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{{Short description|Biotechnology that acquires genome information without sequencing}}

'''Genome profiling''' (GP) is a [[biotechnology]] that acquires [[genome]] information without [[sequencing]]. It can be used for identification and classification of [[organisms]]. It was pioneered by Japanese biophysicist Prof. Koichi Nishigaki and his colleagues at [[Saitama University]] in 1990 and later.<ref>"DNAプロフィーリングー-方法と原理" [DNA profiling—method and principle] (in Japanese). p. S230. In: {{cite journal |title=日本生物物理学会第28回年会13 |trans-title=The 28th Annual Meeting of the Biophysical Society of Japan |language=ja |journal=Seibutsu Butsuri |date=1990 |volume=30 |issue=supplement |pages=S229–S244 |doi=10.2142/biophys.30.supplement_S229 |doi-access=free }}</ref><ref>{{cite journal |last1=Nishigaki |first1=Koichi |last2=Amano |first2=Norihiko |last3=Takasawa |first3=Tsutomu |title=DNA Profiling. An Approach of Systemic Characterization, Classification, and Comparison of Genomic DNAs |journal=Chemistry Letters |date=July 1991 |volume=20 |issue=7 |pages=1097–1100 |doi=10.1246/cl.1991.1097 }}</ref><ref>{{cite journal |last1=Hamano |first1=Keiichi |last2=Takasawa |first2=Tsutomu |last3=Kurazono |first3=Takashi |last4=Okuyama |first4=Yusuke |last5=Nishigaki |first5=Koichi |title=ゲノムプロフイリング-その方法論の確立と実践的評価- |trans-title=Genome Profiling—Establishment and Practical Evaluation of its Methodology— |language=ja |journal=Nippon Kagaku Kaishi |date=1996 |issue=1 |pages=54–61 |doi=10.1246/nikkashi.1996.54 |doi-access=free }}</ref> The term 'DNA profiling' was changed to 'genome profiling' to avoid confusion, as the term 'DNA profiling' had begun to be used for a different technology in the field of forensics.<ref>{{cite journal |last1=Daves |first1=Anne |title=The Use of DNA Profiling and Behavioural Science in the Investigation of Sexual Offences |journal=Medicine, Science and the Law |date=April 1991 |volume=31 |issue=2 |pages=95–101 |doi=10.1177/002580249103100202 |pmid=2062204 }}</ref> In GP, small fragments of [[genomic DNA]] are randomly amplified (random [[polymerase chain reaction|PCR]]) and the random PCR products are subjected to [[temperature gradient gel electrophoresis|temperature-gradient gel electrophoresis]] (TGGE) to generate a species-specific mobility pattern (genome profile). From this, species identification dots (spiddos) are assigned.<ref name="Naimuddin Commonly conserved genetic">{{cite journal |last1=Naimuddin |first1=M. |title=Commonly conserved genetic fragments revealed by genome profiling can serve as tracers of evolution |journal=Nucleic Acids Research |date=15 May 2002 |volume=30 |issue=10 |pages=42e–42 |doi=10.1093/nar/30.10.e42 |pmid=12000847 |pmc=115296 }}</ref> This approach is clearly superior because it does not require prior knowledge of any [[gene]] sequence. It is clear that random PCR can produce commonly conserved genetic fragments (ccgf), which make it possible to measure the difference between [[organisms]].<ref name="Naimuddin Commonly conserved genetic"/> The GP method has been successfully applied to a wide range of organisms, from [[viruses]] and [[bacteria]] to animals and plants, for identification and classification.<ref name="Nishigaki Discoveries by the genome">{{cite journal |last1=Nishigaki |first1=Koichi |title=Discoveries by the genome profiling, symbolic powers of non-next generation sequencing methods |journal=Briefings in Functional Genomics |date=6 December 2024 |volume=23 |issue=6 |pages=775–797 |doi=10.1093/bfgp/elae047 |pmid=39602495 }}</ref> Its unique merit is in the ultra-high performance to obtain the final results (identification and classification), since GP, in principle, requires only a single random PCR plus μTGGE experiment (~2 h task in all)

