124:
metal chelate affinity chromatography. Subsequent studies have revealed that among amino acids constituting proteins, histidine is strongly involved in the coordination complex with metal ions. Therefore, if a number of histidines are added to the end of the protein, the affinity of the protein for the metal ion is increased and this can be exploited to selectively isolate the protein of interest. When a protein with a His-tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized, the histidine residue chelates the metal ion and binds to the carrier. Since other proteins do not bind to the carrier or bind only very weakly, they can be removed by washing the carrier with an appropriate buffer. The poly-histidine tagged protein can then be recovered by eluting it off the resin.
20:
109:
138:
199:
each protein and the chosen purification strategy; it may be necessary to test multiple constructs with the tag at different positions. Although polyhistidine tags are considered to typically not alter the properties of a protein, it has been demonstrated that addition of the tag can cause unwanted effects, such as influencing the protein's oligomeric state.
299:
or by passing over a resin bed in a column format. The resin is then washed with buffer to remove proteins that do not specifically interact with bound cation and the protein of interest is eluted off the resin using buffer containing a high concentration of imidazole or a lowered pH. The purity and amount of protein can be assessed by methods such
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198:
of a protein and is attached via a short flexible linker, which may contain a protease cleavage site. Less commonly, tags can be added at both the N- and C-termini or inserted at an intermediate part of a protein, such as within an exposed loop. The choice of tag position depends on the properties of
298:
or any combination of these. At this stage, the lysate contains the recombinant protein among many endogenous proteins originating from the host cells. The lysate is exposed to affinity resin bound to a carrier matrix coupled with a divalent cation, either by direct addition of resin (batch binding)
250:
immobilized metal ions. The compound added at high concentration replaces virtually all carrier-bound protein which is thus eluted from the carrier. Imidazole is the side chain of histidine and is typically used at a concentration of 150 - 500 mM for elution. Histidine or histamine can also be used.
351:
Hexahistadine CyDye tags have been developed, which use nickel covalent coordination to EDTA groups attached to fluorophores in order to create dyes that attach to the polyhistidine tag. This technique has been shown to be useful for following protein migration and trafficking and may be effective
156:
Polyhistidine tags most commonly consist of six histidine residues. Tags with up to twelve histidine residues or dual tags attached via short linker are not uncommon though and may improve purification results by enhancing binding to the affinity resin, allowing for increased stringency of washing
123:
Proteins can coordinate metal ions on their surface and it is possible to separate proteins using chromatography by making use of the difference in their affinity to metal ions. This is termed as immobilized metal ion affinity chromatography (IMAC), as originally introduced in 1975 under the name
231:
are most frequently used for this purpose. The choice of cation is generally a compromise between binding capacity and purity. Nickel is often used as it offers a good balance between these factors, while cobalt can be used when it is desired to increase the purity of purification as it has less
249:
For releasing the His-tagged protein from the carrier, a compound is used that has a structure similar to the His-tag and which also forms a coordination complex with the immobilized metal ions. Such a compound added to the His-tagged protein on the carrier competes with the protein for the
342:
assay. Polyhistidine tagging has several advantages over other tags commonly used for pull-down assays, including its small size, few naturally occurring proteins binding to the carrier matrices and the increased stability of the carrier matrix over monoclonal antibody matrices.
447:, and is more likely to be a soluble protein with no bias in charge distribution compared to the His tag. The arrangement of histidines in the HAT tag allows high accessibility compared to the His tag, and it binds efficiently to the immobilized metal ion.
330:. These impurities can be eliminated using additional purification steps or by expressing the recombinant protein in a deficient strain of cells. Alternatively, cobalt charged IMAC resins which have less affinity for endogenous proteins can be used.
152:) The polyhistidine tag is added using primers containing the tag coding sequence as an overhang on the forward primer. After PCR amplification, the tag is present on the N-terminus of the gene, which can then be sub-cloned into an expression vector.
165:
the gene of interest into a vector containing a polyhistidine tag sequence. Many vectors for use with various expression systems are available with polyhistidine tags in a variety of positions and with differing protease cleavage sites, other tags
240:
In order to elute His-tagged protein from the carrier there are several potential methods, which can be used in combination if necessary. In order to avoid denaturation of proteins, it is generally desirable to use as mild a method as possible.
434:
The HN tag has alternating histidine and asparagine (HNHNHNHNHNHN) and is more likely to be presented on the protein surface than
Histidine-only tags. The HN tag binds to the immobilized metal ion more efficiently than the His tag.
