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

AUTOMATED DNA SEQUENCING CHEMISTRY

345

Other sources of error in DNA sequencing are caused by

mispriming. Not uncom-

monly, there will be a secondary site on the template where the primer can bind and be

extended by the polymerase (Fig. 10.15). This adds a bit of low-level, specific noise to the

primary sequencing data. Another source of ambiguity arises when the sample is het-

erozygous or a mixture. The basic point is that most of

these

errors can be partially or

even totally corrected if the software is clever enough to search out these possibilities. As

more raw sequence data are obtained, and ultimately corrected into finished sequence, it

should be possible to go back to the raw data and refine the algorithms used to process it.

In short, the automated analysis of DNA sequencing data ought to be able to improve it-

self continually with time. The ideal software, which does not

yet exist,

would actually

give use the probability of each of the four bases occurring at a given position. At a given

site the result might be

A

0.01

G 0.98

C

0.00

T

0.01

This would be the best data to feed back into artificial intelligence

approaches to refine

the software further. A nice step in this direction is Phil Green’s phred algorithm which

automatically calls sequences and assigns a quality score,

q, to each base,

q

10 log

p

Figure 10.16

Two solid state DNA sequencing schemes.

(a ) From a plasmid DNA miniprep. (

b )

AUTOMATED DNA SEQUENCING CHEMISTRY

347

erate more than 10

5

bp of raw DNA

sequence per day per worker. Lanes read to 600 and

700 bases are common. The entire process

is fully automated from colony picking

to

DNA sample preparation to gel loading and running, to the raw sequence analysis and en-

try into a database. Only gel casting and sequence editing are still manual.

A number of different approaches are being tested to see if the throughput of ladder

sequencing can be further improved. Here we

will describe some or the more promising

or more novel attempts. The basic issues are

how to extend a ladder to longer sizes, how

to perform the fractionation more rapidly, how

to increase the number of different sam-

ples that can be handled simultaneously, and how to read the data more rapidly. A number

of the approaches share the feature that they

use very thin samples. The advantages

of

such gels for increasing

speed

were described earlier. A disadvantage is that thin

gels

APPROACHES TO DNA SEQUENCING BY MASS SPECTROMETRY

349

As detectors are improved, it is to be expected that larger numbers of samples will be

loaded on each gel by using closer spaced and narrower lanes. One limitation with the

current ALF system is its single color detection; yet the high sensitivity afforded by hav-

ing the laser in the plane of the gel is a clear advantage. In principle, one could use multi-

ple lasers in the gel, at different positions, and each could be accompanied by a suitable

detector array. Ansorge has developed such an instrument, which clearly will have higher

throughput since each lane will then be available for multicolor sequencing.

One method of diminishing sample size while retaining sensitive detection is to use a

fluorescent microscope as the detector. In Chapter 7 the power of confocal scanning laser

microscopy for FISH was described. This microscope also makes an excellent detector

for direct scanning of fluorescent-labeled DNA samples in gels. The advantage of the

confocal microscope is that it gathers emission very efficiently from a very narrow verti-

cal slice through the sample. Light that emanates from above or below this plane is not

imaged. Thus the confocal microscope can detect fluorescence from inside a capillary or

thin slab without background due to

scattering from the interface between the capillary

and the gel, or the interface between the capillary and the external surroundings. This is a

major improvement. One consequence is that the capillaries are scanned off line, in order

to take full advantage of the scanning speed of the microscope. Dense bundles of capillar-

ies can be made, loaded in parallel

with multiple headed syringes, run in parallel, and

scanned together (Fig. 10.19). The increase throughput ultimately achievable with this ap-

proach may be considerable. A potential limitation is the difficulty in making gel-filled

capillaries. This will be alleviated somewhat as it becomes possible to use liquid (non-

crosslinked) gels instead of solid (crosslinked) gels. This is because solid gels must be

polymerized within the capillary, while

liquid gels can just be poured into the capillary.

The alternative to capillaries is to

use large thin gel slabs. This simplifies the optics

needed for on-line detection of the DNA.

In anticipation of the considerable demands

placed on detector systems by fast running, thin gels, a number of alternative new detec-

tors are being developed as possible readout devices for fluorescence-based DNA se-

quencing.

APPROACHES TO

DNA

SEQUENCING

BY

MASS

SPECTROMETRY

A separate approach to improving ladder sequencing is to change the way in which the la-

beled DNA fragments are detected. Here considerable attention has been given to mass

spectrometry. There are actually three ways in which mass spectrometry might be used, in

principle, to assist DNA sequencing. In the simplest case the mass spectrometer is used as

a detector for a mass label attached to the DNA strand in lieu of a fluorescent label.

Alternatively, the mass of the DNA molecule itself can be measured. In this case the mass

spectrometer replaces the need for gel

electrophoresis; it separates the DNA molecules

and detects their sizes. The most ambitious and difficult potential use of mass spectrome-

try would involve a fragmentation analysis of the DNA and the determination of all of the

resulting species. In

this

way the mass spectrometer would replace all of the chemistry

and electrophoresis steps in conventional ladder sequencing. We are a long way from ac-

complishing this. In this section we will

discuss

each of these potential applications of

mass spectrometry to enhance DNA sequencing.

