Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 12

Figur

e 12.23

Patterns of hybridization seen when an immobilized tar

get sequence and a control sequence are probed successi

v e ly with adjacent

radiola-

beled

octanucleotides chosen as complements to the tar

get sequence.

T a ken from Strezoska et al. (1991).

DATA ACQUISITION

AND ANALYSIS

415

a comparable negative control sequence. Oligonucleotides were selected on the

basis

of

the known sequence; others were added to serve as

negative

controls. The

results

are

fairly convincing. As shown in Figure

12.23, the

discrimination between

true positives

and negatives is quite good in most of the individual hybridizations. Of course the

obvi-

ous criticism of this experiment is that with a sequence known in advance, the test is not a

truly objective one.

To address these concerns, Drmanac and Crkvenjakov performed a second pilot test of

SBH on three closely related unknown sequences containing a total of 343 bases. The de-

sign of the test was based on an uninvolved third party who analyzed these sequences and

designed a set of oligonucleotides in which only

about half

corresponded

to

the se-

quences in the target samples. In addition the challenge was to determine all three un-

known samples and not generate erroneous composites of them by errors in reconstruc-

tion. The test was a total success—all three

unknown sequences

were

correctly

determined. However, one caveat needs to be considered. Because all 65,536 8-mers were

not provided, this automatically supplies enormous amounts of information about the true

sequence. Any compound omitted from the set provided is automatically a true negative.

Just this information alone restricts the possible sequences tremendously, even before a

single experiment has been done. Thus, while the experimental results that have been

achieved are impressive, it cannot yet

be said that

a

definitive test of SBH for

de

novo

DNA sequencing has been done. Indeed, in defense of all who work in this field, it will

probably not be possible to test the

methods definitively until the gamble

is

taken to

make, directly on chips or in bulk for distribution, all of the 65,536 8-mers.

DATA ACQUISITION AND ANALYSIS

Three different methods have been used thus far to detect hybridization in pilot SBH ex-

periments. In each case quantitative data are needed so that positive signals can be dis-

criminated as clearly as possible from

background. Southern used image plate analyzers

to examine radioisotope decay for the results shown in Figure 12.22. Others have used

autoradiograms quantitated with a CCD camera. These approaches were discussed

in

Chapter 9. Fluorescent probes have been used by Fodor and by Mirzabekov. Here a CCD

camera can be used in conjunction with a fluorescence microscope to record quantitative

signals. Alternatively, a confocal scanning fluorescence microscope can be used. Other

approaches such as mass spectrometry (see Chapter 11) are under development. The very

notion of an oligonucleotide or sample chip raises the expectation that it should be possi-

ble to find a way to read out the amount of hybridization by a direct electronic method.

Kenneth Beattie and Mitchell Eggers

have developed one approach to this by

detecting

the mass of bound sample as it changes the local impedance on a silicon surface. In prin-

ciple, one ought to be able to enhance such detection by providing the DNA probes or tar-

gets with attachments that generate more dramatic effects through altered conductivity, as

a source of electrons or holes, or through magnetic properties. Perhaps the ultimate no-

tion, as shown by the purely hypothetical example in Figure 12.24, would be to use the

stability of the duplex formed in hybridization to directly manipulate

elements

of

a

nanoscale chip and thus lead to a detectable electrical signal.

OBSTACLES TO SUCCESSFUL SBH

417

Figure 12.26

Overcoming branch point ambiguities by the

use

of

several

homologous

but

not

identical DNA sequence targets.

clones. This is what is done for ordinary shotgun ladder sequencing, except that for SBH

the clones would probably have to be even smaller. In these clones unique sequences will

lie outside and between the repeats that cause branching ambiguities. Matching up these

unique sequences not only places the clones in the proper order, it also resolves the am-

biguous internal

arrangement of

sequences on a clone with three

repeats,

since

the

order

is determined by the identity of these sequences on the flanking clones. This looks like a

powerful approach, but it requires a great deal of experimental redundancy

with

little

overall gain.

A second strategy for resolving branch point ambiguities

is shown

in

Figure

12.26.

