DNA–DNA hybridization

Technique used to measure similarity in DNA sequences

This article is about the specific use in genomics. For the general phenomenon, see Nucleic acid thermodynamics § Hybridization.

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In genomics, DNA–DNA hybridization is a molecular biology technique that measures the degree of genetic similarity between DNA sequences. It is used to determine the genetic distance between two organisms and has been used extensively in phylogeny and taxonomy.[1]

## Method

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The DNA of one organism is labelled, then mixed with the unlabelled DNA to be compared against. The mixture is incubated to allow DNA strands to dissociate and then cooled to form renewed hybrid double-stranded DNA. Hybridized sequences with a high degree of similarity will bind more firmly, and require more energy to separate them. An example is they separate when heated at a higher temperature than dissimilar sequences, a process known as "DNA melting".[2][3][4]

To assess the melting profile of the hybridized DNA, the double-stranded DNA is bound to a column or filter and the mixture is heated in small steps. At each step, the column or filter is washed; then sequences that melt become single-stranded and wash off. The temperatures at which labelled DNA comes off 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.[5]

A method was introduced to hybridize a large number of DNA samples against numerous DNA probes on a single membrane. The samples would need to be separated into individual lanes within the membrane, which would then be rotated to allow simultaneous hybridization with multiple DNA probes.[6]

## Uses

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When several species are compared, similarity values allow organisms to be arranged in a distance matrix, which can produce a phylogenetic tree. It is therefore, one possible approach to carrying out molecular systematics.[citation needed]

### In microbiology

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DNA–DNA hybridization (DDH) is used as a primary method to distinguish bacterial species as it is difficult to visually classify them accurately.[7] This technique is not widely used on larger organisms where differences in species are easier to identify. In the late 1900s, strains were considered to belong to the same species if they had a DNA–DNA similarity value greater than 70% and their melting temperatures were within 5 °C of each other.[8][9][10] In 2014, a threshold of 79% similarity has been suggested to separate bacterial subspecies.[11]

DDH is a common technique for bacteria, but it is labor intensive, error-prone, and technically challenging. In 2004, a new DDH technique was described. This technique utilized microplates and colorimetrically labelled DNA to decrease the time needed and increase the amount of samples that can be processed.[12] This new DDH technique became the standard for bacterial taxonomy.[13]

### In zoology

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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.[14][15]

### In radioactivity

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In 1969, one such method was performed by Mary Lou Pardue and Joseph G. Gall at the Yale University through radioactivity where it involved the hybridization of a radioactive test DNA in solution to the stationary DNA of a cytological preparation, which is identified as autoradiography.[16]

## Replacement by genome sequencing

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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.[17][better source needed][better source needed] 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.[18]

### In silico methods

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The modern approach is to carry out DNA–DNA hybridization in silico utilizes completely or partially sequenced genomes.[19] Digital DDH (dDDH) is developed at the DSMZ and uses the GBDP (Genome Blast Distance Phylogeny) algorithm to produce DDH-analogous methods. DSMZ offers several web services based on dDDH. dDDH does not suffer from DDH's issues with paralogous genes, large repeats, reduced genomes, and low-complexity regions. Among other algorithmic improvements, it solves the problem with paralogous sequences by carefully filtering them from the matches between the two genome sequences.[19]

dDDH has been used for resolving difficult taxa such as Escherichia coli, Bacillus cereus group, and Aeromonas.[20] The Judicial Commission of International Committee on Systematics of Prokaryotes has admitted dDDH as taxonomic evidence.[21]

## See also

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* DNA melting
* Temperature gradient gel electrophoresis

