DNA–DNA hybridization#In silico methods

{{Short description|Technique used to measure similarity in DNA sequences}}

{{ about|the specific use in genomics|the general phenomenon|Nucleic acid thermodynamics#Hybridization }}

{{Multiple issues|

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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.{{cite book |author=Erko Stackebrandt |url=https://books.google.com/books?id=eIf6RQeOZPoC |title=Molecular Identification, Systematics, and Population Structure of Prokaryotes |date=8 September 2010 |publisher=Springer Science & Business Media |isbn=978-3-540-31292-5 |pages=}}

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. An example is they separate when heated at a higher temperature than dissimilar sequences, a process known as "DNA melting".{{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}}{{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}}{{Cite journal |last1=Forster |first1=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 |journal=Nucleic Acids Research |volume=13 |issue=3 |pages=745–761 |doi=10.1093/nar/13.3.745 |issn=0305-1048 |pmc=341032 |pmid=2582358}}

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.{{Cite journal |last1=Hood |first1=D. W. |last2=Dow |first2=C. S. |last3=Green |first3=P. N. |year=1987 |title=DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs |journal=Journal of General Microbiology |volume=133 |issue=3 |pages=709–720 |doi=10.1099/00221287-133-3-709 |doi-access=free |issn=0022-1287 |pmid=3655730}}

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.{{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}}

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|date=June 2019}}

=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.{{Cite journal |last1=Auch |first1=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 |journal=Standards in Genomic Sciences |language=en |volume=2 |issue=1 |pages=117–134 |doi=10.4056/sigs.531120 |issn=1944-3277 |pmc=3035253 |pmid=21304684}} 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.{{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}}{{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}}{{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|hdl = 10261/49238|hdl-access = free}} In 2014, a threshold of 79% similarity has been suggested to separate bacterial subspecies.{{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^T, the type strain (U5/41^T) 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 |doi-access=free }}

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.{{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}} This new DDH technique became the standard for bacterial taxonomy.{{Cite journal |last1=Huang |first1=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 |journal=Frontiers in Microbiology |volume=9 |page=1974 |doi=10.3389/fmicb.2018.01974 |issn=1664-302X |pmc=6113361 |pmid=30186277|doi-access=free }}

=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.[http://evolution.berkeley.edu/evolibrary/article/_0/history_26 Genetic Similarities: Wilson, Sarich, Sibley, and Ahlquist]{{cite journal| title=The Phylogeny of the Hominoid Primates, as Indicated by DNA–DNA Hybridization| author=C.G. Sibley| author2=J.E. Ahlquist| name-list-style=amp| journal=Journal of Molecular Evolution| volume=20| pages=2–15| year=1984| doi=10.1007/BF02101980| pmid=6429338| issue=1| bibcode=1984JMolE..20....2S| s2cid=6658046}}

= 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.[https://www.pnas.org/content/pnas/64/2/600.full.pdf 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.]

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.{{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 }}{{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.{{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}}

=''In silico'' methods=

The modern approach is to carry out DNA–DNA hybridization in silico utilizes completely or partially sequenced genomes.{{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 |doi-access=free }} 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.

dDDH has been used for resolving difficult taxa such as Escherichia coli, Bacillus cereus group, and Aeromonas.{{cite journal |last1=Riojas |first1=Marco A. |last2=McGough |first2=Katya J. |last3=Rider-Riojas |first3=Cristin J. |last4=Rastogi |first4=Nalin |last5=Hazbón |first5=Manzour Hernando |title=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 |journal=International Journal of Systematic and Evolutionary Microbiology |date=1 January 2018 |volume=68 |issue=1 |pages=324–332 |doi=10.1099/ijsem.0.002507 |pmid=29205127 |doi-access=free}} The Judicial Commission of International Committee on Systematics of Prokaryotes has admitted dDDH as taxonomic evidence.{{cite journal |last1=Arahal |first1=David R. |last2=Bull |first2=Carolee T. |last3=Busse |first3=Hans-Jürgen |last4=Christensen |first4=Henrik |last5=Chuvochina |first5=Maria |last6=Dedysh |first6=Svetlana N. |last7=Fournier |first7=Pierre-Edouard |last8=Konstantinidis |first8=Konstantinos T. |last9=Parker |first9=Charles T. |last10=Rossello-Mora |first10=Ramon |last11=Ventosa |first11=Antonio |last12=Göker |first12=Markus |title=Judicial Opinions 123–127 |journal=International Journal of Systematic and Evolutionary Microbiology |date=27 April 2023 |volume=72 |issue=12 |doi=10.1099/ijsem.0.005708|pmid=36748499 |hdl=10261/295959 |hdl-access=free }}