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

BOX 9.1

(Continued)

Figure 9.7

Making a dense array of samples by offset spotting of a more dilute array.

applications. Most dense arrays that have been made thus far for mapping projects are

random. That is, no information is available about any of the DNA samples at the time

the

array

is made. As the map develops, the array becomes much more informative,

but the

x, yindexes of each sample have only historical significance.

Once

a map has been completed, it is convenient to reconfigure the array

of

sam-

ples so that they are placed in the actual order dictated by the map. While this is not

absolutely necessary, it does allow for visual inspection of a hybridization to be in-

stantly interpretable in many cases. There is no difficulty in instructing a robot to re-

configure an array. This procedure needs to be done only once, however

slow

the

process, and then replicas of the new configuration can be made rapidly. The great mo-

tivation

for achieving

more

compressed arrays is sample storage. Many

clones

and

most

DNA

samples are stored at low temperature. Large libraries of

samples

can

rapidly saturate all available temperature-controlled laboratory storage

space,

espe-

cially if a large number of replicas is made for subsequent distribution.

select out human material from hybrid cell lines or to reduce the complexity of a probe to

desirable levels. Some of these techniques were described in Chapter 4;

others

will be

dealt with in Chapter 14. It is worth noting that randomly chosen oligonucleotides often

make very effective probes for fingerprinting. For example, a randomly chosen 10-mer

should detect 1 out of every 25 cosmid clones. A key issue that we will

address

later in

this chapter is whether it is possible to increase the efficiency of handling probes by pool-

ing them instead of using them individually.

SCREENING

BY

PCR

VERSUS HYBRIDIZATION

A key variable in contemporary mapping efforts is whether the connections between probes

and samples are made by hybridization or by PCR. The two techniques have compensating

disadvantages

and

advantages,

and

the choice of which to use will depend on the nature of

the samples available and the complexity of the mapping project. We will consider these dif-

ferences at the level of the target, the probe, and the organization of the project.

The sensitivity of PCR at detecting small amounts of target is unquestionably greater

than hybridization. The advantage of being able to use smaller samples

is

that

with

presently

available methods,

it

is much easier to automate the preparation

of relatively

SCREENING

BY

PCR

VERSUS

HYBRIDIZATION

297

DNA that will be detected by one method or the other. With PCR this

concentration

can

be almost arbitrarily low, so long as there are not contaminants that will give an unaccept-

able PCR background.

Hybridization has the advantage when a large number of

physically

discrete

samples

(or pools) must be examined simultaneously. We have already described the use of dense

arrays of samples on filters to process in parallel large numbers of targets in hybridization

against a single probe. These arrays can easily contain 10

4 samples. In comparison, typi-

cal PCR reactions must be done in individually isolated liquid samples. Microtitre plates

handle

around 10

2

of these. Devices

have

been built

that

can

do

thousands of

simultane-

ous PCRs, but these are large; with objects on such a scale one could easily handle 10

5

samples at once by hybridization.

At the level of the probe, PCR is more demanding than hybridization. Hybridization

probes can be made by PCR or by radiolabeling any DNA sample available. Typical PCR

analyses require a knowledge of the DNA sequence of the target. In contrast, hybridiza-

tion requires only a sample

of the DNA that corresponds to one element

of

the

target.

PCR primers require custom synthesis, which is still expensive and time-consuming al-

though recent progress in automated synthesis has lowered the unit cost of these materials

considerably. PCR with large numbers of different primers is not very convenient because

in most current protocols the PCR reactions must be done individually or at most in small

pools. Pooling of probes (or using very complex probes) is a powerful way to speed up

mapping, as we will illustrate later in this chapter. However, in PCR, primers are difficult

to pool. The reason is that with increasing numbers of PCR primers, the possible set of

reactions rises as the square of the number of primers: Each primer, in principle, could

amplify with any other primer if a suitable target were present. Thus the expected back-

ground will increase as the square of the number of primers. The desired signal will in-

crease only linearly with the number of primers. Clearly this rapidly becomes a losing

proposition. In contrast, it is relatively easier to use many hybridization probes simultane-

ously, since here the background will increase only linearly with the number of probes.

