Isolation of Rare Sequences Utilizing PCR

Isolation of Rare Sequences Utilizing PCR

The power of PCR can be used to simplify the cloning of genes and to isolate special rare DNA or RNA molecules out of large populations containing many different sequences. First let us consider the cloning of a gene if a tiny portion of its protein product can be isolated. The amino acid sequence of a stretch of the protein must be determined. This is used to design oligonucleotides that hybridize to the top and bottom strands of the ends of the region encoding the peptide and can be used in PCR to amplify the region from cDNA template. Of course, redundancy in the genetic code necessitates that each oligonucleotide be a mixture as discussed earlier. For use with PCR, however, there is less need to minimize the degeneracy. Even though the proper DNA sequence is represented by only a tiny fraction of each oligonucleotide mixture, just these oligonucleotides will be functional in PCR amplification of the desired region of DNA. The other oligonu - cleotides may hybridize to the template cDNA, but they are unlikely to give rise to any PCR products, and it is even less likely that such products would be of the same size as the desired PCR product.

The PCR amplified product of a portion of the gene can then be used to probe a cDNA library contained on plasmid or phage clones. This will reveal the clones likely to contain an intact version of the desired gene.

PCR is also useful for the isolation of very rare sequences present in complex mixtures of sequences. During the chemical synthesis of DNA, the use of mixtures of nucleotide precursors for some synthesis cycles enables large random populations to be synthesized. Consider the determination of the optimum binding sequence of a DNA-binding protein. One strand of DNA can be synthesized with unique sequences at each end and containing a totally random interior region. First, strands complementary to the chemically synthesized molecules must be constructed. Due to the random sequences involved, the complemen-

Figure 10.10 Use of PCR to amplify a small amount of DNA present in acomplex mixture.

tary strands cannot be synthesized chemically. They are made enzymati-cally be hybridizing a DNA primer to the unique sequence at one end of the chemically synthesized strand. Next, DNA pol I is used to elongate and complete the synthesis of an exact complement (Fig. 10.10). Then, the small population of DNA molecules with sequences capable of binding the protein are isolated, for example, by electrophoretic sepa-ration of protein-DNA complexes from DNA incapable of binding the protein. This tiny amount of DNA is amplified by PCR using primers complementary to the unique sequences at the ends. After amplifica-tion, the selection and amplification steps can be repeated, and finally the DNA can be cloned and sequenced.

Simple variations on the basic idea described above can be developed for the isolation of RNA capable of binding a protein or specific support on an exchange column. It is also possible to devise methods for the selection of DNA or RNA molecules that possess hydrolytic activities.

Mutagenic steps can be introduced in the PCR amplification steps so that variants of the selected molecules can be created. In this way, true in vitro evolution experiments can be done. Living cells are not required.