To O

Figure 10.18 Denaturing gradient gel extraction track profiles.

their central role in the process of gene expression, there are strong selective pressures, making it unlikely that they will undergo rapid changes. This leads to the presence of extended regions of the molecule that are highly conserved (little changed) and which therefore are useful for establishing distant phylogenetic relationships. On the other hand, they also have an adequate number of variable regions that can be used to examine closer relationships. Thus, 16S rRNA can be used as an evolutionary clock, or chronometer, to measure the phylogenetic distance between taxa at a variety of levels based on the number of nucleotide differences—representing stable mutations—that have occurred over time.

One method to sequence 16S rRNA begins with extraction of a cell's RNA. A small DNA oligonucleotide primer (15 to 20 nucleotides in length) that is complementary in its base sequence to a conserved region of the 16S rRNA molecule is added. Reverse transcriptase can then be used to generate complementary DNA (cDNA), which in turn is amplified using PCR. The nucleotide sequences of these cDNA are then determined, and the original sequence of the rRNA is deduced from them. Another common approach is to extract and sequence the DNA of the gene that codes for the 16S rRNA rather than the RNA itself.

The 16S rRNA sequences of thousands of species have now been determined. Using advanced computer algorithms, short (6 to 10 nucleotides) signature sequences have been found that are highly consistent within groups of organisms. Also, phylogenetic trees can be generated in which the evolutionary distance between two groups is indicated by the length of the connecting lines (Figure 10.19).

Was this article helpful?

0 0

Post a comment