Both PCR and hybridization schemes lend themselves to

large

scale

organized

projects, but the implications and mechanics are very different. With hybridization, filter

replicas of an array can be

sent out to a set of distant users. However, the power

of

the

array increases, the more about it one knows. Therefore, for an array to have optimal im-

pact, it is highly desirable that all results of probe hybridizations against it be compiled in

one centrally accessible location. In practice, this lends itself

to schemes in place in

Europe where the array hybridizations are actually done in central locations, and data are

compiled there. Someone who wishes to interrogate an array with a probe, mails in that

probe to a central site.

With

PCR screening,

the key

information

is the

sequences

of

the

DNA

primers.

This information can easily be compiled and stored on a centrally

accessible

database.

Users simply have to access this database, and either make the primers needed or obtain

them from others who have already made them. This allows a large number of individual

laboratories to use the map, as it develops, and to participate in the mapping without any

kind of elaborate distribution scheme for samples and without centralized experimental

facilities. The PCR-based screening approach has thus far been

more

popular

in

the

United States.

In the long run it would

be nice to have a hybrid approach that

blends the

advantages

of both PCR and hybridization. One way to think about this

would be to use small

primers

or

repeated

sequence

primers

to reduce

the

complexity

of

complex

probes

by

TIERED SETS OF SAMPLES

299

sert size, the ordering should be reasonably effective. However, the key point in the over-

all strategy is that one need not make the YAC contig in advance. Cross-connecting the

various sets of samples in parallel will eventually provide all of the information needed to

order all of them.

The example shown in Figure 9.9 is a somewhat difficult case because the size step

taken, from 500 kb clones to 40 kb clones, is a big one. It would be better to have one or

two tiers of samples in between; say 250 kb YACs and 100 kb P1 clones. This will com-

pensate quite a bit for the inevitable irregularities in the distribution and coverage of each

of the libraries in particular regions. An analogy that may not be totally farfetched is that

the intermediate tiers of samples can help strengthen

the ordering process in the

same

manner as intermediate levels in a neural net can enhance its performance (see Chapter

15).

YAC and cosmid libraries involve large numbers of clones, and it would be very ineffi-

cient to handle such samples one at a time. We have already shown that cosmids can be

handled

very

efficiently as dense arrays of DNAs on filters. YACs are less easily handled

in this manner. The amount of specific sample DNA is much less, since typical YACs are

single-copy clones in a 13-Mb genome background,

while cosmids are multicopy

in a

4.8-Mb background. Thus hybridization screening of arrays of YAC clones has not always

been very successful. It would greatly help if there were an effective way to purify the

YAC DNA away from the yeast genomic DNA. Automated procedures already have been

developed to purify total DNA from many YAC-bearing strains at once (Box 9.1). Now

what is needed is a simple automatable method for plucking the YACs out of this mixture.

An alternative to purification would be YAC amplification. As described earlier in this

chapter, this is possible but not yet widely used, and

the amount of amplification is

still

only modest. Perhaps the most effective method currently available for increasing our

sensitivity of working with YAC DNA is PCR.

In Chapter 14 we will illustrate how

human-specific PCR, based on repeating sequence primers like

Alu ’s, can be used

to see

just the human DNA in a complex sample. Almost every YAC is expected to contain mul-

tiple

Alu

repeats. It is possible to do hundreds of PCR reactions simultaneously with com-

monly available thermal cyclers, and ten to perhaps a hundred times larger number

of

samples is manageable with equipment that has been designed and built specially for this

purpose.

Figure 9.9

Division of a YAC into a contig of cosmids by taking advantage of other YACs known

to be contiguous.

302

ENHANCED METHODS FOR PHYSICAL MAPPING

STP

or STRP.