== Procedure and theory ==
The GP procedure is outlined in the following steps
[[File:Whole process of GP.png|thumb|Whole process of GP: Beginning with random PCR which provides with DNA fragments from genomic DNA. Then, obtained DNAs are subjected to μTGGE where sequence-specific DNA melting profiles (genome profile) appear (schematically drawn; original photo in Panel C left). As increasing the temperature, DNA melting proceeds to: double-stranded, partially melted, and single-stranded depending on each DNA sequence. The initial melting point of each DNA is plotted (white dot) which is converted to spiddos (species identification dot(s): Panel C right) by use of internal references (shown in double circles in photo).|300x300px]]

===Random PCR===
A set of [[genomic DNA]] and a single primer are subjected to a modified [[Polymerase chain reaction|PCR]] operated at a lower annealing temperature (i.e. ~30 °C) than conventional PCR, allowing less stable template-primer hybrid structures to initiate the elongation reaction. Random PCR requires only a single short primer, whereas conventional PCR requires two types of primers (forward and reverse), and these sequences must be predetermined and specific to each template sequence. The primer pfM12 (dAGAACGCGCCTG) is known to be used universally for any kind of organism.<ref name="sak" />

Although random PCR leads to the generation of [[DNA]] fragments that are not intentionally designed, the products are theoretically predictable based on knowledge of the template and primer sequences<ref name=sak>{{cite journal |last1=Sakuma |first1=Yoshito |last2=Nishigaki |first2=Koichi |title=Computer Prediction of General PCR Products Based on Dynamical Solution Structures of DNA |journal=The Journal of Biochemistry |date=October 1994 |volume=116 |issue=4 |pages=736–741 |doi=10.1093/oxfordjournals.jbchem.a124589 |pmid=7883746 }}</ref><ref>{{cite journal |last1=Nishigaki |first1=K. |last2=Saito |first2=A. |last3=Takashi |first3=H. |last4=Naimuddin |first4=M. |title=Whole genome sequence-enabled prediction of sequences performed for random PCR products of Escherichia coli |journal=Nucleic Acids Research |date=May 2000 |volume=28 |issue=9 |pages=1879–1884 |doi=10.1093/nar/28.9.1879 |pmid=10756186 |pmc=103271 }}</ref>

===Micro-TGGE (μTGGE)===
Random PCR products are subjected to [[Temperature gradient gel electrophoresis|temperature-gradient gel electrophoresis]] to separate fragments by size and melting behavior. As the gel temperature increases, each double-stranded DNA fragment denatures (melts) at a specific temperature depending on its sequence. This causes a transition in mobility on the gel, resulting in a specific mobility pattern.<ref>{{cite journal |last1=Biyani |first1=Manish |last2=Nishigaki |first2=Koichi |title=Hundredfold productivity of genome analysis by introduction of microtemperature-gradient gel electrophoresis |journal=Electrophoresis |date=January 2001 |volume=22 |issue=1 |pages=23–28 |doi=10.1002/1522-2683(200101)22:1<23::AID-ELPS23>3.0.CO;2-Z |pmid=11197172 }}</ref>

