The magic and challenge of DNA probes as diagnostic reagents

Veterinary Microbiology, 24 (1990) 217-233 Elsevier Science Publishers B.V., Amsterdam

217

The magic and challenge of DNA probes as diagnostic reagents David GiUespie Hahnemann University, Department of Neoplastic Diseases, Broad and Vine, Philadelphia, PA 19102, U.S.A.

ABSTRACT Gillespie, D., 1990. The magic and challenge of DNA probes as diagnostic reagents. Vet. Microbiol., 24:217-233. Molecular basis of hybridization and effect of different conditions, such as probe size, salt concentration, temperature, detergent concentration, time of washing, sa~aple preparation and sample purtity, on hybridization is discussed. Different formats of hybridization such as in solution hybridization, solid phase hybridization, and methods for probe preparation are presented. Polymerase chain reaction for the amplification of target and alternative methods for signal amplification are introduced in this review.

INTRODUCTION

An overvieuw on nucleic acid probes is presented as it might be used in clinical veterinary medicine. A particular emphasis is placed on the different kinds of molecular hybridization processes that have been developed to answer different kinds of questions. I will describe how conditions are decided for molecular hybridization. There are many different formats for molecular hybridization and there are many different sets of conditions which can be used. The message of this review is that given a particular question, the format and the conditions follow directly from the kind of question being asked. This is referred to here as the magic and challenge of nucleic acid probes, because the process of molecular hybridization as the reaction of a probe with an intended target can be so rapid, specific and stable that it seems sometimes to be inexplicable by mere science. Imagine that a single RNA or DNA molecule of a specific sequence can be detected in a sample and distinguished from all of 10~°-1012 other molecules in the sample, even from those which differ by only a single mutation. And yet, despite the power of molecular hybridization, the application to clinical medicine has been minimal because of the challenges faced in reducing exotic technology to routine, high throughput practice. 0378-1135/90/$03.50

© 1990 - - Elsevier Science Publishers B.V.

218

D. G1LLESPIE

The term "target" describes nucleic acids contained within a specimen, nucleic acids which stably interact with a probe, as opposed to the myriad of other nucleic acids which also are contained in that specimen but which do not stably interact with the probe. The term "intended target" defines the specific nucleic acid sequence in the specimen with which the probe was designed to interact. The two terms are not always equivalent. The word "probe" describes labeled nucleic acids that are prepared either synthetically, enzymatically or chemically, whose sequence is known or whose sequence properties are known and whose sequence properties force it to hybridize more or less specifically with the intended target, specificity depending on a variety of definable conditions. One kind of probe carries as a distinguishing feature, a label of some kind, either a radioactive atom, an adduct which can attract an enzyme, an enzyme itself, etc. I will call this kind of probe a "label-probe." Other kinds of probes exist, and some probes carry affinity ligands. These probes, when hybridized with intended targets, provide reactive groups on the hybrid which allow the hybrid to be purified through attachment to a solid support which binds the affinity ligand, are referred as "capture-probes". MOLECULAR HYBRIDIZATION

The process of nucleic acid probing, molecular hybridization, involves the interaction between a molecule of label-probe and a molecule of intended target to form a molecular hybrid, a duplex, ideally to the exclusion of any interaction between the label-probe and any other molecule in the specimen. A typical probing mixture consists of 109 to 1012 molecules of probe and about the same number of nucleic acid molecules from the specimen. But with regard to the intended target, there can be as few as one or as many as a 110 X 10 6 molecules. So the range of target molecules that needs to be detected within the specimen is great, whereas the number of probe molecules added to a hybridization reaction is always very large. The magic is that label-probe can hybridize with this intended target even if there is only one molecule present, and this hybridization can occur to the exclusion of stable interaction with any other nucleic acid molecule in the specimen. That sort of specificity can be obtained by adjusting conditions and by adjusting the nature of the probe as long as there is at least one nucleotide in the sequence of the intended target which distinguishes it from the sequence of every other nucleic acid molecule in the specimen. The reason that a probe can hybridize extraordinarily and specifically with a target has to do with the fact that the chemistry of the interaction is relatively simple, relative, for example, to an antibody-antigen interaction. Secondly, and again as opposed to an antibody-antigen interaction, the forces that maintain the hybrid of probe and target are extraordinarily stable to purification. A nucleic acid hybrid is about l 0 9 times more stable than an anti-