## References

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1. ^ Erko Stackebrandt (8 September 2010). Molecular Identification, Systematics, and Population Structure of Prokaryotes. Springer Science & Business Media. ISBN 978-3-540-31292-5.
2. ^ Sinden, Richard R. (1994). DNA structure and function. San Diego: Academic Press. pp. 37–45\. ISBN 0-12-645750-6. OCLC 30109829.
3. ^ Tools and techniques in biomolecular science. Aysha Divan, Janice Royds. Oxford: Oxford University Press. 2013. ISBN 978-0-19-969556-0. OCLC 818450218.`{{cite book}}`: CS1 maint: others (link)
4. ^ Forster, A. C.; McInnes, J. L.; Skingle, D. C.; Symons, R. H. (1985-02-11). "Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin". Nucleic Acids Research. 13 (3): 745–761\. doi:10.1093/nar/13.3.745. ISSN 0305-1048. PMC 341032. PMID 2582358.
5. ^ Hood, D. W.; Dow, C. S.; Green, P. N. (1987). "DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs". Journal of General Microbiology. 133 (3): 709–720\. doi:10.1099/00221287-133-3-709. ISSN 0022-1287. PMID 3655730.
6. ^ Socransky, S. S.; Smith, C.; Martin, L.; Paster, B. J.; Dewhirst, F. E.; Levin, A. E. (October 1994). ""Checkerboard" DNA-DNA hybridization". BioTechniques. 17 (4): 788–792\. ISSN 0736-6205. PMID 7833043.
7. ^ Auch, Alexander F.; von Jan, Mathias; Klenk, Hans-Peter; Göker, Markus (2010). "Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison". Standards in Genomic Sciences. 2 (1): 117–134\. Bibcode:2010SGenS...2..117A. doi:10.4056/sigs.531120. ISSN 1944-3277. PMC 3035253. PMID 21304684.
8. ^ Brenner DJ (1973). "Deoxyribonucleic acid reassociation in the taxonomy of enteric bacteria". International Journal of Systematic Bacteriology. 23 (4): 298–307\. doi:10.1099/00207713-23-4-298.
9. ^ 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 (4): 463–464\. doi:10.1099/00207713-37-4-463.
10. ^ Tindall BJ, Rossello-Mora R, Busse H-J, Ludwig W, Kampfer P (2010). "Notes on the characterization of prokaryote strains for taxonomic purposes". International Journal of Systematic and Evolutionary Microbiology. 60 (Pt 1): 249–266\. doi:10.1099/ijs.0.016949-0. hdl:10261/49238. PMID 19700448.
11. ^ 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. PMC 4334874. PMID 25780495.
12. ^ Mehlen, André; Goeldner, Marcia; Ried, Sabine; Stindl, Sibylle; Ludwig, Wolfgang; Schleifer, Karl-Heinz (November 2004). "Development of a fast DNA-DNA hybridization method based on melting profiles in microplates". Systematic and Applied Microbiology. 27 (6): 689–695\. Bibcode:2004SyApM..27..689M. doi:10.1078/0723202042369875. ISSN 0723-2020. PMID 15612626.
13. ^ Huang, Chien-Hsun; Li, Shiao-Wen; Huang, Lina; Watanabe, Koichi (2018). "Identification and Classification for the Lactobacillus casei Group". Frontiers in Microbiology. 9: 1974. doi:10.3389/fmicb.2018.01974. ISSN 1664-302X. PMC 6113361. PMID 30186277.
14. ^ Genetic Similarities: Wilson, Sarich, Sibley, and Ahlquist
15. ^ 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\. Bibcode:1984JMolE..20....2S. doi:10.1007/BF02101980. PMID 6429338. S2CID 6658046.
16. ^ Pardue, Mary Lou, and Joseph G Hall. “Molecular Hybridization of Radioactive DNA to the DNA of Cytological Preparations.” Kline Biology Tower, Yale University, 13 Aug. 1969.
17. ^ Marks, Jonathan (2007-05-09). "DNA hybridization in the apes—Technical issues". Archived from the original on 2007-05-09. Retrieved 2019-06-02.
18. ^ 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\. doi:10.1111/j.1399-302x.2004.00168.x. PMID 15491460.
19. ^ a b 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\. doi:10.1186/1471-2105-14-60. PMC 3665452. PMID 23432962.
20. ^ Riojas, Marco A.; McGough, Katya J.; Rider-Riojas, Cristin J.; Rastogi, Nalin; Hazbón, Manzour Hernando (1 January 2018). "Phylogenomic analysis of the species of the Mycobacterium tuberculosis complex demonstrates that Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium microti and Mycobacterium pinnipedii are later heterotypic synonyms of Mycobacterium tuberculosis". International Journal of Systematic and Evolutionary Microbiology. 68 (1): 324–332\. doi:10.1099/ijsem.0.002507. PMID 29205127.
21. ^ Arahal, David R.; Bull, Carolee T.; Busse, Hans-Jürgen; Christensen, Henrik; Chuvochina, Maria; Dedysh, Svetlana N.; Fournier, Pierre-Edouard; Konstantinidis, Konstantinos T.; Parker, Charles T.; Rossello-Mora, Ramon; Ventosa, Antonio; Göker, Markus (27 April 2023). "Judicial Opinions 123–127". International Journal of Systematic and Evolutionary Microbiology. 72 (12). doi:10.1099/ijsem.0.005708. hdl:10261/295959. PMID 36748499.

## Further reading

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* Graur, D. & Li, W-H. 1991 (2nd ed. 1999). Fundamentals of Molecular Evolution.