Mass spectroscopy is almost as sensitive a detector as fluorescence, with some instru-

ments having sensitivities

of the order

of

thousands of atoms or molecules, and special

Computer

Amplifier

>

590

Mirror

nm

580

nm

Spatial filter

555

Lens

nm

525

Long pass filter

nm

PMT

Dichroic beam splitter

Spectral filter

Mirror

Laser (488 nm)

Dichroic beam splitter

Objective

Detection zone

Translation stage

Separation

Buffer reservoir

capillaries

—

+

High-voltage

power supply

(b)

Figure 10.19

Apparatus for DNA sequencing by capillary electrophoresis.

(a ) An array of gel-

filled capillaries

used for DNA sequencing. (

b ) On-line

detection by

confocal scanning fluores-

APPROACHES TO DNA SEQUENCING BY MASS SPECTROMETRY

351

techniques such as ion cyclotron resonance mass spectrometry having even greater sensi-

tivity. However, the

principal

potential advantages of mass spectra over fluorescence

is

that

isotopic

labeling

leads

to

much less of a perturbation of electrophoretic properties

than fluorescent labeling, and the number of easily used isotopic labels far exceeds the

number of fluorophores that could be used simultaneously. Mass spectrometers actually

measure the ratio of mass to charge; the best instruments have a mass to charge resolution

of better than 1 part in a million. Thus asking a mass spectrometer to distinguish between,

say,

two

isotopes like

34S and

36S is not very demanding if these isotopes reside in small

molecules.

One basic strategy in using mass spectrometry as a DNA sequencing detector simply

replaces

the

fluorophore with

a stable isotope. Two approaches have been explored. In

one

case

four

different

stable

isotopes of sulfur would be used as a 5

label

incorporated,

for

example, as thiophosphate. In the other

case a metal chelate is attached at the 5

-end

of the primer, and different stable metal isotopes are used. Some of the possibilities are

shown in Table 10.1. Since many of the divalent ions in the table have very similar chem-

istry, chelates can be built that, in principle, would bind many different elements. Thus,

when all the isotopes are considered, there is the possibility of doing analyses with more

than 30 different colors. Whether sulfur or metal isotopes are used, the sample must be

vaporized and the DNA destroyed so that the only mass detected is that of a small mole-

cule

or

single atom containing

the isotope. With sulfur labeling, one possible role

for

mass spectrometry is as an on-line detector for capillary electrophoresis. DNA fragments

are eluted from the capillary into a chamber where the sample is burned, and the resulting

SO 2 is ionized and detected.

With metal labeling, a much more complex process is used to analyze the sample by

mass

spectrometry. This is a

technique called resonance ionization spectroscopy (RIS),

and it is illustrated in Figure 10.20. Here mass spectrometry would serve to analyze a fil-

ter blot, or a thin gel, directly, off line. In RIS just the top few microns of a sample are ex-

amined. Either a strong laser beam or an ion beam is used to vaporize the surface of the

sample, creating a mixture of atoms and ions. The beam scans the surface in a raster pat-

tern. Any ions produced are pulled away by a strong electric field. Then a set of lasers is

used

to

ionize a particular element of interest; in our case this is the metal atom used

as

the label. Because ionization energies are higher than the energy in any single laser pho-

ton, two or more lasers must

be used in tandem to pump the atom up to its ionization

state. Then

it is detected by

mass spectrometry. The same set of lasers can be used to

Figure 10.20

Resonance ionization mass spectrometry (RIS).

(a ) Schematic design of the instru-

ment used to scan

a surface. (

b ) Three electronic states used for

the resonance ionization of metal

atoms.

APPROACHES TO DNA SEQUENCING BY MASS SPECTROMETRY

353

TABLE 10.2 Stable Metal Isotopes Bound by Metallothionein

26

Fe

54

Fe

50

Sn

112

Sn

56 Fe

114

Sn

57 Fe

115

Sn

58 Fe

116

Sn

117

Sn

Co

56 Co

118

Sn

27

119

Sn

Ni

58 Ni

120

Sn

28

60 Ni

122 Sn

61 Ni

124

Sn

62Ni

64 Ni

Au

197 Au

79

29Cu

63Cu

80 Hg

196 Hg

65 Cu

198 Hg

199 Hg

30

Zn

64 Zn

200

Hg

66 Zn

201

Hg

67Zn

202 Hg

68Zn

204

Hg

70 Zn

Pb

204

Pb

Ag

107 Ag

82

206

Pb

47

109 Ag

207 Pb

208 Pb

48

Cd

106 Cd

108 Cd

Bi

209

Bi

110

Cd

83

111

Cd

112 Cd

113 Cd

114

Cd

read each gel or blot more than once. Alternative schemes for the

use of

metals as

labels

in DNA sequencing exist. One is described in Box 10.1

The second way to use mass spectrometry to analyze DNA sequences ladders is to at-

tempt to place the DNA molecules that constitute the sequencing

ladder

into the

vapor

confusing and leads to a loss

in

experimental sensitivity. Two

methods show great

promise for placing macromolecules

into

the gas phase. In one, called

electrospray (ES),

erated

if

it has

a charge. These two procedures

appear

to work very well for proteins.

They

also

work

very well on oligonucleotides,

and a

few examples of high-resolution