219

DNA PROBES AS DIAGNOSTIC REAGENTS

body-antigen complex. It is possible with DNA probes to detect a single intended target molecule because the probe-target hybrid can be rigorously purified and will not fall apart. The association of two strands of nucleic acid uses the specific base pairing rule of Watson and Crick, where adenine pairs with thymidine and guanidine pairs with cytidine. The requirement for Watson-Crick base pairing provides a uniqueness in the specificity of the interaction between the probe and the target. A given probe sequence 20 nucleotides in length will be represented once, on the average, in a random-sequence nucleic acid one trillion nucleotides long. Put another way, if a cell contains a billion nucleotides worth of genetic information, there is only a one in one thousand chance that the probe sequence will be found by chance in that cell's genome. A typical m a m m a l i a n cell contains around one billion nucleotides in its genome. Therefore, when a 20 nucleotide long probe complementary to a known genome sequence is used and if all 20 nucleotides are required for hybridization, it is likely that that sequence will only be represented by the intended target. I emphasize the word likely. This is strictly a probability assessment. There is a chance that an unintended target could carry the same sequence by chance or by evolutionary design. Accepting the fact that a probe sequence can be unique, will the probe uniquely interact with the intended target to the exclusion of all other targets? To approach this we need to understand the mechanism of nucleic acid interactions. The interaction between a probe and a target involves three steps as illustrated in Fig. 1. First it involves a large number of more or less random collisions between nucleic acid molecules. Most of those are non-productive, MOLECULAR HYBRIDIZATION(G:C, A:T) XGAATTC XXXXXXAAGCTTXXX XCTTAAGXXXXXXTTCGAAXXX

~,Gf

I

r

I

**

j

AGCTTXXXxxxGAATTCX 2

~

-xG I ~

°o,,

(denatured ONA)

XAAGCTTXX TTCGAAXXx Xx X XXx "~ CTTAAGX

nucleation complex

Fig. 1. T h e interaction between p r o b e a n d target.

helix (native

DNA)

220

D. GILLESPIE

that is, they are interactions between regions of nucleic acid molecules that have less than about five complementary nucleotides. These interactions are either not stable or are freely reversible; unless stabilized, they are non-productive and do not lead to the formation of a hybrid. The second step is the productive collision between a short region of the probe with a complementary region of the target, forming what Wetmur and Davidson ( 1968 ) defined as the nucleation complex. The nucleation complex is a small duplex region about six nucleotides long which causes the quasi-stable formation of a complex having a half-life long enough so that other parts of the probe and target that are complementary to each other now zip up rapidly. The nucleation complex does not involve a length which provides sequence uniqueness. The vast majority of these quasi-stable interactions will occur with unintended targets. If there is no complementary flanking region the nucleation complex will dissociate and the probe will collide with other targets until it collides with the intended target and the zipping up process takes place. That may sound as if the formation of a stable hybrid would take an agonizingly long time. The fact is that even with the large number of specimen nucleic acid molecules that have to be "scanned", the molecular hybridization process in practice can be driven to completion on the order of 10 minutes or so, regardless of how few intended targets exist. To do that, it is necessary to use an excess of probe. And as I will describe later, that presents some problems in purification of the hybrid. But if a large amount of probe is used, two advantages are gained. First, the hybridization reaction will be "driven" by the probe so the reaction will go very quickly. And secondly, all of the available target molecules will become hybridized with probe, a condition known as "saturation" of target. That means that the amount of hybrid formed reflects exactly the amount of target present and provides a way to quantitate the number of targets present. I will be discussing two different uses of molecular hybridization. One is detection of a sequence and other is its quantitation, the measurement of the number of intended targets within a specimen. The most specific probes are short oligonucleotide DNA probes. Oligonucleotide probes are synthesized chemically in a totally automated format. Generally such probes have ends which can be labeled. For a radioactively labeled probe which is relatively stable, a single radioactive phosphate can be added to the 5' end of the oligonucleotide with gamma 32p-ATP and polynucleotide kinase. For oligonucleotide probes of higher specific activity, several radioactive nucleotides can be added to the 3' end with terminal transferase, forming a radioactive "tail". However, tails can participate in hybridization reactions of their own, decreasing specificity. Also, high specific activity tails made with 32p cleave themselves with every radioactive decay, reducing stability during storage. Oligonucleotide probes labeled at the 5' end yield probe-target hybrids of relatively low radioactivity. Long DNA or RNA probes which are usually 1000

