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

Other types of mispairing are

less destabilizing, but there may be a

way

to

eliminate

these, as we will discuss, momentarily. In standard hybridization sequencing, a terminal

mismatch is the least destabilizing event, and thus it leads to the greatest source of ambi-

guity or background. For an octanucleotide complex, an average terminal mismatch leads

to a 6 °C lowering in

T m . For stacking hybridization,

a

terminal

mismatch on the side

away from the preexisting duplex is the least destabilizing

event. For

a

pentamer, this

leads to a drop in

T

m

of 10 °C. These considerations predict that the discrimination power

of

stacking

hybridization

in

favor of perfect duplexes might be greater

than

ordinary

SBH. They encourage attempts to modify the notion of stacking hybridization so that it

becomes a general, stand-alone method for DNA sequencing.

OTHER APPROACHES FOR ENHANCING SBH

Once an oligonucleotide has formed a duplex

with the target,

it

ought

to

be

possible

to

use enzymatic steps to proofread the

accuracy of the

hybridization

and

to

read

further

DNA sequence information from the target. For example, the 3

-end of the oligonu-

cleotide could serve as a primer for DNA polymerase to extend. What is needed is a suffi-

ciently stable primer-template complex to allow the polymerase to function at a suitable

temperature. An issue that needs to be explored is whether 8-mers are sufficient for this

purpose. There are also potential background problems

that

will

need

to

be

addressed.

This general approach has been used quite successfully with longer primers and DNA

polymerase extension to detect specific alleles adjacent to the primer in a method called

genetic bit analysis (Nikiforov et al., 1994). An alternative method for proofreading and

extending a sequence read could use DNA ligase to attach a stacked oligonucleotide next

to an initial duplex. This would

have the potential advantage that ligase requires

proper

base pairing and might increase the discrimination of the stacking hybridization. In both

cases, and in other schemes that can be contemplated, the label is introduced as a result of

the enzymatic reaction. This eliminates much of the current background

in

SBH

that

arises from imperfect hybridization products. Some specific examples of how these pro-

cedures can be implemented in practice will be described in the next section.

A second, general way to

enhance the power of SBH is to use

gapped

oligonu-

cleotides. Two examples of this are shown below:

AGCN

4 GAC

AGCI

4 GAC

The first case uses a mixture of 256 possible 10-mers that share the

same

six

external

bases. The second uses a single

10-mer, but its four central bases are inosine

(I)

which

can base pair with A, C, or T. In both of these cases the stability of the duplex is increased

because it has more base pairs: one can read

six bases of sequence

but with

the

stability

of a decanucleotide duplex. However, of even greater significance is the fact that the ef-

fective reach of the oligonucleotide is increased. Branch point ambiguities are less serious

with these gapped molecules than with ungapped oligonucleotides with the same number

of

well-defined

bases.

William

Baines

has

simulated

SBH

experiments

with

gapped

POSITIONAL SEQUENCING BY HYBRIDIZATION (PSBH)

425

N

2AGCTTAAGN

2

The advantage of this approach is that any mismatches at the ends of the internal 8-base

probe sequence are converted to internal mismatches in the actual 12-base probe used.

Another way to enhance the power of SBH is to use the same kinds of pooling and

multicolor detection schemes that we discussed in Chapters 9 and 11 for fast

physical

mapping and enhanced ladder DNA sequencing. There is every reason to use groups of

oligomers simultaneously in hybridization to sample arrays, or groups of samples simul-

taneously, to oligonucleotide chips. Simulations are needed to help design the most effec-

tive strategies to do this. However, very simple arguments show that a considerable in-

crease in throughput ought to be achievable. Earlier we calculated that less than 0.4% of

the probes or targets score positive in a single hybridization. Performing 16 hybridiza-

tions in binary pools will therefore not entail much risk of ambiguities. Doing this in a

single color would result in up to a fourfold increase in throughput. Multiple colors could

be used to increase the throughput much more.

Alternatively, multiple colors might be used to help resolve branch point ambiguities.

Suppose that one had a way of labeling a target with

two colors, such that

the

ratio of

these colors depended on the

location of the target within

a much larger clone. One

way

to think about doing this is placing the label in the target by a single cycle of primer ex-

tension, varying the relative concentrations of two different labeled dpppN’s during the

extension. When fragments of this target are hybridized to a oligonucleotide chip, the ra-

tio of the labels will tell, roughly, where in the sequence the particular oligonucleotide is

located (Fig. 12.33).

POSITIONAL SEQUENCING BY HYBRIDIZATION (PSBH)

Here we describe a scheme that was developed and refined in our own laboratories as an

alternate form of SBH. It is called positional sequencing by hybridization (PSBH). It has

a number of potential advantages over conventional SBH but also presents its own set of

different obstacles that must be overcome to make the total scheme a practical reality.

