untitled

Expressed sequence tags and microarray

technology

Expressed sequence tags (ESTs) provide an alternative

to whole genome sequencing and are particularly use-

ful for finding genes that are expressed in specific con-

ditions (in other words, the genes that are “switched

on” at different times or in different circumstances).

This technology can also be used as a quick and

inexpensive approach to finding new genes and for

constructing genome maps.

ESTs are small lengths of DNA (about 200 –500

nucleotides) produced by sequencing one or both

ends of an expressed gene. The technique involves

sampling the pool of messenger RNA (the product of

gene expression) and using this mRNA as a template

to synthesize a complementary DNA (cDNA), using the

enzyme reverse transcriptase. cDNA is much more

stable than mRNA, and it has the additional advantage

of containing only the coding regions, because introns

have been removed by splicing during the natural pro-

cessing of messenger RNA.

Once a single strand of cDNA has been formed

from the RNA template, this RNA is digested by a

ribonuclease. Then a complementary strand of DNA

is synthesized, to produce double-stranded DNA, as

shown in Fig. 9.16.

Large numbers of ESTs can be generated easily and

can subsequently be assembled into contigs or compared

with the many ESTs of various organisms now avail-

able on electronic databases, providing a means of

identifying different types of gene. As an illustration

of the power of such techniques, Martina et al. (2003)

180 CHAPTER 9

Gene/protein expression

Metabolism

Cell/organism defense

Cell structure

Cell signaling/cell communication

Transport mechanisms

Cell fate

Cell division

Unknown function

No homology

0 5 10 15 20 40 60 80

(a) % ESTs

Gene/protein expression

Metabolism

Cell/organism defense

Cell structure

Cell signaling/cell communication

Transport mechanisms

Cell fate

Cell division

Unknown function

No homology

0 5 10 15 20 40 60 80

(b) % ESTs

13

7 8 7 3 5 3 5 4

2

1

2

0.3

1

2

0.4

6

5

60

66

39

66

2

2

0.3

1

2

0.4

14

5

15

7

11

7

3

5

9

5

4

2

Laccaria mycelium

Pisolithus mycelium

Pisolithus mycorrhizas

Pisolithus mycelium

Fig. 9.17Functional classification of expressed sequence tags (ESTs) from (a) mycelial cultures of two mycorrhizal fungi
or from (b) symbiotic tissue of Pisolithusmycorrhizas compared with mycelial cultures of Pisolithus. (From Peter et al.
2003, with permission from the New Phytologist Trust.)