Sometimes the protein product of a gene is available in pure form. This happy circumstance can be used to facilitate cloning of the gene. Portions of the protein can be sequenced to determine a potential DNA sequence that could have encoded this portion of the protein. An oligonucleotide with this sequence can then be used to screen a collection of clones, which is called a library, to detect those containing complementary sequences. The screening is done as described in the previous chapter. Occasionally, a clone is found in the libraries which hybridizes to the screening oligonucleotide, but which is not the correct clone. This results from the chance occurrence of a sequence complementary to the probing oligonucleotide. These incorrect positives can be detected by screening with a second oligonucleotide that should hybridize to a different part of the gene encoding the protein in question. Only the desired clones should hybridize to both oligonucleotides.

The redundancy in the genetic code prevents simple reverse translation from an amino acid sequence to a DNA sequence. The difficulty caused by the redundancy can be partially overcome by using portions of the protein’s sequence containing amino acids whose codon redundancy is low. This is possible since both tryptophan and methionine have unique codons. Consider the sequence met-cys-his-trp-lys-met. Only one codon specifies an internal methionine, while the cysteine, histidine, and lysine are each specified by only two possible codons. Therefore one of only 1 × 2 × 2 × 1 × 2 × 1 = 8 sequences encoded the six amino acids (Fig. 1).

Fig1. Reverse translating to obtain the sequences that could have encoded a short peptide.

The eight necessary oligonucleotides can be synthesized simultaneously by machine by incorporating either of the two ambiguous nucleotides at the necessary positions. This is accomplished simply by supplying at the correct time a mixture of the two nucleotides to the synthesis solution.

Purification of the protein necessary for the oligonucleotide probing approach often is straightforward. Conventional purification need not be performed, however. Since all that is needed for the cloning is determination of portions of the amino acid sequence, purification and detection methods need not preserve the protein’s native structure. SDS gel electrophoresis, for example, can be used as a final step in the purification of the protein. The protein in the correct band in the gel can be eluted and a portion of its amino terminal sequence determined by gas phase and mass spectrometry. As little as 10^-12 moles of protein are sufficient for determining enough of the sequence that oligonucleotide probes can be designed to identify clones carrying the gene.

مواضيع ذات صلة

Enzymatic DNA Sequencing

Chemical DNA Sequencing

Targeting of Nuclear Proteins

Ribosome Disorders

The Process of Translation

RNA Granules and Membraneless Organelles

RNA Interference

Balancing Synthesis of Ribosomal Components

Regulation of Ribosome Synthesis

Proportionality of Ribosome Levels and Growth Rates

The Signal Recognition Particle and Translocation

Verifying the Signal Peptide Model