Ttttttttttt

cDNA

cDNA

Denaturation in the first PCR cycle

TTTTTTTTTTT 5'

5' Anchor

Forward GSP

Reverse GSP

Amplification

3' RACE

Figure 2.1 The procedure for characterizing a full-length cDNA from partial knowledge of a protein sequence. Top: general structure of a eukaryotic mRNA with two exons, after splicing, capping and polyadenylation. Middle: mRNA-cDNA hybrid synthesized by reverse transcription. Bottom: single-stranded cDNA acting as a template for PCR amplification. Positions are indicated for primers used to amplify cDNA ends in 3'- and 5'-RACE. GSP, gene-specific primer (degenerate), developed from partial knowledge of the amino acid sequence of the protein; UTR, untranslated region.

Figure 2.2 Outline of a gene-discovery procedure in a non-model organism, commencing with protein purification and leading to gene characterization. MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry.

work backwards from protein structure to gene characterization, and in some ecological applications this may be the only way to begin genomic explorations.

2.1.2 Differential screening

Liang and Pardee (1992) first proposed that genes whose expression differs between two populations of cells can be discovered by a PCR-based technique called differential display of mRNA. When animals or plants are subjected to a certain treatment, for example drought stress, their cells will have a higher or lower abundance of the mRNAs for those genes that respond to the treatment, compared with untreated controls. These differential genes can be detected and visualized by a PCR technique that amplifies complementary DNA sequences prepared by reverse transcription from the mRNA pool. The differential display PCR takes advantage of the poly(A) tail to anchor a poly(T) primer at the end of the cDNA. The other primer is a short oligonucleotide, 6 or 7 bp long, with an arbitrary sequence; this primer will anneal somewhere near the end of the cDNA strand, depending on the sequence. Because of the specific (but unknown) annealing site of the forward primer, amplified products from different cDNAs will differ in size and so can be resolved on a highresolution electrophoresis gel. The presence or absence of a band in one treatment compared to the other is evidence of a differentially expressed

Drought Control stress

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