DNA PROBES AS DIAGNOSTIC REAGENTS

221

to 2000 nucleotides long can have a radioactive atom every 40 or 50 nucleotides. When they are hybridized with a target, the hybrid may have 100 radioactive atoms per target. However, the specificity will be lower because a 1000 nucleotide probe will have 980 different sequences each 20 nucleotides long and the r a n d o m chance of finding a target complementary to one of these 980 sequences in a typical m a m m a l i a n cell is about 1 in 2. More importantly, the chance that related sequences exist by evolutionary design is greater. The trade between sensitivity and specificity is an essential part of the design of any DNA probing experiment. If the specimen contains more than one million target molecules per assay volume either oligonucleotides or long probes can provide the required sensitivity and the major issue to be addressed is specificity, but if the specimen contains fewer than 100 000 targets, longer probes are necessary or an "amplification" step is required. Oligonucleotides or long probes can also be labeled nonradioactively, with affinity ligands which bind enzymes or with enzymes themselves. While nonradioactive probes having the same sensitivity as 32p have been reported, they are experimental and not for routine use. Nonradioactive probes should be viewed as providing only one-tenth the signal of 32p probes. The stability of a hybrid between various targets and a given probe varies with the degree of "mismatching" in the hybrids; unpaired bases and "non Watson-Crick" base pairs. Furthermore, any environmental condition that destabilizes hydrogen bonds increases the specificity or "stringency" of the molecular hybridization reaction. For example, a mismatched hybrid may be stable at low temperatures, because a smaller number of hydrogen bonds per base are required to stabilize the hybrid. As the temperature is raised and more stress is put on the hybrid, more hydrogen bonds per base are required to stabilize the structure. For example, at 65 °C a hybrid will denature even though there is only a single mismatch, whereas the perfectly matched hybrid will be stable at 70 ° C. Experimentally, it has been shown that oligonucleotide probes can distinguish between a wild type sequence and a mutant sequence where the m u t a n t is a single nucleotide substitution (Wallace et al., 1981 ). A new strategy has been developed which improves this even further and allows one to use longer probes with nearly absolute specificity (Wallace et al., 1981 ). If, in addition to the label-probe, a second unlabeled "competitor" probe is added, which has a sequence exactly complementary to the mutant sequence, it will tend ot hybridize preferentially to the mutant sequence and not to the wild-type sequence. It will exclude interaction between label-probe and the m u t a n t target. Although label-probe might tend to interact with the mutant sequence, the presence of competitor oligonucleotide will prevent that interaction. The discrimination between a wild type sequence and a mutant sequence becomes greater. The competition technique also allows the use of longer probes of at least 25 to 30 nucleotides. Other conditions influence stringency. For example, low salt concentration