Here the notion is to determine the DNA sequence of several similar but not identical

samples. Because of sequence variations among the samples, exact recurrences in

one

sample will not necessarily be

exact in all the others. Any

imperfections

in

the

repeats

will break the branch point ambiguities in all of the samples because they can be aligned

by homology. In principle, one could use different individuals of the same species and

take advantage of natural sequence polymorphism. However, simulations show that

the

most effective application of this approach would use samples that have about 10% diver-

gence on average. In practice, this may mean that it would

be more useful to compare

three to five similar species, like human and

chimp,

rather

than

compare

individuals

within a species. Here, as in the previous method, the cost of resolving branch point am-

biguities is a considerable increase in the number of samples that have to be examined.

However, the additional information that will be obtained will be highly interesting if the

species are well chosen.

OBSTACLES

TO SUCCESSFUL

SBH

The base composition dependence of the melting temperature,

T m poses a very serious

challenge to simple and effective implementations of SBH. If a temperature is chosen that

allows effective discrimination between perfect matches and mismatches in G

C-rich

compounds,

many

A

T-rich sequences may not

form

enough

duplex

to

be

detected.

Alternatively, if one chooses a low enough temperature to stabilize the weakest A

T-rich

duplexes, there will not be enough discrimination against mispairing in G

C-rich com-

pounds, and many false positives will result. There are many possible ways to circumvent

this problem; quite a few of them are being

tested,

but no

generally

acceptable

solution

has yet been demonstrated in practice.

Ed Southern has been

experimenting

with

the

use

of

high

concentrations

of

tetra-

OBSTACLES TO SUCCESSFUL SBH

419

Figure 12.28

Adjusting the concentration of different oligonucleotides can compensate for differ-

ence in their melting temperatures. Adapted from Mirzabekov et al.

Instead of attempting to compensate

for effects of sequence on the stability of du-

plexes, one can just measure the hybridization across a range of temperatures. This does

not increase the number of samples needed. Instead, one would effectively be recording a

melting profile for each sample with the

entire set of oligonucleotides. This

would in-

crease the experimental time by a factor

of ten or more, which is tolerable. In the long

run, once extensive data on the thermal

stability of each of the 8-mer complexes are

known, it may be possible to use a much

simpler approach. The set of compounds could

be split into

groups, each studied at a

different optimal temperature. In

principle, this

probably

easily circumvented in the sample chip strategy.

Here the target could be at-

tached

at

random but frequent places

to the surface under

denaturing conditions. This

would

not

be expected to interfere

with oligonucleotide

hybridization very much. It

problem of secondary structure is likely to

be

more serious

when

oligonucleotide chips

are used. The effect of such structures will

be

to cause a gap

in

the readable sequence.

Figure 12.29

An example of a hairpin that is too stable to be detected in SBH.

if the target tries to pair

with itself. There is undoubtedly

room

for much

development

here and much clever chemistry. A second approach would be to

use probes with un-

charged backbones. Then low ionic strength conditions can be used to suppress target

secondary structure without affecting target-probe interactions. One example of such

compounds is polypeptide nucleic acids (PNAs; see Chapter 14). Another example is

phosphotriesters in which the oxygen that is normally charged in natural nucleic acids is

esterified with an alkyl group. However, this creates an addition optically active center at

each phosphorous, which leads to

severe stereochemical

complexities

unless

optically

pure phosphotriesters are available.

The effects of secondary

structure or unusual DNA structures

are

significant but not

yet known in any great depth. In Chapter 2 we discussed the peculiar features of a cen-

tromere-associated repeat where the single strands may have a more stable secondary

structure than the duplex. In Chapter 10

we illustrated the

abnormally stable

hairpin

formed by a particular short

DNA sequence. Whether these cases are representive of 1%

of all the DNA sequences, or more or less, is simply unknown at the present time. About

the only way we will be able to uncover such idiosyncratic behavior, understand it, and

learn to deal with it, is to make large oligonucleotide arrays and start to study them.

Unfortunately, this appears to be one of those cases in science where a timid approach is

likely to be misleading. At some point we will have to dive in.

SBH IN COMPARATIVE DNA SEQUENCING

Some of the difficulties just described with full de novo SBH approaches have led some

experts to doubt that SBH will ever mature into a widespread user-friendly method. For

this reason much effort has been concentrated on developing SBH for comparative (or

differential) DNA sequencing where one assumes that a reference sequence is known and

the objective is to compare it with another sample and look for any potential differences.

Comparative sequencing is needed in checking existing sequence data for errors. It is the

type of sequencing required for horizontal studies in which many members of a popula-

tion are examined. This is needed in genetic map construction, genetic diagnostics, the

search for disease genes, in mutation detection, and for more biological objectives includ-

ing ecology, evolution, and profiling gene expression. Some of these applications are dis-

cussed in Chapters 13 and 14.