PSBH relies totally on stacking hybridization. It uses an array of probes constructed as

follows, where

X n

refers to a single specific DNA sequence of length

n, and

Y n is the com-

428

FUTURE DNA SEQUENCING WITHOUT LENGTH FRACTIONATION

Once the target has been ligated to the probe, it can serve as a substrate for the acquisi-

tion of additional DNA sequence data. For example, as shown in Figure 12.36, the 3

-end

of the probe can be used as a primer to read the next base of the target by extension with a

single, labeled terminator. Alternatively, any of the single nucleotide addition methods de-

scribed at the beginning of this chapter can now be used on each immobilized target mol-

ecule as in Genetic Bit Analysis (Nikoforov et al., 1994). It would also be possible to do

plus/minus sequencing on each immobilized target if one had sufficient quantitation with

four colors to tell the amounts of each base incorporated. The basic idea is that the probe

array can serve to localize a

large number of different target molecules, simultaneously,

and determine a bit of their sequence. Most probes will capture only a single target, and

each of these complexes can then be sequenced in parallel. This should combine some of

the best features of ladder

and hybridization sequencing. It should produce sequence

reads on each target molecule

that are long

enough to resolve all the common branch

point ambiguities, except for those caused by true interspersed repeating sequences.

A major limitation in the

PSBH approach we have described thus far is that it only

reads the sequence at one end of the target. This would seem to limit its application to rel-

atively

short targets. However,

one can circumvent this problem, in principle, by making

a nested set of targets, as shown in Figure 12.37. One has to be careful in choosing the

strategy

for

constructing

these

samples, since

the ends of the DNAs must still be able to

be ligated.

Thus dideoxy

terminators could be

used, but they would have to be replaced

single-base addition early in the chapter. Alternatively, chemical cleavage

could be used,

as described when genomic DNA sequencing was used to locate

m C’s (Chapter 11). The

POSITIONAL SEQUENCING BY HYBRIDIZATION (PSBH)

429

scheme discussed in the previous section. A particularly simple way to

mark the

location

of a branch point ambiguity is to combine a fixed end label and

an internal

label, as

SOURCES AND

ADDITIONAL READINGS

431

The resulting captured set of samples is spatially resolved and can now be subjected to

Sanger extension reactions to generate a

set

of sequence

ladders. The

appeal

of this

method is that a set of samples can be processed all at once in

a single tube without any

need for prior fractionation. Capture has been shown to be efficient with mixtures of up to

25 samples (Broude et al., 1994), and high-quality sequencing ladders have been prepared

from mixtures of up to eight samples (Fu et al., 1995). The real promise of this approach

probably lies in the preparation of samples

for

MALDI MS

DNA sequencing

(Chapter

SOURCES AND ADDITIONAL

READINGS

Broude, N. E., Sano, T., Smith, C. L., and Cantor, C. R. 1994. Enhanced DNA sequencing by hy-

bridization.

Proceedings of the National Academy of Sciences USA

91: 3072–3076.

Chee, M., Yang, R., Hubbell, E., Berno, A., Huang, X. C., Stern, D., Winkler, J., Lockhart, D. J.,

Morris, M. S., and Fodor, S. P. A. 1996. Accessing genetic information with high-density DNA

arrays.

Science

274: 610–614.

Chetverin, A., and Kramer, F. R. 1994. Oligonucleotide array: New concepts and possibilities.

Bio/Technology

12: 1093–1099.

Dubiley, S. et al. 1997. Fractionation, phosphorylation and ligation on oligonucleotide microchips

to enhance sequencing by hybridization.

Nucleic Acids Research

25: 2259–2265.

Fu, D-J., Broude, N. E., Koster, H., Smith, C. L., and Cantor, C. R. 1995. Efficient preparation of

short DNA sequence ladders potentially suitable for MALDI-TOF DNA sequencing.

Genetic

Analysis

12: 137–142.

Fu, D.-J., Broude, N. E., Koster, H., Smith, C. L., and Cantor, C. R. 1995. A DNA sequencing strat-

egy that requires only five bases of known terminal sequence for priming.

Proceedings of

the

National Academy of Sciences USA

92: 10162–10166.

Guo, Z., Liu, Q., and Smith, L. M. 1997. Enhanced discrimination of single nucleotide polymorpo-

hisms by artificial mismatch hybridization.

Nature Biotechnology

15: 331–335.

