Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 11

382

STRATEGIES FOR LARGE-SCALE DNA SEQUENCING

the library. Any clones that are detected by this hybridization are not included in the next

set of 100 to be sequenced. By continuing in this way, most duplication can be avoided.

Unfortunately, there will also be a tendency

to

discard

cDNAs from

gene

families,

so

many of the members of these families will be underrepresented or missed. As an alterna-

tive to handling the clones as arrays, one can carry out

a solution

hybridization

of the

entire cDNA library with an excess of sequenced clones, discard all the samples that

hybridize, and regrow the remainder. This effectively replaces a screen by a physical

selection process.

A more complex approach to compensating for uneven cDNA distribution is to try to

equalize or normalize the library. The distribution of mRNAs in a typical cell is shown in

Table 11.1. Roughly speaking there are three classes of messages: a few very common

species, then approximately equal total amounts of species

20

times

more

rare,

and

species another factor of 20 rarer still. The goal is to try to even out these differences. The

approach used is to anneal the library to itself and remove all the double-stranded species

that are formed. We will do this by allowing the reannealing to occur at a very high

C

0 t:

Typically

C

0 t 250 is

used. From

Chapter 3,

we

can

write

for

the fraction

of

single

strand remaining in a hybridization:

f

1

1

C 0 t1/2

1 n

C

t k /2N

1 C

t/C

C

t1/2 C

t

s

0

0

0

t1/2

0

0

2

where all the quantities in this equation have

been defined in Chapter

3. When

C 0 t

C 0 t

1/2

, we can approximate this result as

f

C 0 t1/2

s

C

0

t

Note

that

the

C 0 t1/2value for a

given

sequence

depends

on

the

ratio of genome

size,

N,

and the number of times the sequence is represented,

n.

For a cDNA library,

N

is the total

complexity of the DNA sequences represented in the library, and

n is the number of times

a given sequence is represented. Thus, for highly frequent cDNAs,

N

/n will be

small so

that

the

C 0 t1/2will be small, and these species will

renature relatively more rapidly. Note

that the amount of a particular cDNA remaining after extensive annealing will be propor-

tional to its original abundance

n

and to

its

hybridization

rate,

which

will

scale as

N

/n.

Thus, at very long times in the reaction, a relatively even distribution of cDNAs should be

produced. We can evaluate the expected results for an attempt

to

normalize

the typical

cell mRNAs shown in Table 11.1. This is given in Table 11.2.

TABLE 11.1

Distribution of mRNA in a Typical Cell

Species

Percent

Number of

Relative

C

0

t1/2

Species

Frequency

Common

10

10

330

0.08

Medium

45

1000

15

1.7

Rare

45

15,000

1

25.

384

STRATEGIES FOR LARGE-SCALE DNA SEQUENCING

TABLE

11.3

cDNA Library Normalization

Probes

Original Library

Normalized (HAP-FT)

HAP-Bound

Human C

0 t 1 DNA

10%

2%

2.6%

Elongation factor 1a

4.6%

0.04%

3.7%

a-Tubulin

3.7%–4.4%

0.045%

6%

b-Tubulin

2.9%

0.4%

0.85%

Mitochondrial 16S rRNA

1.3%

1%

Myelin basic protein

1%

0.09%

g-Actin

0.35%

0.1%

1.3%

Hsp 89

0.4%

0.05%

0.14%

CH13-cDNA#8

0.009%

0.035%

Source:

Data adapted from Soares et al. (1994).

lost if entire cDNA sequences were used

for

hybridization

because,

as

described

in

Chapter 3, their rate constants for duplex formation (

k ’s) are larger and because there are

2

more places to nucleate potential duplex. The trick used in the scheme of Figure 11.18 is

to start with single-stranded cDNA clones and produce

a short

duplex

at

the 3

-end of

each cDNA clone by primer extension in the presence

of

chain terminators. By

focusing

on this region, one

will ensure that the new

strands

synthesized

preferentially come from

3 noncoding flanking regions where even closely related genes have significant diver-

gence, since the sequences are not translated and presumably

have little

function. Any

cDNAs that have not successfully templated the synthesis of a short duplex are discarded

by chromatography on hydroxyapatite, which specifically binds only duplexes, under the

conditions used. These duplex-containing clones are then eluted, melted, and allowed to

self-anneal to high

C 0 t. Now any

clones

with duplexes are

removed,

and the

clones that

have remained as single strands represent the normalized library. These are then amplified

and sequenced.

Some actual results using the scheme of Figure 11.18 are given in Table 11.3. It is ap-

parent that the equalization is far from perfect. However, it represents a major improve-

ment over nonnormalized libraries, and materials made in this

way

are

currently being

used extensively for cDNA sequencing. Two additional schemes for cDNA normalization

are described in Box 11.1. It is not clear at the present time just which schemes will ulti-

mately be widely adopted.

LARGE-SCALE cDNA SEQUENCING

In the past three years at least five separate efforts have

been made to collect massive

amounts of cDNA sequence. One of these is a collaboration between the Institute for

Genome Research (TIGR) and Human Genome Sciences, Inc. At least initially, this effort

took an anatomical approach. Libraries of cDNAs from as many different major tissues as

possible were collected, and large numbers of clones from each of these were sequenced.

The second approach was orchestrated by Incyte Pharmaceuticals, Inc. Here the emphasis

was on cell physiology. Sets of cDNA libraries were collected

from

pairs

of

cells

in

known, related physiological states, such

as

activated or

unactivated

macrophages.

A

fixed number of cDNAs, 5000 in the earliest studies, was randomly selected

for

each of

the cell pairs and sequenced. In this way information was obtained

about the frequencies

of common cDNAs in

addition to the sequence

information from all

classes

of

cDNAs.

WHAT IS MEANT BY A COMPLETE GENOME SEQUENCE?

389

BOX 11.2

(Continued)

Figure 11.21

Steps involved in making an hncDNA library. Interspersed repeat elements in

hnRNA are represented in the upper line by solid boxes

(R ). Arrows

indicate

primers. Vertical

lines crossing the arrows symbolise the primers with sites for

Eco

R I

and

Bam

H I restriction

endonucleases (

E ) or ( B ). EC is 5 GAGAATTC(C)203

. The open boxes with

similar

symbols