DNA–DNA hybridization: Difference between revisions
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== Method == |
== Method == |
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{{unreferenced section|date=June 2019}} |
{{unreferenced section|date=June 2019}} |
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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: i.e. they separate when heated at a higher temperature than dissimilar sequences, a process known as "[[DNA melting]]". |
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: i.e. they separate when heated at a higher temperature than dissimilar sequences, a process known as "[[DNA melting]]".<ref>{{Cite book |last=Sinden |first=Richard R. |url=https://www.worldcat.org/oclc/30109829 |title=DNA structure and function |date=1994 |publisher=Academic Press |isbn=0-12-645750-6 |location=San Diego |pages=37-45 |oclc=30109829}}</ref><ref>{{Cite book |url=https://www.worldcat.org/oclc/818450218 |title=Tools and techniques in biomolecular science |date=2013 |publisher=Oxford University Press |others=Aysha Divan, Janice Royds |isbn=978-0-19-969556-0 |location=Oxford |oclc=818450218}}</ref><ref>{{Cite journal |last=Forster |first=A. C. |last2=McInnes |first2=J. L. |last3=Skingle |first3=D. C. |last4=Symons |first4=R. H. |date=1985-02-11 |title=Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin |url=https://pubmed.ncbi.nlm.nih.gov/2582358 |journal=Nucleic Acids Research |volume=13 |issue=3 |pages=745–761 |doi=10.1093/nar/13.3.745 |issn=0305-1048 |pmc=PMC341032 |pmid=2582358}}</ref> |
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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 |
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; 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.<ref>{{Cite journal |last=Hood |first=D. W. |last2=Dow |first2=C. S. |last3=Green |first3=P. N. |date= |year=1987 |title=DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs |url=https://pubmed.ncbi.nlm.nih.gov/3655730 |journal=Journal of General Microbiology |volume=133 |issue=3 |pages=709–720 |doi=10.1099/00221287-133-3-709 |issn=0022-1287 |pmid=3655730}}</ref> |
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One method was introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single membrane. These samples would have to be separated in their own lanes inside the membranes and then the membrane would have to be rotated to a different angle where it would result in simultaneous hybridization with many different DNA probes.<ref>{{Cite journal|last1=Socransky|first1=S. S.|last2=Smith|first2=C.|last3=Martin|first3=L.|last4=Paster|first4=B. J.|last5=Dewhirst|first5=F. E.|last6=Levin|first6=A. E.|date=October 1994|title="Checkerboard" DNA-DNA hybridization|journal=BioTechniques|volume=17|issue=4|pages=788–792|issn=0736-6205|pmid=7833043}}</ref> |
One method was introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single membrane. These samples would have to be separated in their own lanes inside the membranes and then the membrane would have to be rotated to a different angle where it would result in simultaneous hybridization with many different DNA probes.<ref>{{Cite journal|last1=Socransky|first1=S. S.|last2=Smith|first2=C.|last3=Martin|first3=L.|last4=Paster|first4=B. J.|last5=Dewhirst|first5=F. E.|last6=Levin|first6=A. E.|date=October 1994|title="Checkerboard" DNA-DNA hybridization|journal=BioTechniques|volume=17|issue=4|pages=788–792|issn=0736-6205|pmid=7833043}}</ref> |
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===In microbiology=== |
===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.<ref>{{Cite journal |last=Auch |first=Alexander F. |last2=von Jan |first2=Mathias |last3=Klenk |first3=Hans-Peter |last4=Göker |first4=Markus |year=2010 |title=Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison |url=https://environmentalmicrobiome.biomedcentral.com/articles/10.4056/sigs.531120 |journal=Standards in Genomic Sciences |language=en |volume=2 |issue=1 |pages=117–134 |doi=10.4056/sigs.531120 |issn=1944-3277 |pmc=PMC3035253 |pmid=21304684}}</ref> This technique is not widely used on larger organisms where differences in species are easier to identify. In the late 1900's, 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 eachother.<ref name="Brenner1979">{{cite journal|author = Brenner DJ|title=Deoxyribonucleic acid reassociation in the taxonomy of enteric bacteria|journal=International Journal of Systematic Bacteriology|volume=23|issue=4|pages=298–307|year=1973|doi=10.1099/00207713-23-4-298|doi-access=free}}</ref><ref name="Wayne1987">{{cite journal|vauthors = Wayne LG, Brenner DJ, Colwell RR, Grimont PD, Kandler O, Krichevsky MI, Moore LH, ((Moore WEC)), ((Murray RGE)), Stackebrandt E, Starr MP, Trüper HG|year = 1987|title = Report of the ad hoc committee on reconciliation of approaches to bacterial systematics|journal = International Journal of Systematic Bacteriology|volume = 37|issue = 4|pages = 463–464|doi = 10.1099/00207713-37-4-463|doi-access = free}}</ref><ref name="Tindall2010">{{cite journal|vauthors = Tindall BJ, Rossello-Mora R, ((Busse H-J)), Ludwig W, Kampfer P|title = Notes on the characterization of prokaryote strains for taxonomic purposes|journal = International Journal of Systematic and Evolutionary Microbiology|volume = 60|issue = Pt 1|pages = 249–266|doi = 10.1099/ijs.0.016949-0|pmid = 19700448|year = 2010|doi-access = free}}</ref> In 2014, a threshold of 79% similarity has been suggested to separate bacterial subspecies.<ref name="doi:10.1186/1944-3277-9-2">{{cite journal|vauthors = 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|title=Complete genome sequence of DSM 30083<sup>T</sup>, the type strain (U5/41<sup>T</sup>) of ''Escherichia coli'', and a proposal for delineating subspecies in microbial taxonomy|journal=Standards in Genomic Sciences|volume=9|pages=2|year=2013|doi=10.1186/1944-3277-9-2|pmid=25780495|pmc=4334874}}</ref> |