===Spiddo extraction and analysis===
Species identification dots (spiddos) are extracted from the genome profile as initial melting points for DNA bands. The positions of these bands are determined by the DNA sequence.<ref name="Naimuddin Commonly conserved genetic"/><ref>{{cite journal |last1=Nishigaki |first1=Koichi |last2=Husimi |first2=Yuzuru |last3=Masuda |first3=Masaaki |last4=Kaneko |first4=Kiyomitu |last5=Tanaka |first5=Toyosuke |title=Strand Dissociation and Cooperative Melting of Double-Stranded DNAs Detected by Denaturant Gradient Gel Electrophoresis |journal=The Journal of Biochemistry |date=1984 |volume=95 |issue=3 |pages=627–635 |doi=10.1093/oxfordjournals.jbchem.a134651 |pmid=6202679 }}</ref> In other words, spiddos are theoretically predictable and can be connected to the template sequence.<ref name="Wada Tachibana Melting fine structure">{{cite journal |last1=Wada |first1=Akiyoshi |last2=Tachibana |first2=Hideki |last3=Ueno |first3=Shizue |last4=Husimi |first4=Yuzuru |last5=Machida |first5=Yasunori |title=Melting fine structure of DNA fragments of known base sequence from ΦX174 |journal=Nature |date=September 1977 |volume=269 |issue=5626 |pages=352–353 |doi=10.1038/269352a0 |pmid=904689 |bibcode=1977Natur.269..352W }}</ref><ref>{{cite journal |last1=Fixman |first1=Marshall |last2=Freire |first2=Juan J. |title=Theory of DNA melting curves |journal=Biopolymers |date=December 1977 |volume=16 |issue=12 |pages=2693–2704 |doi=10.1002/bip.1977.360161209 |pmid=597576 }}</ref><ref>{{cite journal |last1=Wada |first1=Akiyoshi |last2=Yabuki |first2=Sadato |last3=Husimi |first3=Yuzuru |last4=Brahms |first4=J. G. |title=Fine Structure in the Thermal Denaturation of DNA: High Temperature-Resolution Spectrophotometric Studie |journal=Critical Reviews in Biochemistry |date=January 1980 |volume=9 |issue=2 |pages=87–144 |doi=10.3109/10409238009105432 |pmid=6777116 }}</ref> Therefore, spiddos contain a type of information known as SIOWS (sequence-inherent information obtained without [[sequencing]]), which is unique and essential for GP technology based on DNA melting theory.<ref name="Wada Tachibana Melting fine structure"/><ref>{{cite journal |last1=Poland |first1=Douglas |title=Recursion relation generation of probability profiles for specific-sequence macromolecules with long-range correlations |journal=Biopolymers |date=September 1974 |volume=13 |issue=9 |pages=1859–1871 |doi=10.1002/bip.1974.360130916 |pmid=4415504 }}</ref>

===Genome distance===
Using spiddo information, the difference between two [[genome]]s can be calculated in terms of pattern similarity score (PaSS). This parameter has been successfully used for [[species]] identification and classification, as well as for measuring the degree of [[mutation]].<ref>{{cite report |type=Preprint |last1=Nishigaki |first1=Koichi |title=Method and theory of genome profiling (GP) developed for identification and classification of organisms |date=2025 |doi=10.26434/chemrxiv-2024-n9q36-v3 |doi-access=free }}</ref><ref>{{cite journal |last1=Kouduka |first1=Mariko |last2=Sato |first2=Daisuke |last3=Komori |first3=Manabu |last4=Kikuchi |first4=Motohiro |last5=Miyamoto |first5=Kiyoshi |last6=Kosaku |first6=Akinori |last7=Naimuddin |first7=Mohammed |last8=Matsuoka |first8=Atsushi |last9=Nishigaki |first9=Koichi |title=A Solution for Universal Classification of Species Based on Genomic DNA |journal=International Journal of Plant Genomics |date=5 February 2007 |volume=2007 |article-number=27894 |doi=10.1155/2007/27894 |doi-access=free |pmid=18253463 |pmc=1893011 }}</ref>

== Applications ==
[[File:Application of GP in Taxonomy.png|thumb|337x337px|Application of GP in Taxonomy: Plants , insects, and fish are classified by GP (right half), showing excellent congruences with the classical phenotype-based taxonomic results (left half). Spiddos data are shown rightmost, which are ensemble of coordinate points determined by mobility- and melting temperature axes]]
GP has been applied to a variety of [[taxa]], including viruses, bacteria, fungi, protozoa, insects, fish, animals and plants. An early study by Kouduka et al. reported the congruence between GP-based clustering and classical, [[phenotype]]-based [[taxonomy]] for insects, fish, and plants. Further investigation has revealed that insects can be more easily classified using GP than with traditional [[sequencing]]-based approaches, such as [[18S ribosomal RNA|18S]] rDNA sequencing.<ref>{{cite journal |last1=Ahmed |first1=Shamim |last2=Komori |first2=Manabu |last3=Tsuji-Ueno |first3=Sachika |last4=Suzuki |first4=Miho |last5=Kosaku |first5=Akinori |last6=Miyamoto |first6=Kiyoshi |last7=Nishigaki |first7=Koichi |title=Genome Profiling (GP) Method Based Classification of Insects: Congruence with That of Classical Phenotype-Based One |journal=PLOS ONE |date=31 August 2011 |volume=6 |issue=8 |article-number=e23963 |doi=10.1371/journal.pone.0023963 |doi-access=free |pmid=21912611 |pmc=3166070 |bibcode=2011PLoSO...623963A }}</ref> A GP-based genome database has been proposed<ref name="Watanabe Saito Takeuchi database">{{cite journal |last1=Watanabe |first1=Takehiro |last2=Saito |first2=Ayumu |last3=Takeuchi |first3=Yusuke |last4=Naimuddin |first4=Mohammed |last5=Nishigaki |first5=Koichi |title=A database for the provisional identification of species using only genotypes: web-based genome profiling |journal=Genome Biology |date=28 January 2002 |volume=3 |issue=2 |article-number=research0010.1 |doi=10.1186/gb-2002-3-2-research0010 |doi-access=free |pmid=11864372 |pmc=65688 }}</ref> and is ongoing, in which organisms are properly located in genome sequence space (the closer the similarity, the closer the distance).