222

D. GILLESPIE

or the addition of hydrogen bond breakers (e.g. formamide or chaotropic salts) increase stringency. For very stringent condition, it is theoretically best to work at fairly low salt concentrations. However, high salt concentrations speed molecular hybridization, so there is a trade in deciding between conditions of specificity and rate of hybridization. The range of salts that are usually used for molecular hybridization are between 0.1 and 1.0 M. For accelerated hybridization that cannot be driven with an excess of probe, even higher salt concentrations can be used. Then the specificity will have to be adjusted by increasing temperature or including hydrogen bond breakers. The disadvantage of working at a high temperature is the instability of nucleic acids during hybridization. Accelerated hybridizations of high stringency at low temperatures can be performed with high salt and hydrogen bond breakers. This is one reason for formamide-based nucleic acid hybridization (Bonner et al., 1967; Gillespie and Gillespie, 1971 ) and newer techniques based on guanidine thiocyanate, GuSCN (Thompson and Gillespie, 1987 ). PURIFICATION OF HYBRIDS WITH IMMOBILIZED TARGETS

One challenge of nucleic acid probing is the purification of probe-target hybrids. The primary object of hybrid purification is to purify that hybrid away from unreacted label-probe. A molecular hybridization signal is probe hybridized with intended target, identified by the label on the probe. Unhybridized label-probe represents noise. There is always unhybridized labelprobe that passes through any hybrid purification scheme. The interpretation of any hybridization experiment must include knowledge about the magnitude of signal versus noise. The rest of this discussion will focus on methods for purifying molecular hybrids. I will introduce methods which are used in research, then move to methods which are more appropriate for clinical diagnostics. The first and still most popular procedure for purifying a probetarget hybrid is to first immobilize the target on a solid support, then to immerse the solid support containing target molecules in a solution containing probe. Enough time is allowed for probe to react specifically with intended target, then the solid support is removed and washed. If everything works perfectly, the only label on the solid support is from probe hybridized with the intended target. However, there is always a noise from label-probe which remains attached to the solid support through direct interaction with the support or through molecules other than the intended target. The usual strategy for reducing this noise is to change the washing conditions by changing salt concentration, detergent concentration, temperature, time of washing, etc. That strategy has led to conditions which yield lower and lower noise, but no such strategy has eliminated noise. The conventional label-probe for hybridization with immobilized targets is a long DNA probe labeled with 32p by the nick the nick translation procedure

DNA PROBES AS DIAGNOSTIC REAGENTS

223

(Rigby et al., 1977; Maniatis et al., 1982). DNA, usually a cloned sequence, is incubated with deoxynucleotide triphosphates, DNA dependent DNA polymerase and low concentrations of DNase, available in kit form. One or more deoxynucleoside triphosphates are labeled with 32p in the alpha phosphate. DNase produces single-strand "nicks" at random places in the template DNA, providing starting points for DNA synthesis. DNA products usually consist of short, labeled chains (approximately 500 nucleotides long), which are double-stranded probes about 108 to 109 cpm/ug. Such probes need to be denatured before use. They will reanneal with themselves during hybridization, a process which tends to reduce signal. On the other hand, since the template is often a circular DNA and since DNase produces multiple, randomly placed starting points, reannealing can form "networks" which tend to increase signal. There are several versions of hybridizing with an immobilized target. One is simply to "dot" a pure nucleic acid on a membrane (Gillespie and Spiegelman, 1965; Kafatos et al., 1979; Meinkoth and Wahl, 1984). Denatured DNA will bind to nitrocellulose or nylon membrane in the presence of moderate concentrations of NaC 1, to some nylons in the presence of NaOH and to nitrocellulose in the presence of sodium iodide. Denatured RNA will bind to nitrocellulose or nylon in the presence of high salt concentrations. Commercial vacuum manifolds permit dotting multiple samples. Immobilized targets need to be "fixed" onto some membranes by heating. The fixed target is then hybridized with probes dissolved in solution. If probe is used in weight excess over complementary target sequences, all available target molecules will be saturated with probe and the amount of probe hybridized will be directly proportional to the amount of target present. However, such techniques are not quantitative because not all target molecules are purified, because not all purified target molecules are irreversibly immobilized, especially with short target fragments, and because not all immobilized target molecules are available for hybridization with probe. These problems are not adequately addressed with reference probes or targets. Therefore, the result indicates relative amounts of target when comparing two or more specimens. Furthermore, probe will hybridize with unintended targets and will interact directly with the solid support, creating a "noise" level which is difficult to correct for. Consequently, dot hybridization techniques are usually used for identification and detection of target nucleic acids and for relative quantitation. It is also possible to immobilize nucleic acids directly from crude cell lysate (White and Bancroft, 1982; Gillespie and Bresser, 1983 ). Specimens are solubilized with NaOH, detergents plus enzymes or chaotropic salts and filtered through nitrocellulose membranes. Nucleic acids bind the membrane, while most of cell debris do not. The efficiency of immobilization of targets from crude lysates is lower than with pure DNA or RNA but losses from extractions are not experienced. Starting from crude lysate, loss of sensitivity during