258:
When the pH decreases, the histidine residue is protonated and can no longer coordinate the metal tag, allowing the protein to be eluted. When nickel is used as the metal ion, it is eluted at around pH 4 and cobalt at around pH 6.
539:
to Roche. The original patent expired on 11 Feb 2003, and is now public property; current claims to royalties are based on a much narrower set of more recent patents. Suitable tag sequences are available free for commercial
91:
are used to remove N-terminal His-tags (e.g., Qiagen TAGZyme). Furthermore, exopeptidase cleavage may solve the unspecific cleavage observed when using endoprotease-based tag removal. Polyhistidine-tags are often used for
1473:
Chaga G, Bochkariov DE, Jokhadze GG, Hopp J, Nelson P (December 1999). "Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose".
78:
The total number of histidine residues may vary in the tag from as low as two, to as high as 10 or more His residues. N- or C-terminal His-tags may also be followed or preceded, respectively, by a suitable
380:
of the tag. This allows for the selective enrichment of polyfluorohistidine tagged peptides in the presence of complex mixtures of traditional polyhistidine tags by altering the pH of the wash buffers.
752:
Khan F, He M, Taussig MJ (May 2006). "Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on ni-nitrilotriacetic acid surfaces".
27:. The sample and subsequent buffers are manually poured into the column and collected at the bottom end after flowing through the resin bed (in light blue at the base of the column).
1457:, Tchaga GS, Jokhadze GG, "Polynucleotides encoding metal ion affinity peptides and related products", issued 13 February 2007, assigned to Takara Bio USA Inc.
310:
Affinity purification using a polyhistidine-tag usually results in relatively pure protein. Protein purity can be improved by the addition of a low (20-40 mM) concentration of
170:. However, if an appropriate vector is unavailable or the tag needs to be inserted at a location other than the proteins N- or C-terminus, the gene of interest can be either
491:
Hochuli E, Bannwarth W, Döbeli H, Gentz R, Stüber D (1988). "Genetic
Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent".
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is used in which histidine is replaced by 4-fluorohistidine. The fluorinated analog is incorporated into peptides via the relaxed substrate specificity of
314:
to the binding and/or wash buffers. However, depending on the requirements of the downstream application, further purification steps using methods such as
267:
When a strong chelating agent such as EDTA is added, the protein is detached from the carrier because the metal ion immobilized on the carrier is lost.
714:
Grisshammer R, Tucker J (October 1997). "Quantitative evaluation of neurotensin receptor purification by immobilized metal affinity chromatography".
207:
Various carrier matrices bound to a solid resin support are on the market and these can be subsequently charged with a metal cation. Derivatives of
148:) The polyhistidine tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. (
550:
Porath J, Carlsson J, Olsson I, Belfrage G (December 1975). "Metal chelate affinity chromatography, a new approach to protein fractionation".
223:
Several metal cations have high affinities for imidazole, the functional group of the His-tag. Divalent cation M (M = Mn, Fe, Co, Ni, Cu, Zi
339:
215:
are most frequently used for this purpose, with differing matrices having certain advantages and disadvantages for various applications.
1405:"Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair"
679:
Mohanty AK, Wiener MC (February 2004). "Membrane protein expression and production: effects of polyhistidine tag length and position".
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1019:"Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments"
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can be used to add the tag to a gene. A common approach is to add the coding sequence for the polyhistidine tag to the
322:
chromatography may be required. IMAC resins typically retain several prominent endogenous proteins as impurities. In
280:
Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in
1656:
157:
and separation from endogenous proteins. The tag can be added to a gene of interest using methods common to most
75:. Various purification kits for histidine-tagged proteins are commercially available from multiple companies.
471:
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gels with fluorescent probes bearing metal ions can be used for detection of a polyhistidine tagged protein.
319:
962:"Internal (His)₆-tagging delivers a fully functional hetero-oligomeric class II chaperonin in high yield"
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for instance, a prominent example is FKBP-type peptidyl prolyl isomerase, which appears around 25 kDa on
315:
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1208:"Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification"
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179:
175:
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862:
Applications of
Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification
860:
Bornhorst JA, Falke JJ (2000-01-01). "Purification of proteins using polyhistidine affinity tags".
19:
1356:"Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification"
1073:
Riguero V, Clifford R, Dawley M, Dickson M, Gastfriend B, Thompson C, et al. (October 2020).