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In 2014, a threshold of 79% similarity has been suggested to separate bacterial subspecies.<ref name="doi:10.1186/1944-3277-9-2">{{cite journal|vauthors = 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|title=Complete genome sequence of DSM 30083<sup>T</sup>, the type strain (U5/41<sup>T</sup>) of ''Escherichia coli'', and a proposal for delineating subspecies in microbial taxonomy|journal=Standards in Genomic Sciences|volume=9|pages=2|year=2013|doi=10.1186/1944-3277-9-2|pmid=25780495|pmc=4334874}}</ref> DNA–DNA hybridization has not been tested much worldwide because it could take years to get results and it's not always that easy to perform in routine laboratories. However in 2004, there has been a new method tested out by digesting melting profiles with Sau3A in microplates in order to get a faster DNA–DNA hybridization test result.<ref>{{Cite journal|last1=Mehlen|first1=André|last2=Goeldner|first2=Marcia|last3=Ried|first3=Sabine|last4=Stindl|first4=Sibylle|last5=Ludwig|first5=Wolfgang|last6=Schleifer|first6=Karl-Heinz|date=November 2004|title=Development of a fast DNA-DNA hybridization method based on melting profiles in microplates|journal=Systematic and Applied Microbiology|volume=27|issue=6|pages=689–695|doi=10.1078/0723202042369875|issn=0723-2020|pmid=15612626}}</ref> |
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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.<ref>{{Cite journal|last1=Mehlen|first1=André|last2=Goeldner|first2=Marcia|last3=Ried|first3=Sabine|last4=Stindl|first4=Sibylle|last5=Ludwig|first5=Wolfgang|last6=Schleifer|first6=Karl-Heinz|date=November 2004|title=Development of a fast DNA-DNA hybridization method based on melting profiles in microplates|journal=Systematic and Applied Microbiology|volume=27|issue=6|pages=689–695|doi=10.1078/0723202042369875|issn=0723-2020|pmid=15612626}}</ref> This new DDH technique became the standard for bacterial taxonomy.<ref>{{Cite journal |last=Huang |first=Chien-Hsun |last2=Li |first2=Shiao-Wen |last3=Huang |first3=Lina |last4=Watanabe |first4=Koichi |date=2018 |title=Identification and Classification for the Lactobacillus casei Group |url=https://www.frontiersin.org/articles/10.3389/fmicb.2018.01974 |journal=Frontiers in Microbiology |volume=9 |doi=10.3389/fmicb.2018.01974 |issn=1664-302X |pmc=PMC6113361 |pmid=30186277}}</ref> |
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===In zoology=== |
===In zoology=== |
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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.<ref>{{cite web|author = Marks, Jonathan | title = DNA hybridization in the apes—Technical issues | url=http://personal.uncc.edu/jmarks/DNAHYB/Dnahyb2.html | date=2007-05-09 | access-date = 2019-06-02 | archive-url = https://web.archive.org/web/20070509132131/http://personal.uncc.edu/jmarks/DNAHYB/Dnahyb2.html | archive-date = 2007-05-09 }}</ref>{{better source needed|date=June 2019}}{{better source needed|date=June 2019}} 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.<ref>{{cite journal| title=Use of checkerboard DNA–DNA hybridization to study complex microbial ecosystems| author=S.S. Socransky| author2=A.D. Haffajee| author3=C. Smith| author4=L. Martin| author5=J.A. Haffajee| author6=N.G. Uzel| author7=J. M. Goodson| journal=Oral Microbiology and Immunology| year=2004| volume=19| issue=6| pages=352–362| doi=10.1111/j.1399-302x.2004.00168.x| pmid=15491460}}</ref> |
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.<ref>{{cite web|author = Marks, Jonathan | title = DNA hybridization in the apes—Technical issues | url=http://personal.uncc.edu/jmarks/DNAHYB/Dnahyb2.html | date=2007-05-09 | access-date = 2019-06-02 | archive-url = https://web.archive.org/web/20070509132131/http://personal.uncc.edu/jmarks/DNAHYB/Dnahyb2.html | archive-date = 2007-05-09 }}</ref>{{better source needed|date=June 2019}}{{better source needed|date=June 2019}} 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.<ref>{{cite journal| title=Use of checkerboard DNA–DNA hybridization to study complex microbial ecosystems| author=S.S. Socransky| author2=A.D. Haffajee| author3=C. Smith| author4=L. Martin| author5=J.A. Haffajee| author6=N.G. Uzel| author7=J. M. Goodson| journal=Oral Microbiology and Immunology| year=2004| volume=19| issue=6| pages=352–362| doi=10.1111/j.1399-302x.2004.00168.x| pmid=15491460}}</ref> |
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==In silico methods== |
==''In silico'' methods== |
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{{primary sources|section|date=June 2019}} |
{{primary sources|section|date=June 2019}} |
