DNA hybridization · comparison of liquid
`and solid phase formats
`
`H. SODERLUND 1
`
`SUMMARY
`
`RESUME
`
`In nucleic acid hybridization an oligo- or polynucleotide probe Is allo·
`wed to anneal to its complementary strand which possibly is present in
`the sample. This offers an extremely specific way to Identify and quan·
`tlfy given genes and thus, for instance, given microbes. The annealing
`reaction is, however, slow since the reactants are present at very low
`concentrations and the diffusion rate of DNA Is slow. To overcome this
`problem high concentrations of probe are used in order to• drive• the
`reaction In a pseudo-first order fashion. As a result a positive hybridiza·
`tion is easily masked by the large excess of unreacted probe molecules,
`uneless a powerful fractionation system is used which removes the free
`probes. A frequently used method is to immobilize the nucleic acids of
`the sample on a solid support which then after the reaction is easy to
`wash. The solid support introduces, however, a diffusion barrier which
`signifanctly reduces the reaction rate. Thus kinetically solution phase
`reactions are preferrable to solid phase ones. In this communication a
`test format is described in which the advantages of both solid and solu·
`tion phase assays are combined. Two probes are used, one carrying a
`detectable lable (the detector probe) and the other an affinity moiety,
`e.g. biotin (the capture probe). After hybridization in solution a sand·
`wich hybrid is formed In which the target nucleic acid Is annealed to the
`two probes. By exposing the reaction mixture to an affinity matrix carr·
`ying the other moiety of the affinity pair, e.g. avidin, the capture probe
`becomes bound only when associated to the capture probe via the
`sandwich hybrid. Thus a quantitative measure of target Is obtained In a
`test format which is both easy to use and relatively fast. The affinity
`capture method Is also applicable to the quantification of PCR
`products.
`
`Hybrldation de l'ADN: comparaison des methodes en phase liquide et
`en phase solide. - Dans une reaction d'hybridation entre brins d'acide
`nucleique, les deux molecules s'assemblent l'une a l'autre en amor,;:ant
`une reaction d'appariement de bases tres rapide. La cinetique de reac(cid:173)
`tion est de ce fait d'ordre 2. Les concentrations des reactifs peuvent etre
`Ires faibles, de l'ordre de la femtomole, et conduisent a des reactions len·
`tes. Lors des experiences d'hybridation, ce probleme est resolu par !'ad(cid:173)
`dition d'un large exces de la sonde, avec dans ce cas une cinetique de
`pseudo ordre 1 et une vitesse de reaction augmentee. Se pose alors le
`probleme de la separation de l'exces de sonde de la sonde hybridee. La
`sonde libre, d'une concentration d'un million de fois superieure, devra
`etre separee de l'hybride a l'aide d'une technique biochimique. L'acide
`nucleique cible est le plus souvent immobilise sur un support solide
`avant hybridation, assurant ainsi une separation efficace mais cela
`entraine !'apparition de reactions non specifiques et la diminution de la
`vitesse d'hybridation due a la presence de barrieres de diffusion. Les
`auteurs ont combine la rapidite de reaction en solution avec la puissance
`de separation des techniques en pha,se solide. En ajoutant a la sonde de
`revelation marquee une deuxieme sonde dite de capture, un hybride
`sandwich est realise au cours de la reaction d'hybridation. Grace ii cette
`sonde de capture, les hybrides peuvent etre separes de la sonde libre
`avec l'efficacite requise. Le choix de la methode depend d'un certain
`nombre de facteurs tels que le but de !'analyse, la concentration de
`l'analyte, l'efficacite-cout, les limitations de temps et le marqueur utilise.
`Les possibilites d'automatisation sont importantes des qu'il est question
`de tests diagnostiques de routine.
`
`INDEX TERMS: Hybridization - Nucleic acid probes • Laboratory
`diagnosis.
`
`MOTS CLES: Hybrldation - Son de de capture • Phase solide.
`
`The possibility to identify and quantify nucleic acids,
`either DNA or RNA, in various biological samples is of
`great value both in basic and applied research. For
`this aim, the hybridization reaction, in which two
`complementary nucleic acids strands anneal to form
`a double-stranded structure, is used. This reaction is
`very specific, since the probability of random nucleo(cid:173)
`tide sequences to be complementary over signifi(cid:173)
`cant regions is practically non-existing. The hybridi(cid:173)
`zation reaction obeys second order reaction kine(cid:173)
`tics. In many cases the concentration of the nucleic
`acid to be analyzed, i.e. the target, is extremely low.