792:"Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options"
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93:
24:
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Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a
535:
The use of the tag for academic users was unrestricted; however, commercial users had to pay
444:
369:
212:
1526:
1307:"Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions"
1075:"Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins"
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803:
559:
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and the resulting cell pellet lysed either by physical means or by means of detergents and
1263:, Methods in Molecular Biology, vol. 1615, New York, NY: Springer, pp. 247–255,
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The polyhistidine-tag can also be used for detecting a protein via anti-polyhistidine-tag
232:
affinity for endogenous proteins; binding capacity however is lower compared with nickel.
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Hengen P (July 1995). "Purification of His-Tag fusion proteins from
Escherichia coli".
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913:"Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology"
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55:) residues, often at the N- or C-terminus of the protein. It is also known as a
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Raducanu VS, Isaioglou I, Raducanu DV, Merzaban JS, Hamdan SM (August 2020).
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Zhao C, Hellman LM, Zhan X, Bowman WS, Whiteheart SW, Fried MG (April 2010).
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Paul DM, Beuron F, Sessions RB, Brancaccio A, Bigotti MG (February 2016).
864:. Methods in Enzymology. Vol. 326. Academic Press. pp. 245–254.
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601:
Porath J (August 1992). "Immobilized metal ion affinity chromatography".
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The HAT tag is a peptide tag (KDHLIHNVHKEEHAHAHNK) derived from chicken
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and other immuno-analytical methods. Alternatively, in-gel staining of
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Majorek KA, Kuhn ML, Chruszcz M, Anderson WF, Minor W (October 2014).
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Louche A, Salcedo SP, Bigot S (2017), Journet L, Cascales E (eds.),
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containing a polyhistidine tag sequence or various methods based on
71:, although the use of histidines and its vectors are distributed by
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or other expression systems. Typically, cells are harvested via
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Ring CM, Iqbal ES, Hacker DE, Hartman MC, Cropp TA (May 2017).
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The HQ tag has alternating histidine and glutamine (HQHQHQ).
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1515:(Protein Science Society Archive # 019/ Article in Japanese)
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1016:
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Bacterial
Protein Secretion Systems: Methods and Protocols
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637:"His-tag: The timeless standard for protein purification"
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that facilitates removal of the polyhistidine-tag using
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A polyfluorohistidine tag has been reported for use in
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1205:
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1206:
Andersen KR, Leksa NC, Schwartz TU (November 2013).
190:Most commonly, a polyhistidine tag is fused at the
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1157:Chen X, Nomani A, Patel N, Hatefi A (June 2017).
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142:Examples of methods for adding polyhistidine tags
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1257:"Protein–Protein Interactions: Pull-Down Assays"
661:: CS1 maint: bot: original URL status unknown (
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643:. Archived from the original on 2 December 2021
112:Three views of the X-ray structure of Ni(NTA)(H
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917:Cold Spring Harbor Perspectives in Biology
87:. This extra sequence is not necessary if
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911:Hughes RA, Ellington AD (January 2017).
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368:translation systems. In this system, an
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47:that typically consists of at least six
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35:, best known by the trademarked name
1360:Organic & Biomolecular Chemistry
229:transition metal imidazole complexes
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23:A simple gravity flow column for Ni-
1409:The Journal of Biological Chemistry
1163:Protein Expression and Purification
716:Protein Expression and Purification
681:Protein Expression and Purification
603:Protein Expression and Purification
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790:Singh MI, Jain V (2013-05-15).
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1124:Trends in Biochemical Sciences
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161:. The most basic method is to
1:
1488:10.1016/S0021-9673(99)01008-0
1136:10.1016/S0968-0004(00)89045-3
870:10.1016/s0076-6879(00)26058-8
477:
472:Glutathione S-transferase-tag
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132:
1728:Molecular biology techniques
1269:10.1007/978-1-4939-7033-9_20
1092:10.1016/j.chroma.2020.461505
817:10.1371/journal.pone.0063922
615:10.1016/1046-5928(92)90001-D
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7:
1476:Journal of Chromatography A
1079:Journal of Chromatography A
929:10.1101/cshperspect.a023812
641:Cube Biotech Knowledge Site
462:Maltose-binding protein-tag
450:
352:for measuring distance via
213:nitrilotriacetic acid (NTA)
16:Molecular biology technique
10:
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438:
393:, which can be useful for
67:. The tag was invented by
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1175:10.1016/j.pep.2017.03.012
693:10.1016/j.pep.2003.10.010
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376:and lowers the overall pK
1513:Ni - NTA affinity column
1422:10.1074/jbc.ra120.014132
1323:10.1016/j.ab.2009.12.028
395:subcellular localization
245:Competition with analogs
209:iminodiacetic acid (IDA)
182:primers as an overhang.
