Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

DNA Library Screening
Synthesis of Oligonucleotide Probes

Obviously, the application of membrane Hybridization depends on the availability of complementary radioactive probes. An oligonucleotide used as a probe must be sufficiently long so that its nucleotide sequence is unique (the only possible one) exclusively for the given clone and does not match any other clone. This condition is usually met for oligonucleotides about 20 NUCLEOTIDES long, since a 20-nucleotide sequence occurs once every 4²⁰≈10¹² nucleotides. Given that all known genomes are much shorter (≈3x10⁹ nucleotides for humans), a specific 20-nucleotide sequence typically appears only once in such a system.

Oligonucleotides of the required length can be chemically synthesized, after which a radioactive label is attached to them using polynucleotide kinase, which transfers a ³²P-labeled phosphate group from ATP to the 5'-end of each oligonucleotide.

A problem arises when deciding exactly which nucleotide sequence to synthesize in each specific case. On the one hand, if the entire Amino Acid Sequence of a protein (or part of it) is known, the DNA probe Structure can be deduced using METABOLISM/28.html">The Genetic Code. However, due to the degeneracy of the genetic code (i.e., the fact that many Amino Acids are encoded by more than one codon), such an amino acid sequence analysis requires including in the probe all possible oligonucleotide sequences that could theoretically encode that amino acid sequence. Furthermore, within this mixture of oligonucleotides, only a single one will hybridize perfectly with the clone of interest.

In recent years, this approach has been greatly simplified by the fact that researchers now have access to fully sequenced genomes of humans and several important model organisms, such as the mouse, the fruit fly Drosophila, and the nematode roundworm Caenorhabditis elegans.

Using appropriate computer software, a researcher can search the genomic database for The nucleotide sequence that corresponds to a specific region of The amino acid sequence of the protein under study. If such a sequence is found, a DNA probe synthesized on its basis will hybridize perfectly with the clone encoding the target protein.

The Chemical synthesis of single-stranded DNA probes for a specific nucleotide sequence can be carried out through a series of reactions shown in Figure 94.

The first 3'-nucleotide (monomer 1) is attached to a Glass substrate via its 3' hydroxyl group. The 5' hydroxyl of the first monomer is ready to bind the second monomer. The second nucleotide of the sequence (monomer 2) is modified by adding 4',4'-dimethoxytrityl (DMT) to its 5'-hydroxyl in order to block this hydroxyl and prevent it from participating in nonspecific reactions. In addition, a highly reactive phosphoramidite MeO-P-N(IP)₂ (Me = methyl group, N(IP)₂ = diisopropylamine group) is attached to the 3'-hydroxyl of the second monomer.

When these two monomers are mixed in the presence of a weak acid, they bind due to The formation of a phosphodiester bond involving trivalent phosphorus.

Oxidation of such a dimer using an iodine mixture increases the phosphorus valence to 5, followed by the release of the 5'-end through the removal of the DMT group with zinc bromide (ZnBr₂).

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Figure 94 - Chemical synthesis of oligonucleotides

The third monomer is attached in a similar manner. Repeating this Procedure makes it possible to synthesize an oligonucleotide of the desired length. Then, using an alkaline environment, all methyl groups are removed from the phosphates, and the oligonucleotide (monomer 1) is cleaved from the glass substrate.

Currently, the oligonucleotide synthesis process is automated. Researchers can program the Synthesis of specific sequences up to 100 nucleotides in length.

An alternative method for preparing probes is to use the Polymerase Chain Reaction (PCR), which will be discussed in detail below.