224

D. GILLESPIE

immobilization and hybridization takes place for two reasons. One is the immobilization of other molecules including proteins. They can bind to target, interfering with immobilization and hybridization. Furthermore, they can stick to the membrane and to probes and increase noise. While target immobilizations from lysates were popular and are still used to some extent, in general most people shy away from t h e m because there are too many ways to interpret a positive result. A second way of performing immobilized target hybridization is Southern blot hybridization (Southern, 1975 ). This procedure involves transfer of nucleic acids to membranes after they were fractionated by electrophoresis in agarose. This technique is commonly used in research and is being used in a limited way in clinical diagnostics. Subsequently, Thomas (1980) described transfer of RNA to membranes from agarose gels. This procedure is referred to as Northern blot hybridization. For these procedures, nucleic acids from specimen are purified, DNA is cleaved with a restriction endonuclease, introduced into a slot in an agarose gel and subjected to electrophoresis to fractionate the nucleic acids according to size. Nucleic acids in the gel are denatured. A membrane is then laid over the gel and the liquid is sucked from the agarose gel through the membrane. Most of the nucleic acid in the gel is sucked along with the liquid out of the gel and sticks to the membrane forming an image of nucleic acids on the membrane in the same orientation they assumed in the gel. This collection of immobilized nucleic acids includes intended targets as well as other nucleic acids. After hybridization with probe, the position of hybrids on the membrane can be determined by autoradiography. As with dot hybridization, in addition to signal from hybridization with the intended target, there will be noise from hybridization of probe with other target and from probe sticking directly to the membrane. However, unlike dot hybridization, there is an added piece of information in a Southern transfer, which is the position of the nucleic acid on the membrane. A hybridization signal should occur at a predictable position in the gel. If the target were RNA, that position would be predictable because the size of the intended target would be known. If the target were DNA, the size of fragments with a particular restriction endonuclease would be known. The advantage of this technique over dot hybridization is that signal from hybridization with the intended target is localized while noise is spread more or less uniformly across the filter. This signal localization is an extra piece of information that is obtained from a Southern transfer that is not obtained from a dot hybridization. Results are less ambiguous and more informative than dot hybridization, since information relating to the size as well as the presence and amount of a target is produced. The last version of hybridization with immobilized targets is "in situ" hybridization (Gall and Pardue, 1969; Harper and Saunders, 1981; Brahic et al., 1984; Lawerence and Singer, 1985). In this procedure, cells are immobi-