1311:Analytical Biochemistry
25:affinity chromatography
1604:10aa<length<50aa
728:10.1006/prep.1997.0766
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120:
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445:lactate dehydrogenase
374:histidine-tRNA ligase
370:expanded genetic code
263:Removal of metal ions
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94:affinity purification
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754:Analytical Chemistry
505:10.1038/nbt1188-1321
360:Fluorohistidine tags
276:Protein purification
172:directly synthesised
98:genetically modified
1415:(34): 12214–12223.
978:2016NatSR...620696P
808:2013PLoSO...863922S
564:1975Natur.258..598P
81:amino acid sequence
1372:10.1039/C7OB00844A
1224:10.1002/prot.24364
966:Scientific Reports
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57:hexa histidine-tag
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1733:Protein structure
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558:(5536): 598–599.
159:purification tags
128:Practical choices
33:polyhistidine-tag
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89:exopeptidases
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1584:(EQKLISEEDL)
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722:(1): 53–60.
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645:. Retrieved
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417:Similar tags
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316:ion exchange
309:
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77:
64:
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32:
30:
1572:(YPYDVPDYA)
1550:Protein tag
647:December 2,
457:Protein tag
411:native-PAGE
1712:Categories
1590:(WSHPQFEK)
1566:(DYKDDDDK)
1455:US 7176298
1292:2023-06-08
1085:: 461505.
478:References
391:antibodies
219:Metal ions
196:C-terminus
192:N-terminus
133:Tag length
100:proteins.
41:amino acid
1636:Isopeptag
1588:Strep-tag
1169:: 11–17.
1109:221373404
537:royalties
385:Detection
340:pull-down
312:imidazole
104:Principle
61:6xHis-tag
49:histidine
43:motif in
1682:CLIP-tag
1677:SNAP-tag
1657:BCCP-tag
1631:Spot-tag
1596:(CCPGCC)
1578:(HHHHHH)
1564:FLAG-tag
1496:10669292
1441:32647010
1390:28517015
1341:20036207
1287:28667618
1242:23852738
1212:Proteins
1193:28315745
1101:32861092
1053:25044180
1004:26856373
947:28049645
898:11036646
836:23691118
796:PLOS ONE
774:16642995
701:14711520
657:cite web
451:See also
407:SDS-PAGE
366:in vitro
328:SDS-PAGE
301:SDS-PAGE
296:lysozyme
294:such as
163:subclone
65:His6 tag
45:proteins
39:, is an
1697:TXN-tag
1692:MBP-tag
1687:HUH-tag
1672:HaloTag
1667:GFP-tag
1662:GST-tag
1626:SBP-tag
1611:iCapTag
1582:Myc-tag
1576:His-tag
1432:7443479
1381:6010304
1332:2832190
1233:4086167
1184:5479735
1144:7667882
1044:4286991
995:4746591
974:Bibcode
938:5204324
889:2909483
827:3655076
804:Bibcode
736:9325139
623:1422221
588:4271836
560:Bibcode
523:7229670
513:9518666
439:HAT tag
324:E. coli
292:enzymes
37:His-tag
1641:SpyTag
1616:NE-tag
1594:TC tag
1570:HA-tag
1494:
1461:
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552:Nature
520:
511:
467:SpyTag
430:HN tag
422:HQ tag
73:Qiagen
1621:S-tag
1105:S2CID
584:S2CID
517:INIST
509:S2CID
399:ELISA
69:Roche
63:, or
1492:PMID
1437:PMID
1386:PMID
1337:PMID
1283:PMID
1273:ISBN
1238:PMID
1189:PMID
1140:PMID
1097:PMID
1083:1629
1049:PMID
1000:PMID
943:PMID
894:PMID
874:ISBN
832:PMID
770:PMID
732:PMID
697:PMID
663:link
649:2021
619:PMID
580:1678
576:PMID
540:use.
303:and
211:and
1484:doi
1480:864
1427:PMC
1417:doi
1413:295
1376:PMC
1368:doi
1327:PMC
1319:doi
1315:399
1265:doi
1228:PMC
1220:doi
1179:PMC
1171:doi
1167:134
1132:doi
1087:doi
1039:PMC
1031:doi
990:PMC
982:doi
933:PMC
925:doi
884:PMC
866:doi
822:PMC
812:doi
762:doi
724:doi
689:doi
611:doi
568:doi
556:258
501:doi
409:or
318:or
225:etc
194:or
180:PCR
176:PCR
168:etc
144:. (
96:of
59:,
53:His
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