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

APPROACHES TO DNA SEQUENCING BY MASS SPECTROMETRY

355

BOX 10.1

MULTIPHOTON DETECTION (MPD)

In contrast to RIS, a much simpler, nondestructive detector has been developed by

Andy Druckier that has the capacity to analyze many different metal isotopes simulta-

neously, but these must be short lived radioisotopes. The principle used in this exquis-

itely sensitive detector is shown in Figure 10. 24. It is based on certain special ra-

dioisotopes that emit three particles in close succession. First a positron and an

electron are emitted simultaneously, at 180-degree angles. Shortly thereafter a gamma

ray is emitted. The gamma energy depends on the particular isotope in question.

Hence many different isotopes can be discriminated by the same detector. The electron

and positron events are used to gate the detector. In this way the background produced

by gamma rays from the environment is extremely low. The potential sensitivity of

APPROACHES TO DNA SEQUENCING BY MASS SPECTROMETRY

357

Figure 10.26

Sequencing

a

compression region

in the beta globin

gene

by

gel

electrophoresis

(top

) and MALDI-TOF mass

spectrometry (

bottom

). Note

in

the gel data a poorly resolved set of

G’s topped by what appears to be a C, G heterozygote. The true sequence, GGGGC, is obvious in

the mass spectrum. Provided by Andi Braun. Figure also appears in color insert.

MS, however, all that is measured is the ratio of mass to charge, so any secondary struc-

ture effects are invisible. Thus the resulting sequence ladder is unambiguous.

A second mass analyzer that has been used for

analysis of DNA is Fourier

transform

ion cyclotron resonance (FT-ICR) mass spectrometry. Here ions are placed in a stable cir-

cular orbit, confined by a magnetic field. Since they continuously circulate, one can re-

peatedly detect the DNA, and thus very high experimental sensitivity is

potentially

achievable. The other great advantage of FT-ICR instruments is that they have extraordi-

nary resolution, approaching 1/10

6 in some cases. This produces extremely complex spec-

tra because of the effects of stable isotopes like

13C. However, since the mass differences

caused by such isotopes are known in advance, the bands they create can be used to assist

spectral assignment and calibration. FT-ICR can be used for direct examination of Sanger

ladders, or it can be used for more complex strategies as described below. The major dis-

advantages of FT-ICR are the cost and complexity of current instrumentation

and the

greater complexity of the spectra. An example of

a

FT-ICR spectrum

of

an

oligonu-

cleotide is shown in Figure 10.27.

The third strategy in mass spectrometric sequencing

is the traditional one

in

this field.

A sample is placed into the vapor phase and then fragmentation is stimulated either by

collision with other atoms or molecules or by laser irradiation. Most fragmentation under

these

conditions

appears to occur

by successive release of mononucleotides

from

either

358

DNA

SEQUENCING: CURRENT TACTICS

100

6117 6118

6116

6119

50

6120

6115

6121

0

6122 6123

6114

6116

6118

6120

6122

6124

Figure 10.27

Mass (m/z)

An example of FT-ICR mass spectrometry of an oligonucleotide. Here the complex

set of bands seen for the parent ion of a single compound are

molecules

with

different numbers

of

13C atoms. Provided by Kai Tang.

end of the DNA chain. While this approach

is feasible and has

yielded

the sequence

of

one 50-base DNA fragment, the resulting spectra are so

complex that at least at present

this approach does not appear likely to become a routine tool for DNA analysis.

One great potential power of mass spectroscopy for direct analysis of DNA ladders is

speed. In principle, at most a few seconds of analysis

would be needed to collect suffi-

cient data to analyze one ladder. This is much faster than any current extrapolation of

electrophoresis rates. The major ultimate challenge may be finding a way to introduce

samples into a mass spectrometer fast enough to keep up with its sequencing speed.

RATE-LIMITING STEPS IN CURRENT DNA SEQUENCING

Today, laboratories that can produce 10

4 to 10

5

raw base pairs of DNA sequence per per-

son per day are virtually unanimous in their conclusion

that at this rate, analysis of the

data is the rate-limiting step. So long as one has a good supply of samples worth sequenc-

ing, the rate of data acquisition with current automated equipment is not a barrier. A few

laboratories have equipment that operates at ten times this rate. This has not yet been used

in a steady production mode for sequence determination,

but once it enters this mode,

there is every reason to believe that data analysis will

continue to be rate limiting. Some

of the potential enhancements we have described for ladder sequencing promise almost

certainly to yield an additional factor of 10 improvement in throughput over the next few

years, and a factor of 100, eventually, is not inconceivable. This will surely exacerbate the

current problems of data analysis.

The rapid rate of acquisition of DNA sequence data makes it critical that we improve

our methods of designing large-scale DNA sequencing projects and develop improved

abilities for on-line analysis of the data. This analysis includes error correction, assembly

of raw

sequence

into finished sequence, and

interpreting

the significance

of the sequenc-

SOURCES AND ADDITIONAL READINGS

359

SOURCES AND

ADDITIONAL

READINGS

Ansorge, W., Zimmermann, J., Schwager, C., Stegemann, J., Erfle, H., and Voss, H. 1990. One la-

bel, one tube, Sanger DNA sequencing in one and two lanes on a gel.