DNA PROBES AS DIAGNOSTIC REAGENTS

225

lized on a microscope slide and targets are immobilized within the cell. The principle of this technique is that instead of purifying nucleic acids to study, one takes a specimen which consists of individual cells or preferably sections from tissue which have recognizable, morphological landmarks, and deposit those cells or tissue sections on a microscope slide. The microscope slide is the solid support in this method. The immobilized section is placed in a solution containing probe, the probe is allowed to enter the cells in the section and hybridize with intended targets and then the unreacted probe is washed away. The incredible feature is that these sections are amenable to molecular hybridization in the sense that the probe can get in and in the sense that the target does not come out dissolved in solution. The target can be either DNA or RNA. In a section containing morphologically identifiable cells, some cells stand out by showing label from probe hybridized with cellular targets. The labeled cells have a certain morphology. Whether the label is over nucleus or cytoplasm can be learned. Like dot hybridizations, there is no structural information obtained with regard to the target and there is no good way to be convinced that the apparent signal is a hybridization reaction as opposed to label sticking to cells, except knowledge of the behavior of these targets, knowledge that the intended targets should reside should reside in certain cells and in certain subcellular locations. In situ hybridization has two general uses in clinical diagnosis. In situ hybridization can be used as a cytogenetic tool to determine the chromosomal location of single copy genes in metaphase and prophase spreads. Chromosomal abnormalities such as translocations, amplifications and even gene deletions can be detected. Second, in situ hybridization can be used as a tool in clinical pathology to detect virus-infected cells or cells producing a specific mRNA. Unlike other DNA probe techniques which rely on immobilized targets, in situ hybridization is not likely to be replaced with simpler methods and will probably find a permanent and growing niche in clinical pathology. On the other hand, the other formats of immobilized target hybridization will be relegated to research and are inappropriate for clinical diagnosis because sample preparation is too difficult, hybridization takes too long, and sensitivity is inadequate. The d e m a n d for sensitivity varies from one clinical application to another. But on the average we need to be thinking in the range of detecting about 1000 target molecules within a specimen. Clearly there are going to be situations where only one intended target is present in a specimen and there are situations where 1 000 000 targets are present, but I think the reasonable range to prepare for is around 1000 target molecules per assay. The techniques described so far, with the possible exception of in situ hybridization, lack that sensitivity.

226

D. GILLESPIE

SAMPLE PREPARATION AND SOLUTION HYBRIDIZATION

One of the requirements for clinical diagnostics will be to perform hybridization in solution without purifying or immobilizing target nucleic acids. A major difficulty is to put the specimen in a state where the target nucleic acids are amenable to probing without having to purify nucleic acids. One approach is to utilize the combined action of detergents and enzymes and high salt concentrations. Proteolytic enzymes destroy proteins and detergents disrupt weak forces and high salt concentrations break electrostatic interactions. That logic is being used successfully to measure bacteria and mycoplasma. It remains to be seen how widely useful that approach is, because the hybridization conditions are not ideal and because of significant complex formation of lysate and target. An alternative approach is the use of chaotropic salts to disrupt a specimen and present nucleic acids in a state where they can be probed directly in the same solution containing the chaotropic salt. Chaotropic salts were defined initially by Hamaguchi and Geiduschek (1962) as salts which destabilize DNA. Chaotropic ions include the iodide, thiocyanate, perchlorate and trichloracetate anions and probably the guanidinium cation. At high concentrations, chaotropic salts are thought to work by disrupting the water lattice in a solution and thereby destabilizing bonds which depend on water, such as nonpolar bonds. At high concentration, chaotropic salts also compete with electrostatic bonds. Chaotropic salts do not disrupt covalent or disulfide bonds. Chaotropic salts have become popular during the last 10 years in membrane biochemistry for purification of membrane components that cannot be solubilized any other way. Chaotropic salts cause the disruption of cells and subcellular organelles basically by solubilizing the individual molecules of cellular structures; for example, they free RNA and DNA from proteins that they might have been associated with in the cell. Nucleic acids are presented in a way that is suitable for efficient molecular hybridization. One chaotropic salt, guanidine thiocyanate (GuSCN), additionally promotes molecular hybridization and accelerates it some 100-fold (Thompson and Gillespie, 1987). In 2-5 M GuSCN molecular hybridization occurs at temperatures ranging from room temperature to 37 ° C. GuSCN has other advantages; it denatures proteins by eliminating weak forces that are required for activity, it inactivates nucleases, so target nucleic acids are stable during the hybridization procedure, and it also inactivates microorganisms, providing a level of safety. All that is required for this format is to form a fine suspension of the specimen and then dissolve the suspension in about 5 M GuSCN/0.1 M EDTA, pH 7. Then for molecular hybridization, one simply needs to add probe, incubate for minutes or hours, depending on the concentration of probe and measure the amount of probe which has been hybridized to target. The principle to be emphasized with regard to clinical diagnostics

