Selection and isolation of genes

Genetic information is carried in the linear sequence of nucleotides in DNA. Its expression involves the translation of the linear sequence of specific regions of DNA existing in the nucleus of the cell (called coding regions or genes) into a colinear sequence of amino acids (proteins). As an intermediate step, however, DNA must be copied into a different type of polynucleotide known as ribonucleic acid (RNA) which retains all the information of the DNA sequence from which it was copied. Single-stranded RNA molecules are synthesized by a process known as DNA transcription which is regulated by interactions between DNA sequences located upstream of the gene (promoters) and proteins (transcription factors). Thousands of RNA transcripts can be made from the same DNA segment in a given cell. Many of these RNA molecules undergo major chemical changes before they leave the nucleus to serve as the messenger RNA (mRNA) molecules that direct the synthesis of proteins in the cytosol.

Fragments of DNA can be amplified by a process called DNA cloning which consists in inserting the DNA into a plasmid or a bacterial virus and then growing these in bacterial (or yeast) cells. Plasmids are small circular molecules of DNA that occur naturally in bacteria, where they replicate as independent units. As these bacteria divide, the plasmid also replicates to produce an enormous number of copies of the cloned DNA fragment. Although restricted genomic DNA fragments can be cloned to produce genomic libraries, cDNA libraries are most frequently used to isolate and characterize genes necessary for the production of genetically engineered plants. cDNA libraries represent the information encoded in the mRNA of a particular tissue or organism. mRNA molecules are exceptionally labile and difficult to amplify in their natural form. For this reason, the information encoded by the mRNA is converted into a stable DNA duplex (cDNA) via enzymatic reactions catalyzed by reverse transcriptase and DNA polymerase I, and then is inserted into a self-replicating plasmid. The resulting heterogeneous population of cDNA molecules collectively encodes virtually all of the mRNAs sysnthesized by the cell. Once the information is available in the form of a cDNA library, individual processed segments of the original genetic information can be isolated and examined with relative ease.

A representative cDNA library should contain full-length copies of the original population of mRNA. cDNA libraries provide a method by which the transcription and processing of mRNA can be examined and interpreted to produce models for the flow of information responsible for the fundamental characteristics of each organism and tissue type. Comprehensive cDNA libraries can be routinely established from small quantities of mRNA, and a variety of reliable methods are available to identify cDNA clones corresponding to extremely rare species of mRNA. As the enzymatic reactions used to synthesize cDNA have improved, the sizes of cloned cDNAs have increased, and it is often possible to isolate cloned full-length cDNAs corresponding to large mRNAs.

Screening of recombinant clones for the search of agronomically interesting genes can be carried out effectively with only two types of reagents: antibodies and nucleic acid probes. In those instances when both types of reagents are available, nucleic acid probes are preferred because they can be used under a variety of different stringencies that minimize the chance of undesired crossreactions. Furthermore, nucleic acid probes will detect all clones that contain cDNA sequences, whereas antibodies will react only with a subset of these clones (in some cases one in six at best) in which the cDNA has been inserted into the vector in the correct reading frame and orientation.

The higher the concentration of the sequences of interest in the starting mRNA, the easier the task of isolating relevant cDNA clones becomes. It is therefore worthwhile investing some effort to make sure that the richest source of mRNA available is being used. Whenever possible, estimates should be obtained of the frequency with which the mRNA of interest occurs in the starting preparation. mRNAs that represent less than 0.5% of the total mRNA population of the cell are classified as ‘low-abundance’ mRNAs. Using the protocol to generate cDNA libraries explained above, the isolation of cDNA clones from low-abundance mRNAs presents two major problems, first, construction of a cDNA library whose size is sufficient to ensure that the clone of interest has a good chance of being represented and secondly, identification and isolation of the clone(s) of interest. These problems have been overcome by the possibility of amplifying specific segments of DNA by the polymerase chain reaction (PCR) which is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that specifically hybridize to opposite strands and flank the region of interest in the target DNA. [15] Starting from minute amounts of DNA, repetitive series of cycles involving template denaturation, primer annealing, and the extension of the annealed primers by thermostable DNA polymerase results in the exponential accumulation of a specific fragment. In vitro amplification systems have the advantage of being specific, rapid, but above all they allow the detection and amplification of lowabundance transcripts from total RNA. [16] PCR can be also used to produce probes, DNA sequencing and in vitro generation of mutations in DNA molecules.