GP has also been used to confirm the authenticity of fungal culture collections<ref>{{cite journal |last1=Hamano |first1=Keiichi |last2=Ueno-Tsuji |first2=Sachika |last3=Tanaka |first3=Reiko |last4=Suzuki |first4=Motofumi |last5=Nishimura |first5=Kazuko |last6=Nishigaki |first6=Koichi |title=Genome profiling (GP) as an effective tool for monitoring culture collections: A case study with Trichosporon |journal=Journal of Microbiological Methods |date=May 2012 |volume=89 |issue=2 |pages=119–128 |doi=10.1016/j.mimet.2012.02.007 |pmid=22401825 |doi-access=free }}</ref> and to detect irreplaceable samples, such as single-celled P[[Protist|rotists]], [[Radiolaria]] and [[Foraminifera]],<ref>{{cite journal |last1=Kouduka |first1=Mariko |last2=Matsuoka |first2=Atsushi |last3=Nishigaki |first3=Koichi |title=Acquisition of genome information from single-celled unculturable organisms (radiolaria) by exploiting genome profiling (GP) |journal=BMC Genomics |date=December 2006 |volume=7 |issue=1 |article-number=135 |doi=10.1186/1471-2164-7-135 |doi-access=free |pmid=16740170 |pmc=1523345 }}</ref> as well as forensic materials such as body fluids (blood, saliva and semen).<ref>{{cite journal |last1=Suwa |first1=Nagisa |last2=Ikegaya |first2=Hiroshi |last3=Takasaka |first3=Tomokazu |last4=Nishigaki |first4=Koichi |last5=Sakurada |first5=Koichi |title=Human blood identification using the genome profiling method |journal=Legal Medicine |date=May 2012 |volume=14 |issue=3 |pages=121–125 |doi=10.1016/j.legalmed.2012.01.001 |pmid=22285643 }}</ref><ref>{{cite journal |last1=Takasaka |first1=Tomokazu |last2=Sakurada |first2=Koichi |last3=Akutsu |first3=Tomoko |last4=Nishigaki |first4=Koichi |last5=Ikegaya |first5=Hiroshi |title=Trials of the detection of semen and vaginal fluid RNA using the genome profiling method |journal=Legal Medicine |date=September 2011 |volume=13 |issue=5 |pages=265–267 |doi=10.1016/j.legalmed.2011.05.001 |pmid=21684187 }}</ref> For scientific purposes, GP has been used to discover continuous [[mutation]] of body cells,<ref>{{cite journal |last1=Diwan |first1=Deepti |last2=Masubuchi |first2=Yuki |last3=Furukawa |first3=Tatsuya |last4=Nishigaki |first4=Koichi |title=Ordered genome change of plant and animal body cells revealed by the genome profiling method |journal=FEBS Letters |date=July 2016 |volume=590 |issue=14 |pages=2119–2126 |doi=10.1002/1873-3468.12248 |pmid=27277546 }}</ref> discriminate leaf origins from ambient trees<ref>{{cite journal |last1=Diwan |first1=Deepti |last2=Komazaki |first2=Shun |last3=Suzuki |first3=Miho |last4=Nemoto |first4=Naoto |last5=Aita |first5=Takuyo |last6=Satake |first6=Akiko |last7=Nishigaki |first7=Koichi |title=Systematic genome sequence differences among leaf cells within individual trees |journal=BMC Genomics |date=December 2014 |volume=15 |issue=1 |article-number=142 |doi=10.1186/1471-2164-15-142 |doi-access=free |pmid=24548431 |pmc=3937000 }}</ref> and determine the family relationships of mice.<ref>{{cite journal |last1=Sharma |first1=Harshita |last2=Ohtani |first2=Fumihito |last3=Kumari |first3=Parmila |last4=Diwan |first4=Deepti |last5=Ohara |first5=Naoko |last6=Kobayashi |first6=Tetsuya |last7=Suzuki |first7=Miho |last8=Nemoto |first8=Naoto |last9=Matsushima |first9=Yoshibumi |last10=Nishigaki |first10=Koichi |title=Familial clustering of mice consistent to known pedigrees enabled by the genome profiling (GP) method |journal=Biophysics |date=2014 |volume=10 |pages=55–62 |doi=10.2142/biophysics.10.55 |pmid=27493499 |pmc=4629661 }}</ref>