`Hence the reaction rate becomes slow. To overcome
`this problem a large excess of probe molecules is
`added to «drive» the reaction, in a pseudo-first
`order fashion [1]. The large excess of labelled probe
`in the assay introduces the problem of separating
`free probe from hybridized probe. In this communi(cid:173)
`cation methods by which hybridization assays can
`be made fast and convenient, without losing sensiti(cid:173)
`vity are discussed.
`
`1 Orion Corporation, Orion Pharmaceutica, Biotechnology, VALIMOTIE 7,
`00380 HELSINKI, Finland.
`Fleprints : H. Soderlund, address above.
`Conference presented at t/J0 First Congres on Advanced Concept in Bio·
`logy. Paris, December 5-8 1/J, 1989.
`
`Immobilized or soluble target ?
`
`In most applications of nucleic acid hybridization the
`target molecule is immobilized onto a solid support,
`such as a filter. The immobilized target is then allo(cid:173)
`wed to react with the labelled probe. The advantages
`of this approach are :
`-
`the two strands of a double stranded target can(cid:173)
`not reanneal and,
`- excess probe can easily be removed after the
`reaction.
`A major drawback is the immobilization step itself.
`When crude biological samples are contacted with
`the support, non-nucleic acid compounds are bound
`in addition to the target. This often causes back(cid:173)
`ground problems. Another limitation of this approach
`is that the reactions are carried out in a non-homo(cid:173)
`geneous medium, and consequently the hybridiza(cid:173)
`tion reaction is slowered due to accessibility pro(cid:173)
`blems for the reacting molecules [2].
`Both the primary collision between the two DNA
`strands and
`the subsequent winding process
`obviously proceed faster in solution.
`Thus it is preferable to perform the hybridization
`assay in solution. However, the labelled probe is
`
`Ann. Biol. clin., 1990, 48, 489-491
`
`Th is mate,ri.a I w.as co1pied
`at the N LIM a,nd ,may· :be
`!iluibject US Co,py·right Laws
`
`Enzo Exhibit 2013
`Hologic, Inc. v. Enzo Life Sciences, Inc.
`Case IPR2016-00820
`
`
`Exhibit 2013 Page 1
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`

`
`490
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`H. SODERLUND
`
`+
`
`i
`
`L
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`Capture
`
`Target
`
`Probe
`
`Hybrid
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`Affinity matrix Detectable complex
`
`Fig. 1. - The principle of the affinity based hybrid collection method [3].
`
`100
`
`80
`
`~
`Q
`
`~ 60
`i-)
`i;
`"'?
`
`40
`
`20
`
`z
`
`•
`
`Template
`bio-primer
`
`B1otar9et
`
`Hytirid
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`Affinity
`matrix
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`Measurable
`complex
`
`20
`
`Fig. 3. - The principle of the PCR product collection method [8].
`
`Time (h)
`
`Fig. 2. - Comparison of the reaction rate in sandwich hybridization
`when the capture probe Is immobilized on a filter (0), immobilized on
`microparticles (11), or immobilized after the hybridization reaction with
`the help of an affinity label (JI) [ 4 ].
`
`generally present in more than 1 000 fold excess
`over the target. The separation of such an excess of
`free probe from the hybrids is a difficult task.
`
`Affinity based hybrid collection
`
`To advantage of the fractionation power of assays
`with immobilized targets, but to retain the advanta(cid:173)
`ges of solution hybridization, we developed the affi(cid:173)
`nity based hybrid collection method (ABC) [3]. In this
`assay two non-overlapping probes are used to reco(cid:173)
`gnize the target nucleic acid. The use of two probes
`increases the specificity since two regions on the
`target must be recognized simultaneously in order to
`form the sandwich hybrid. The principle of the
`method is shown in Figure 1.
`One of the probes (the capturing probe) is chemi(cid:173)
`cally derivatized to contain a moiety with high affinity
`for another chemical group. We call these com(cid:173)
`pounds affinity pairs. A good pair is e.g. avidin and
`biotin. The other probe (the detector probe) is label(cid:173)
`led with a detectable moiety such as a radioisotope,
`a fluorochrome or an enzyme. The sandwich hybrid
`formed is isolated with the help of the affinity group
`on the capturing probe (Fig. 1 ). Th specificity of the
`affinity pair allows at least an 10.000 fold purification
`of hybrids from free detector probe. This is a prequi(cid:173)
`site for the detection of attomole levels of target.
`The kinetic advantage of the hybridization in solution
`is demonstrated in Figure 2. Here the reaction rate
`using affinity labelled capture DNA is compared to
`that of preimmobilized capture. The data is from
`reference 4.
