DNA–DNA hybridization

DNA–DNA hybridization generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences. It is usually used to determine the genetic distance between two organisms. This has been used extensively in phylogeny and taxonomy.

Method

The DNA of one organism is labeled, then mixed with the unlabeled DNA to be compared against. The mixture is incubated to allow DNA strands to dissociate and renewal forming hybrid double-stranded DNA. Hybridized sequences with a high degree of similarity will bind more firmly, and require more energy to separate them: i.e. they separate when heated at a higher temperature than dissimilar sequences, a process known as "DNA melting".

To assess the melting profile of the hybridized DNA, the double-stranded DNA is bound to a column and the mixture is heated in small steps. At each step, the column is washed; sequences that melt become single-stranded and wash off the column. The temperatures at which labeled DNA comes off the column reflects the amount of similarity between sequences (and the self-hybridization sample serves as a control). These results are combined to determine the degree of genetic similarity between organisms.

Use in zoology

When several species are compared that way, the similarity values allow the species to be arranged in a phylogenetic tree; it is therefore one possible approach to carrying out molecular systematics. Charles Sibley and Jon Ahlquist, pioneers of the technique, used DNA–DNA hybridization to examine the phylogenetic relationships of avians (the Sibley–Ahlquist taxonomy) and primates.[1][2]

Use in microbiology

DNA–DNA hybridization is the gold standard to distinguish bacterial species, with a similarity value greater than 70% indicating that the compared strains belong to distinct species.[3][4][5] In 2014, a threshold of 79% similarity has been suggested to separate bacterial subspecies.[6]

Replacement by genome sequencing

Critics argue that the technique is inaccurate for comparison of closely related species, as any attempt to measure differences between orthologous sequences between organisms is overwhelmed by the hybridization of paralogous sequences within an organism's genome.[7] DNA sequencing and computational comparisons of sequences is now generally the method for determining genetic distance, although the technique is still used in microbiology to help identify bacteria.[8]

The modern approach is to carry out DNA–DNA hybridization in silico using completely or partially sequenced genomes.[9] The GGDC developed at DSMZ is the most accurate known tool for calculating DDH-analogous values.[9] Among other algorithmic improvements, it solves the problem with paralogous sequences by carefully filtering them from the matches between the two genome sequences.

See also

References

  1. ↑ Genetic Similarities: Wilson, Sarich, Sibley, and Ahlquist
  2. ↑ C.G. Sibley & J.E. Ahlquist (1984). "The Phylogeny of the Hominoid Primates, as Indicated by DNA–DNA Hybridization". Journal of Molecular Evolution. 20 (1): 2–15. PMID 6429338. doi:10.1007/BF02101980.
  3. ↑ Brenner DJ (1973). "Deoxyribonucleic acid reassociation in the taxonomy of enteric bacteria". International Journal of Systematic Bacteriology. 23: 298–307. doi:10.1099/00207713-23-4-298.
  4. ↑ Wayne LG, Brenner DJ, Colwell RR, Grimont PD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP, Trüper HG (1987). "Report of the ad hoc committee on reconciliation of approaches to bacterial systematics". International Journal of Systematic Bacteriology. 37: 463–464. doi:10.1099/00207713-37-4-463.
  5. ↑ Tindall BJ, Rossello-Mora R, Busse H-J, Ludwig W, Kampfer P. "Notes on the characterization of prokaryote strains for taxonomic purposes". International Journal of Systematic and Evolutionary Microbiology. 60: 249–266. PMID 19700448. doi:10.1099/ijs.0.016949-0.
  6. ↑ Meier-Kolthoff JP, Hahnke RL, Petersen JP, Scheuner CS, Michael VM, Fiebig AF, Rohde CR, Rohde MR, Fartmann BF, Goodwin LA, Chertkov OC, Reddy TR, Pati AP, Ivanova NN, Markowitz VM, Kyrpides NC, Woyke TW, Klenk HP, Göker M (2013). "Complete genome sequence of DSM 30083T, the type strain (U5/41T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy". Standards in Genomic Sciences. 9: 2. doi:10.1186/1944-3277-9-2.
  7. ↑ DNA hybridization in the apes – Technical issues
  8. ↑ S.S. Socransky; A.D. Haffajee; C. Smith; L. Martin; J.A. Haffajee; N.G. Uzel; J. M. Goodson (2004). "Use of checkerboard DNA–DNA hybridization to study complex microbial ecosystems". Oral Microbiology and Immunology. 19 (6): 352–362. PMID 15491460. doi:10.1111/j.1399-302x.2004.00168.x.
  9. 1 2 Meier-Kolthoff JP, Auch AF, Klenk HP, Goeker M (2013). "Genome sequence-based species delimitation with confidence intervals and improved distance functions". BMC Bioinformatics. 14: 60. PMC 3665452 Freely accessible. PMID 23432962. doi:10.1186/1471-2105-14-60.
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