Utilizing the concept of genome distance, GP has been successfully implemented to detect mutagenic chemicals ([[mutagen]]s).<ref>{{cite journal |last1=Futakami |first1=Masae |last2=Salimullah |first2=Md |last3=Miura |first3=Takashi |last4=Tokita |first4=Sumio |last5=Nishigaki |first5=Koichi |title=Novel Mutation Assay with High Sensitivity based on Direct Measurement of Genomic DNA Alterations: Comparable Results to the Ames Test |journal=The Journal of Biochemistry |date=May 2007 |volume=141 |issue=5 |pages=675–686 |doi=10.1093/jb/mvm074 |pmid=17383979 }}</ref><ref>{{cite journal |last1=Kumari |first1=Parmila |last2=Gautam |first2=Sunita Ghimire |last3=Baba |first3=Misato |last4=Tsukiashi |first4=Motoki |last5=Matsuoka |first5=Koji |last6=Yasukawa |first6=Kiyoshi |last7=Nishigaki |first7=Koichi |title=DNA-based mutation assay GPMA (genome profiling-based mutation assay): reproducibility, parts-per-billion scale sensitivity, and introduction of a mammalian-cell-based approach |journal=The Journal of Biochemistry |date=December 2017 |volume=162 |issue=6 |pages=395–401 |doi=10.1093/jb/mvx043 |pmid=29186523 }}</ref> This technology is termed GPMA (GP-based mutation assay), in which a test [[organism]], such as the [[bacterium]] [[Escherichia coli]], is exposed to mutagenic (physical or chemical) reagents and investigated for sequence changes using genome distance.

== General remarks ==

GP is a unique [[genome]] analysis technique as it can acquire useful information (SIOWS: sequence-inherent information obtained without sequencing, expressed as spiddos) without the time-consuming process of [[DNA sequencing]]. GP is so simple that it can be performed using only basic random [[Polymerase chain reaction|PCR]] and [[Temperature gradient gel electrophoresis|TGGE]] techniques with a single primer. Furthermore, GP is so universal that it can use the same primer ([[Primer (molecular biology)|universal primer]]) for any organism, leading to the acquisition of the universal parameter, spiddos, which can be used to measure genome distance. Genome information can be compactly stored and utilized via spiddos in an internet database.<ref name="Watanabe Saito Takeuchi database"/> If the entire GP procedure could be automated (currently, it involves a manual step), GP technology would be far easier to use and more accessible. On the other hand, the current system (PCR, TGGE, imager and computer) has the advantage of being inexpensive and able to be used for multiple purposes separately. In summary, in the age of [[next generation sequencing]], when sophisticated and complicated procedures prevail, simple measures such as GP are complementary in importance.<ref name="Nishigaki Discoveries by the genome"/>

== References ==
{{Reflist}}

[[Category:Genetics]]
[[Category:Molecular biology techniques]]
[[Category:Biotechnology]]
[[Category:DNA profiling techniques]]
[[Category:Bioinformatics]]

Genome profiling - История изменений

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ru>Monkbot: Monkbot/task 21: Replace page(s) with article-number;