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The modern approach is to carry out DNA–DNA hybridization ''in silico'' |
The modern approach is to carry out DNA–DNA hybridization ''in silico'' utilizes completely or partially [[Whole genome sequencing|sequenced genomes]].<ref name="doi10.1186/1471-2105-14-60">{{cite journal|vauthors = Meier-Kolthoff JP, Auch AF, Klenk HP, Goeker M|title=Genome sequence-based species delimitation with confidence intervals and improved distance functions|journal=BMC Bioinformatics|volume=14|pages=60|year=2013|doi=10.1186/1471-2105-14-60|pmid=23432962|pmc=3665452}}</ref> The [http://ggdc.dsmz.de GGDC] and [https://tygs.dsmz.de/ TYGS] developed at [[DSMZ]] are the most accurate known tools for calculating DDH-analogous values.<ref name="doi10.1186/1471-2105-14-60" /> Among other algorithmic improvements, it solves the problem with paralogous sequences by carefully filtering them from the matches between the two genome sequences. |
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==See also== |
==See also== |
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Revision as of 01:48, 11 March 2023
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In genomics, DNA–DNA hybridization is 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 and has been used extensively in phylogeny and taxonomy.
Method
 |
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: i.e. they separate when heated at a higher temperature than dissimilar sequences, a process known as "DNA melting".[1][2][3]
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; 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.[4]
One method was introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single membrane. These samples would have to be separated in their own lanes inside the membranes and then the membrane would have to be rotated to a different angle where it would result in simultaneous hybridization with many different DNA probes.[5]
Uses
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When several species are compared, similarity values allow organisms to be arranged in a phylogenetic tree; it is therefore one possible approach to carrying out molecular systematics.[citation needed]
In microbiology
DNA-DNA hybridization (DDH) is used as a primary method to distinguish bacterial species as it is difficult to visually classify them accurately.[6] This technique is not widely used on larger organisms where differences in species are easier to identify. In the late 1900's, 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 eachother.[7][8][9] In 2014, a threshold of 79% similarity has been suggested to separate bacterial subspecies.[10]
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.[11] This new DDH technique became the standard for bacterial taxonomy.[12]
In zoology
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.[13][14]
In radioactivity
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.[15]
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.[16][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.[17]
In silico methods
|
The modern approach is to carry out DNA–DNA hybridization in silico utilizes completely or partially sequenced genomes.[18] The GGDC and TYGS developed at DSMZ are the most accurate known tools for calculating DDH-analogous values.[18] 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
- ^ Sinden, Richard R. (1994). DNA structure and function. San Diego: Academic Press. pp. 37–45. ISBN 0-12-645750-6. OCLC 30109829.
- ^ 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) - ^ 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.
{{cite journal}}: CS1 maint: PMC format (link) - ^ 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.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ 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.
- ^ 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. doi:10.4056/sigs.531120. ISSN 1944-3277. PMC 3035253. PMID 21304684.
{{cite journal}}: CS1 maint: PMC format (link) - ^ 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.
- ^ 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.
- ^ 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. PMID 19700448.
- ^ 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.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ 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. doi:10.1078/0723202042369875. ISSN 0723-2020. PMID 15612626.
- ^ Huang, Chien-Hsun; Li, Shiao-Wen; Huang, Lina; Watanabe, Koichi (2018). "Identification and Classification for the Lactobacillus casei Group". Frontiers in Microbiology. 9. doi:10.3389/fmicb.2018.01974. ISSN 1664-302X. PMC 6113361. PMID 30186277.
{{cite journal}}: CS1 maint: PMC format (link) CS1 maint: unflagged free DOI (link) - ^ Genetic Similarities: Wilson, Sarich, Sibley, and Ahlquist
- ^ 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.
- ^ 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.
- ^ Marks, Jonathan (2007-05-09). "DNA hybridization in the apes—Technical issues". Archived from the original on 2007-05-09. Retrieved 2019-06-02.
- ^ 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.
- ^ 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.
{{cite journal}}: CS1 maint: unflagged free DOI (link)
Further reading
- Graur, D. & Li, W-H. 1991 (2nd ed. 1999). Fundamentals of Molecular Evolution.