Nucleic

Acids Research

18: 3419–3420.

Carrilho, E., Ruiz-Martinez, M. C., Berka, J., Smirov, I., Goetzinger, W., Miller, A. W., Brady, D.,

and Karger, B. L. 1996. Rapid DNA sequencing of more than 1000 bases per run by capillary

electrophoresis using replaceable linear polyacrylamide solutions.

Analytical

Chemistry

68:

3305–3313.

Ewing, B., Hillier, L., Wendl, M. C., and Green, P. 1998. Base-calling of automated sequencer

traces using Phred. I. Accuracy assessment.

Genome Research

8: 175–185.

Ewing, B., and Green, P. 1998. Base-calling of automated sequencer traces using Phred. II. Error

possibilities.

Genome Research

8: 186–194.

Glazer, A. N., and Mathies, R. A. 1997. Energy-transfer fluorescent reagents for DNA analyses.

Current Opinion in Biotechnology

8: 94–102.

Grothues, D., Voss, H., Stegemann, J., Wiemann, S., Sensen, C., Zimmerman, J., Schwager, C.,

Erfle, H., Rupp, T., and Ansorge, W. 1993. Separation of up to 1000 bases on a modified A.L.F.

DNA sequencer.

Nucleic Acids Research

21: 6042–6044.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, and A. N. Glazer.

Stable

Fluorescent

Complexes of

Double-Stranded DNA with

Bis-Intercalating Asym-

metric Cyanine Dyes: Properties and Applications.

Nucleic

Acids Research

20, 3803-3812

(1992).

Hultman, T., Stahl, S., Hornes, E., and Uhlen, M. 1989. Direct solid phase sequencing of genomic

and plasmid DNA using magnetic beads as solid support.

Nucleic

Acids

Research

19:

4937–4936.

Jacobson, K. B., Arlinghaus, H. F., Schmitt, H. W., Sacherleben, R. A., et al. 1990. An approach to

the use of stable isotopes for DNA sequencing.

Genomics

8: 1–9.

Kalman, L. V., Abramson, R. D., and Gelfand, D. H. 1995. Thermostable DNA polymerases with

altered discrimination properties.

Genome Science and Technology

1: 42.

Koster, H., Tang, K., Fu, D.-J., Braun, A., van den Boom, D., Smith, C. L., Cotter, R. J., and Cantor,

C. R. 1996. A strategy for rapid and efficient DNA sequencing by

mass spectrometry.

Nature

Biotechnology

14: 1123–1128.

Kustichka, A. J., Marchbanks, M., Brumley, R. L., Drossman, H., and Smith, L. M. 1992. High

speed automated DNA sequencing in ultrathin slab gels.

Bio/Technology

10: 78–81.

Kwiatkowski, M., Samiotaki, M., Lamminmaki, U., Mukkala,

V.-M., and Landegren U. 1994.

Solid-phase synthesis of chelate-labelled oligonucleotides: Application in triple-color ligase-me-

diated gene analysis.

Nucleic Acids Research

22: 2604–2611.

Luckey, J. A., Norris, T. A., and Smith, L. M. 1993. Analysis of resolution in DNA sequencing by

capillary gel electrophoresis.

Journal of Physical Chemistry

97: 3067–3075.

Luckey, J. A., and Smith, L. M. 1993. Optimization of electric field strength for DNA sequencing in

capillary gel electrophoresis.

Analytical Chemistry

65: 2841–2850.

Naeve, C. W., Buck, G. A., Niece, R. L., Pon, R. T., Robertson, M., and Smith, A. J. 1995.

Accuracy of automated DNA sequencing: A multi-laboratory comparison of sequencing results.

BioTechniques

19: 448–453.

360

SOURCES AND ADDITIONAL READINGS

Parker, L. T., Deng, Q., Zakeri, H., Carlson, C., Nickerson, D. A., and Kwok, P. Y. 1995. Peak

height variations in automated sequencing of PCR products using Taq dye-terminator chemistry.

BioTechniques

19: 116–121.

Porter, K. W.,

Briley, J. D., and Shaw, B. R. 1997. Direct PCR sequencing with boronated nu-

cleotides.

Nucleic Acids Research

25: 1611–1617.

Stegemann, J.,

Schwager, C., Erfle, H., Hewitt, N., Voss, H., Zimmermann, J., and Ansorge, W.

1991. High speed on-line DNA sequencing on ultathin slab gels.

Nucleic Acids

Research

19:

675–676.

Tabor, S., and

Richardson, C. C. 1995. A single residue in DNA polymerases of the

Escherichia

coli DNA polymerase I family is critical for distinguishing between deoxyand dideoxyribonu-

cleotides.

Proceedings of the National Academy of Sciences USA

92: 6339–6343.

Yamakawa, H., Nakajima, D., and Ohara, O. 1996. Identification of sequence motifs causing band

compressions on human cDNA sequencing.

DNA Research

3: 81–86.

Yarmola, E., Sokoloff, H., and Chrambach, A. 1996. The relative contributions of dispersion and

diffusion to

band spreading (resolution) in

gel electrophoresis.

Electrophoresis

17: 1416–1419.