These abbreviations refer to sequence tagged polymorphism or poly-

morphic sequence tag. A very simple notion is involved here. If the PCR tag is a

polymorphic

sequence, then

the genetic and physical maps can be

directly

aligned

at the position of this tag. This allows the genetic and physical maps to be built in

parallel. A possible limitation

here is that some of the most

useful

genetic probes

are tandemly repeating sequences, and a certain subset of these, usually very simple

repeats

like

(AC)

n , tend to give extra unwanted amplification products in typical

PCR protocols. However, it seems possible to find slightly more complex repeats,

like

(AAAC)

n , that are

equally useful as genetic probes but

show fewer

PCR

arti-

facts.

EST.

This

stands for

expressed sequence tag. It could really refer to any piece of cod-

ing DNA sequence for which PCR primers have been established. However, in

practice, EST almost always refers to a segment of the DNA sequence of a cDNA.

These

samples

are

usually obtained by starting with an existing cDNA library,

choosing clones at random, sequencing as much of them as can be done in a single

pass, and then using this sequence information to place the clone on a physical map

(through somatic cell genetics or FISH). There are many advantages to such se-

quences as probes. One knows that a gene is involved. Therefore the region of the

chromosome is of potential interest. The bit of DNA sequence obtained may be in-

teresting:

It

may

match something already in the database or

be

interpretable

in

some way (see Chapter 15). In general, the kinds of PCR artifacts observed with

STPs, STRPs, and STARs are much less likely to occur with ESTs.

Despite

their

considerable

appeal

there

are a number of potential problems

in

dealing

with ESTs as mapping reagents. As shown in Figure 9.12, cDNAs are discontinuous sam-

ples of genomic DNA. They will typically span many exons. This can be very confusing.

If the EST crosses a large

intron, the probe will show PCR amplification, but genomic

DNA or a YAC clone will not. A common strategy for EST production uses largely un-

translated DNA sequence at the 3

-ends of the cDNA clones. It is relatively easy to clone

these regions,

and

they

are more polymorphic than the internal coding region.

Furthermore cDNAs from gene families will tend to have rather different

3

-untranslated

regions, and thus one will avoid some of the problems otherwise encountered with multi-

ple positive PCR reactions

from members of a gene family. These 3

-end sequences will

also tend to contain only a single exon in front of the untranslated region. However, all of

these advantages carry a price: The 3

-end sequence

is

less

interesting and

interpretable

than the remainder of the cDNA.

POOLING IN MAPPING STRATEGIES

303

Eugene Sverdlov and his colleagues in Moscow have developed an efficient procedure

for preparing chromosome-specific cDNA libraries. Their three-step procedure is an ex-

tension of simpler procedures that were tried earlier by others. This procedure uses an ini-

tial

Alu

-primed PCR reaction to make a cDNA copy of the hnRNA produced in a hybrid

cell containing just the chromosome of interest. The resulting DNA is equipped with an

oligo-G tail, and then a first round PCR is carried out using an oligo-C containing primer

and an

Alu

primer. Then a second

round of PCR is done

with a nested

Alu

primer. The

PCR primers are also designed so that

the first round introduces one restriction site and

the second round another. The resulting products are then directionally cloned into a vec-

tor requiring both sites. In studies to

date, Sverdlov and his coworkers have found that

this scheme produces a diverse

set

of

highly enriched human cDNAs. Because these

come from

Alu s in hnRNA, they will contain introns, and this gives them potential advan-

tages

as

mapping

probes

when

compared

with conventional cDNAs. As with the 3

-

cDNAs discussed above, cDNA from hnRNAs will be

more

effective

than

ordinary

cDNAs in dealing with gene families and in avoiding cross-hybridization with conserved

exonic sequences in rodent-human cell hybrids.

