised and digested in situ with a third, more frequently cutting enzyme, one that recog-

nizes a four-base sequence. The resulting small DNA fragments are now separated in a

second electrophoretic dimension on polyacrylamide, which fractionates in the 0.1 to

1

kb size range. The result is a systematic pattern of thousands of spots, as shown in Figure

9.29 a. Each spot reveals the distance between the original

Not

I site and the nearest site

Figure 9.29

Example of

the results seen by RLGS. (

a ) Two-dimensional electrophoretic separa-

tion of DNA

fragments. (

b ) Sites where polymorphisms can be detected on a typical fragment.

322

ENHANCED METHODS FOR PHYSICAL MAPPING

for the

second and third enzymes (Fig. 9.29

b ). Any polymorphisms in these distances,

caused either by altered restriction sites or by DNA insertions or deletions, will appear as

displaced

spots in the two-dimensional fractionation. The method

appears to

be

very

powerful

because so many spots can be resolved, and the patterns,

at least

for

mouse

needed because

of the difficulties in handling

megaYACs

and

the

need

to

break

them

down into smaller samples for subsequent manipulations. These finer maps, however, will

be more easy to construct by using preexisting megaYAC

contigs, just as

the preexisting

S. pombe

restriction map allowed the design of efficient strategies

to

order an

S. pombe

cosmid library. BAC, PAC, or cosmid maps are still needed for current direct DNA se-

quencing technology. Direct sequencing from YACs or from genomic DNA is possible, as

we will describe in Chapter 10, but it is not yet reliable enough

to be routinely used in

large-scale sequencing projects. As the genome project concentrates on DNA sequencing,

the notion of a sequence-ready map has become important. Such a map consists of sam-

ples ready for DNA sequencing. Detailed order information on these samples could be

known in advance, or it could be obtained

in the

process

of

DNA

sequencing.

See

Chapter 11 for further discussion.

For species other than those already intensively studied, the best strategies will depend

on the kinds of samples that are available. If

radiation hybrids and mega clone

libraries

are made, these will obviously be valuable resources. If dense genetic maps can be made,

the probes from these will order the megaYACs. If a genetic map is not feasible, FISH

provides a readily accessible alternative. In other cases it may be possible to purify the

chromosomes or fragments efficiently by flow sorting, improved microdissection, or other

tricks to be described in Chapter 14.

The notion of having to make a map of a person for diagnostic purposes is still awe-

somely difficult. Mapping methods are complex and make major demands on both instru-

mentation and skilled personnel. New approaches will be needed before diagnostic map-

ping can be considered at all realistic. The use of radioactive

32 P pervades most current

mapping methods, and this is surely something to be avoided in a technique proposed for

widescale clinical use. One area that may impact heavily on the prospects for diagnostic,

mapping is the development of improved, sensitive, nonradioactive detection techniques.

These will be described as we deal with DNA

sequencing

methods

because

it

is

here

where these methods have first been used or tested.

SOURCES AND ADDITIONAL READINGS

323

SOURCES AND

ADDITIONAL

READINGS

Allshire, R. C. 1995. Elements of chromosome structure and function in fission yeast.

Seminars in

Cell Biology

6: 55–64.

Ashworth, L. K., Hartman, M.-A., Burgin, M., Devlin, L., Carrano, A. V., and Batzer, M. A. 1995.

Assembly of high-resolution bacterial artificial chromosome, P1-derived artificial chromosome,

and cosmid contigs.

Analytical Biochemistry

224: 565–571.

Barillot, E., Lacroix, B., and Cohen, D. 1991. Theoretical analysis

of library screening using a

N -

dimensional pooling strategy.

Nucleic Acids Research

19: 6241–6347.

Bruno, W. J., Knill, E., Balding, D. S., Bruce, D. C.,

Doggett,

N. A., Sawhill, W. W., Stallings,

R. L., Whittaker, C. C., and Torney, D. C. 1995. Efficient pooling designs for library screening.

Genomics

26: 21–30.

Green, E. D., Riethman, H. C., Dutchik, J. E., and Olson, M. V. 1991. Detection and characteriza-

tion of chimeric yeast artificial-chromosome clones.

Genomics

11: 658–669.

Grigoriev, A., Mott, R., and Lehrach, H. 1994. An algorithm to detect chimeric clones and random

noise in genomic mapping.

Genomics

22: 482–486.

Grothues, D., Cantor, C. R., and Smith, C. L. 1994. Top-down construction

of an

ordered

Schizosaccharomyces pombe

cosmid library.

Proceedings of the

National

Academy of

Sciences

USA

91: 4461–4465.

Hatada, I., Hayashizaki, Y., Hirotsune, S., Komatsubara, H., and Mukai, T. 1991. A genomic scan-

ning method for higher organisms using restriction sitas landmarks.

Proceedings of the National

Academy of Sciences USA

88: 9523–9527.

Ling, L., Ma, N. S.-F., Smith, D. R., Miller, D. D., and Moir, D. T. 1993. Reduced occurrence of

chimeric YACs in recombination-deficient hosts.

Nucleic Acids Research

21: 6045–6046.

Mejia, J. E., and Monaco, A. P. 1997. Retrofitting vectors for

Escherichia coli

-based artificial chro-

mosomes (PACs and BACs) with markers for transfection studies.

Genome Research

7:

179–186.

Perlin, M., and Chakravarti, A. 1993. Efficient construction of high-resolution physical maps from

yeast artificial chromosomes using radiation hybrids: Inner product

mapping.

Genomics

18:

283–289.

Perlin, M., Duggan, D., Davis, K., Farr, J., Findler, R., Higgins, M., Nowak, N., Evans, G., Qin, S.,

Zhang, J., Shows, T., James, M., and Richard, C. W. III. 1995. Rapid construction of integrated

maps using inner product mapping: YAC coverage of human chromosome 11.

Genomics

28:

315–327.

Smith, D. R., Smyth, A. P., and Moir, D. T. 1990. Amplification of large artificial chromosomes.

Proceedings of the National Academy of Sciences USA

87: 8242–8246.

Teo, I., and Shaunak, S. 1995. Polymerase chain reaction in situ: An appraisal of an emerging tech-

nique.

Histochemical Journal

27: 647–659.

Wada, M., Abe, K., Okumura, K., Taguchi, H., Kohno, K., Imamoto, F., Schlessinger, D., and

Kuwano, M. 1994. Chimeric YACs were generated at unreduced rates in conditions that sup-

press coligation.

Nucleic Acids Research

22: 1651–1654.

Whittaker, C. C., Mundt, M. O., Faber, V., Balding, D. J., Dougherty, R. L., Stallings, R. L., White,

S. W., and Torney, D. C. 1993. Computations for mapping genomes

with clones.

International

Journal of Genome Research

1: 195–226.