`
`Ann. Biol. clin., 1990, 48, 489-491
`
`The ABC principle is used in the AffiProbe HPV iden(cid:173)
`tification kit (Orion Biotechnology) for the detection
`and typing of human pa~illomavirus. In this test bio(cid:173)
`tinylated capturing and 5S labelled detector probes
`are allowed to hybridize to DNA in a crude cell lysate
`for 3 hours. The hybrids are then captured in microti(cid:173)
`ter wells coated with streptavidin.
`
`The test quantifies the amount of target in the
`sample over a range from 5 x 105 - 5 x 108 (1 atto(cid:173)
`mole - 1 femtomole) in a one day assay [5].
`
`Even if attomole sensitivity is useful in HPV typing, it
`is not sufficient for all applications. So far the most
`frequently used non-radioactive labels do not allow a
`significant improvement in the sensitivity of detec(cid:173)
`tion compared to radioisotopes.
`
`Quantification of PCR-products
`
`A promising approach to solve the sensivity problem
`in hybridization assays is to increase the number of
`target molecules in vitro before the detection. This
`can be done using the polymerase chain reaction
`(PCR) [6]. By 20 cycles of PCR 1.000 molecules of
`target DNA can be amplified to about 108 molecules.
`A number of available detection methods [7] can
`consequently be used with a very high sensitivity in
`regard to the original amount of target. However, it
`should be noted that PCR frequently amplifies un(cid:173)
`specific DNA fragments. To reliably identify and
`quantify the specific PCR product, it should be
`detected by hybridization with a probe recognizing
`the nucleotide sequence internal to the two primers.
`In quantifying the PCR product the method of choice
`depends partly on the same factors as those presen(cid:173)
`ted above for direct detection of polynucleotide
`sequences.
`
`This material \tcas,copied
`at the NLM and may be
`Scu bject US OJµyright Laws
`
`
`Exhibit 2013 Page 2
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`DNA HYBRIDIZATION
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`491
`
`We have modified the principle of affinity collection
`for use in PCR as shown in Figure 3. Here the affinity
`moiety is attached to the 5' end of the PCR primer.
`Thus all molecules synthetized during the PCR pro(cid:173)
`cess will carry e.g. a biotin residue. Using an oligonu-
`
`cleotide probe, hybridization in solution, and affinity
`capture of the formed hybrids, the PCR product is
`conveniently and accurately quantified in less than 1
`hour [8].
`
`REFERENCES
`
`1. Young B.D., Anderson M.L.M. - Quantitative analysis of solution
`hybridization. In : Nucleic acid hybridization, a practical appraoch
`(IRL Press, 1985), 4 7.
`
`2. Flavell R., Birfelder E., Sanders P., Borst P. - DNA-DNA hybridization
`on nitrocellulose filters. 1. General Considerations on non-ideal
`kinetics. Eur. J. Biochem., 1974, 47, 535.
`
`3. Syvanen A.C., Laaksonen M., Soderlund H. - Fast quantification of
`nucleic acid hybrids by affinity hybrid collection. Nucleic Acids Res.,
`1986, 14, 5037.
`
`4. Jungell-Nortamo A .. Syvanen A.C., Luoma P., Soderlund H. - Nucleic
`acid sandwich hybridization: enhanced reaction rate with magnetic
`microparticles as carriers. Mo/. Ce//. Probes, 1988, 2, 281.
`
`5. Ranki M., Jalava T., Raussi J., Soderlund H., Nieminen P., Paavonen J.,
`Kallio A. - Affiprobe HPV test kit in the detection of human papillo(cid:173)
`mavirus DNA from cervical scrapes. In: Howley P., Broker J., Papillo(cid:173)
`maviruses; UCLA Symposia on Molecular and Cellular Biology (Alan
`R. Liss Inc.), 1989.
`6. Mullis K., Faloona F.A. - Specific synthesis of DNA in vitro via a poly(cid:173)
`merase-catalyzed chain reaction. In : Wu R., Grossman L., Moldave
`K., Methods Enzymol., 1987, 155, 335 (Academic Press).
`7. Lebacq P. - Non isotopic probe labelling : advantages and draw(cid:173)
`backs for diagnostic specifications. Ann. Biol. clin., 1990, 47, 502-
`504.
`8. Syvanen A.C., Bengtstrom M., Tenhunen J., Soderlund H. - Quantifi(cid:173)
`cation of polymerase chain reaction products by affinity-based
`hybrid collection. Nucleic Acids Res., 1988, 16, 11327.
`
`This mat:e4"ia I was copied
`at the. N,LM and may be
`!liubject US Copyright Laws
`
`Ann. Biol. clin., 1990, 48, 489-491
`
`
`Exhibit 2013 Page 3