Jurinke, C., van den Boom, D., Jacob, A., Tang, K., Worl, R., and Koster, H. 1996. Analysis of li-

gase chain reaction products via matrix-assisted laser desorption/ionization time-of-flight mass

spectrometry.

Analytical Biochemistry

237: 174–181.

Khrapko, K. R., Lysov. Y. P., Khorlin, A. A., Ivanov, I. B., Yershov, G. M., Vasilenko, S. K.,

Florentiev, V. L., and Mirzabekov, A. D. 1991. A method for DNA sequencing by hybridization

with oligonucleotide matrix.

Journal of DNA Sequencing and Mapping

1: 375–368.

Lane, M. J., Paner, T., Kashin, I., Faldasz, B. D., Li, B., Gallo, F. J., and Benight, A. S. 1997. The

thermodynamic advantage of DNA oligonucleotide

“stacking hybridization”

reactions:

Energetics of a DNA nick.

Nucleic Acids Research

25: 611–616.

Li, Y., Tang, K., Little, D.P., Koster, H., Hunter, R. L., and McIver, R. T. Jr. 1996. High-resolution

MALDI fourier transform mass spectrometry of oligonucleotides.

Analytical

Chemestry

68:

2090–2096.

Livshits, M. A., Florentiev, M. L., and Mirzabekov, A. D. 1994. Dissociation of duplexes formed by

hybridization of

DNA

with

gel-immobilized oligonucleotides.

Journal of

Biomolecular

Structure Dynamics

11: 783–795.

Lysov, Y. P. et al. 1994. DNA sequencing by hybridization to oligonucleotide matrix. Calculation of

continuous

stacking hybridization efficiency.

Journal

of Biomolecular Structure Dynamics

11:

797–812.

432

FUTURE DNA SEQUENCING WITHOUT LENGTH FRACTIONATION

Maskos, U., and Southern, E. M. 1992. Parallel analysis of oligodeoxyribonucleotide (oligonu-

cleotide) interactions. I. Analysis of factors influencing

oligonucleotide

duplex

formation.

Nucleic Acids Research

20: 1675–1678.

Maskos, U., and Southern, E. M. 1992. Oligonucleotide hybridisations on glass supports: A novel

linker for oligonucleotide synthesis and hybridisation properties of oligonucleotides synthesised

in situ.

Nucleic Acids Research

20: 1679–1684.

Milosavljevic, A. et al. 1996. DNA sequence recognition by hybridization to short oligomers:

Experimental verification of the method on the

E. coli

genome.

Genomics

37: 77–86.

Murray, M. N., Hansma, H. G., Bezanilla, M., Sano, T., Ogletree, D. F., Kolbe, W., Smith, C. L.,

Cantor, C. R., Spengler, S., Hansma, P. K., and Salmeron, M. 1993. Atomic force microscopy of

biochemically tagged DNA.

Proceedings

of

the National

Academy

of Sciences

USA

90:

3811–3814.

Nikoforov, T. T., Rendle, R. B., Goelet, P., Rogers, Y.-H., Kotewicz, M. L., Anderson, S., Trainor,

G. L., and Knapp, M. R. 1994. Genetic bit analysis: A solid phase method for typing single nu-

cleotide polymorphisms.

Nucleic Acids Research

22: 4167–4175.

Pease, A. C., Solas, D., Sullivan, E., Cronin, M. T., Holmes, C. P., and Fodor, S. P. A. 1994. Light-

generated oligonucleotide arrays for rapid DNA sequence analysis.

Proceedings

of the National

Academy of Sciences USA

91: 5022–5026.

Ronaghi, M., Uhlen, M., and Nyren, P. (1998). Real-time pyrophosphate detection for DNA se-

quencing.

Science

281: 363–365.

Shalon, D., Smith, S. J., and Brown, P. O. 1996. A DNA microarray system for analyzing complex

DNA samples using two-color fluorescent probe hybridization.

Genome Methods

6: 639–645.

Southern, E. M., Maskos, U., and Elder, J. K. 1992. Analyzing and comparing nucleic acid se-

quences by hybridization to arrays of oligonucleotides: Evaluation using experimental models.

Genomics

13: 1008–1017.

Southern, E. M., Green-Case, S. C., Elder, J. K., Johnson, M., Mir, K. U., Wang, L., and Williams,

J. C. 1994. Arrays of complementary oligonucleotides for analysing the hybridisation behavior

of nucleic acids.

Nucleic Acids Research

22: 1368–1373.

Strezoska, Z., Paunesku, T., Radosavljevic, D., Labat, I., Drmanac, R., and Crkvenjakov, R. 1991.

DNA sequencing by hybridization: 100 bases read by a non-gel

method.

Proceedings of the

National Academy of Sciences USA

88: 10089–10093.