POOLING

IN MAPPING STRATEGIES

All of the above methods for screening

individual samples in a complex library are fine

for finding genes. However, they are very inefficient in ordering a whole library. The

schematic result of a successful screen for a gene-containing clone in an arrayed library is

shown

in Figure

9.13

a.

A single positive clone is detected, presumably containing the

sample

of

interest. However, from the viewpoint of information

retrieval,

this

result is

very weak. A sample array is potentially an extremely informative source of information

about

the

order of the samples it contains. A single positive hybridization extracts the

minimum possible amount of

information

from the array and

requires a time-consuming

(a)

(b)

Figure 9.13

Hybridization to arrays. (

a ) Typical pattern seen when screening a

library for a par-

ticular gene. (

b ) Ideal pattern of hybridization in interrogating a dense sample array.

304

ENHANCED METHODS FOR PHYSICAL MAPPING

experiment. From the viewpoint of information theory, a much better designed interroga-

tion of the array would produce the result shown in Figure 9.13

b. In this ideal black-and-

white case, roughly half the clones would be scored positive and half negative if we are

considering only positive and negative hybridization results. When the amount

of

signal

can

be

quantitated, much more

information is potentially available from a single

test of

the array through all the gray levels seen for each individual clone.

The

practical issue is how

to take advantage of the power of arrays, or pools

of

samples,

in a maximally efficient way so that all of the clones are ordered accurately with a minimum

number of experiments. The answer will depend critically on the nature

of the

errors in-

volved in interrogating arrays or pools. We start by considering fairly ideal cases. A detailed

analysis

of the effects of errors in real applications is

still not available, but

the power of

these approaches is so great that it appears that a reasonable error rate can be tolerated.

The goal is to design probe or sample pools and arrays that allow roughly half of the tar-

gets to be scored positive in each hybridization or PCR. There are a number of different ways

to try to accomplish this. One general approach is to increase the amount of different DNA

sequences in the probes or the targets. The other general approach is to increase the fraction

of potential target DNA that will be complementary to probe sequences. In principle, both

approaches can be combined. A very simple strategy is to use complex probes. For example,

purified large DNA fragments can be used as hybridization probes. DNA from hybrid cell

lines or radiation hybrids can be used as probes. In some or all of these cases, it is helpful to

employ human-specific PCR amplification so that the probe contains sufficient concentra-

tions of the sequences that are actually serving to hybridize with specific samples in the tar-

get.

The logic behind the use of large DNA probes is that they automatically contain conti-

nuity information, and sets of target clones detected in such a hybridization should lie in

the same region of the genome. It is far more efficient, for example, to assign an array of

clones

to

chromosome regions

by hybridizing with

DNA

purified

from

those

regions,

than it

is to assign the regional location of clones one at a time by hybridizing

to a panel

of

cell

lines. The key technical advances that makes these new strategies

possible

are

PCR amplification of desired sequences and suppression hybridization of undesired re-

peated sequences.

An alternative to large DNA probes is simple sequence probes. Oligonucleotides of

lengths 10 to 12 will hybridize with a significant fraction of arrayed cosmids or YACs.

Alternatively, one could use simple sequence PCR primers, as we discussed in Chapter 4.

There is no reason why probe

molecules must be used individually. Instead, one

could

make pools of probes and use

this pool directly in hybridization. By constructing differ-

ent sets of pools, it is possible, after the fact, to sort out which members of

which pools

were responsible for positive hybridization. This turns out to be more efficient in princi-

ple. Any sequences can be used in pools. One approach is to select arbitrary, nonoverlap-

ping sets of single-copy sequences. Another approach

is to build

up pools from

mixtures

of short oligonucleotides.

Another basic strategy for increasing the efficiency of mapping is to use pools of sam-

ples. This

is a necessary part of PCR screening

methods, but

there

is no

reason why

it

that

are involved in pooling strategies in the next few sections. These principles apply

equally whether pools

of probes or pools of samples are

involved.

Basically it would

seem

that one should be

able to combine simultaneously sample

pooling

and probe pool-