When SBH is considered in the context of sequence comparisons, two problems of the

method for de novo sequencing are immediately resolved. It is

not necessary to have a

probe array consisting of all possible 4

n

oligonucleotodes of

length

n. Instead the array

can be customized to look

for

the desired

target and

simple

sequence variations

of that

OLIGONUCLEOTIDE

STACKING

HYBRIDIZATION

421

target. Second, since a reference sequence is known, issues of

branch point ambiguities

are virtually always resolvable by use of the information in that sequence. A particularly

powerful version of SBH for comparative sequence has been developed by Affymetrix,

Inc. Here a probe array is made that corresponds to all possible strings of length

n

con-

tained in the original sequence (for a target with

L

base

pairs,

L

n 1 substrings are

required). For each substring four variants are made corresponding to the expected se-

quence at the middle position of the substring and all three possible single-base variants

there. Thus the array of probes will have 4(

L

n

1)

elements. This

is quite manage-

able with current photolithographic syntheses for targets in the range of 10 kb.

In actual practice this approach was tested on 16.6-kb human mitochondrial DNA us-

ing arrays containing up to 130,000 elements, each of which is a 15to 25-base probe.

(Chee et al., 1996). For convenience these nested targets are arranged, serially, horizon-

tally in the array as shown schematically in Figure 12.30

a, with the four possible variants

for each central eighth base located vertically. The target is randomly sheared into short

fragments (but longer than the length of the probes). A perfectly matched target will hy-

bridize strongly to one member of each vertical set of four probes. A target with a single

mismatch will show strong hybridization only to one particular probe in which the central

base variant matches the sequence perfectly. For all

possible flanking probes, there

will

be one or two internal mismatches between that target and the probe; hence hybridization

will be weak or undetectable. A sample

of

the actual data seen

using this

approach

is

shown in Figure 12.30

b.

It

is impressive. In practice, in most cases a two-color competi-

tive hybridization is used. This allows a

sample of the normal sequence

(in one

color)

to

be compared with a potential variant (in

another

color)

with most

differences

in

se-

quence-dependent hybridization efficiency nulled out.

OLIGONUCLEOTIDE STACKING

HYBRIDIZATION

There are a number of ways

that could potentially increase the length

of sequence

that

can be read with a fixed length oligonucleotide. This is one major way to improve the ef-

ficiency of SBH, since the longer the effective word length, the higher the sequencing

throughput and also the smaller the number of branch point ambiguities. One approach,

specifically designed by Mirzabekov to

help resolve branch

point

ambiguities, is

shown

in Figure 12.31. It is based on the fact that once a duplex has been formed by hybridiza-

tion of the target with an 8-mer, it becomes thermodynamically quite favorable to bind a

second oligomer immediately adjacent to the 8-mer. The extra thermodynamic stabiliza-

tion comes from the stacking between the two adjacent duplexes. This same principle was

discussed earlier in schemes for directed primer walking (Chapter 11). In practice,

Mirzabekov uses pools of ninety 5-mers, chosen specifically

to try to resolve known

branch points. A test of this approach, with a single perfectly matched 5-mer or various

mismatches, is shown in Figure 12.31. It is apparent that the discrimination power of

oligonucleotide stacking hybridization is considerable.

Some calculated

T m ’s for perfect and mismatched

duplexes are given in Table 12.1.

These are based on average

base compositions. The calculations were

performed

using

the equations given in Chapter 3. In the case of oligonucleotide

stacking,

it

is

assumed

that the first duplex is fully formed under the conditions where the second oligomer is be-

ing tested; in practice, this may not always be the case. It is, however, approximately true

for the conditions used for the experiments shown in Figure 12.32. The calculations re-

veal a number of interesting

features

about stacking

hybridization. Note that

the

binding

OLIGONUCLEOTIDE STACKING HYBRIDIZATION

423

Figure 12.31

Basic strategy in oligonucleotide stacking hybridization

Structure

a

n

8

7

6

5

38

33

25

15

33

25

15

3

25

15

3

14

51

46

40

31

46

40

31

21

40

31

21

11

Note: Calculated

T m (° C, average base composition).

a Structures consist of a long target and a probe of length

n . The top three samples

are ordinary hybridization; the bottom three are stacking hybridization.