DNA PROBES AS DIAGNOSTICREAGENTS

227

is that the hybridization step will take place in the solution. This not only increases the speed and simplicity of sample preparation and molecular hybridization, but in the longer run it will be much easier to automate than a system depending on the hybridization with a target fixed to a solid support.

P U R I F I C A T I O N O F H Y B R I D S BY A F F I N I T Y C A P T U R E

Simple methods for sample preparation and molecular hybridization exist. The problem at this point is to purify the probe-target hybrids which are formed. This can be accomplished by affinity capture or sandwich hybridization (Virtanen et al., 1983 ). The principle is to use a solid support with an attached affinity ligand as a matrix for purifying probe-target hybrid. In one version, this ligand is an oligonucleotide which is complementary to the target at a site near or adjacent to the site which hybridizes with the label-probe. The ligand acts as a hook to purify target, and in so doing purifies label-probe which is hybridized with target. The disadvantage of this particular approach comes from affinity ligand which has leached from the solid support and has hybridized with target. That target is no longer capturable because the oligonucleotide site has been occupied by an oligonucleotide that has leached off the support. So the efficiency of capture in this version depends critically upon the stability of the ligand-support bonds. A second disadvantage is label-probe which will attach to solid support and gives noise. And finally, the signal from hybridized label-probe is limited by the amount of target. The solution for the leaching problem is the use of second probe to hybridize with target. What is used here are two probes, one probe carries a label and the second probe, which is used to purify the hybrid, is called a captureprobe. The capture-probe is bifunctional. It contains a region which will hybridize with the intended target and it also contains an adduct which can be captured onto the solid support by recognizing the affinity ligand. Essentially, all capture-probe molecules are captured by the solid support. Some of those capture-probes are also hybridized with target carrying label-probe. Again, some of the affinity ligand on the support can leach out. But chances are very high that affinity ligand that leaks off the solid support will hybridize with a free capture-probe, rather than one which has hybridized with target, since probes are present in vast excess over target. This capture-probe version does not suffer from the efficiency disadvantage of the first affinity capture system. There still is a noise component from label-probe which interacts directly with the support and there still is a signal limitation because the amount of label that is limited by target.

228

D. GILLESPIE

The solution to the noise problem is the following. Simply use a captureprobe which forms a reversible link with the affinity ligand on the membrane (Thompson et al., 1989 ). Hybrids formed in solution are captured, then capture-probes and hybrids are released into solution. Some of the noise remains attached to the membrane, some of the noise is released along with the hybrid. Then this mixture is recaptured on fresh solid support. The same or lower percentage of unreacted label-probe binds to the solid support and that means that with every cycle of capture and release, noise is reduced by some factor which is characteristic of the support. Noise can be virtually eliminated by adjusting the number of cycles of capture and release. Since the capture-probe can be an oligonucleotide, the specificity of hybridization can be controlled by defining the structure of the oligonucleotide capture-probe. It does not matter whether the label-probe is specific, because in order to get a signal, label-probe and capture-probe must both hybridize with the target. This version of solution hybridization has the high specificity of oligonucleotides as well as the relatively high signal of long label-probes. Preferred label-probes are RNA probes, also called a "riboprobes". They are synthesized enzymatically, beginning with basically the same sort of recombinant DNA that was used to generate a nick translation probe, except that in addition to an inserted sequence, which is the probe sequence, at one or both ends of the insert there are promoter sequences which recognize DNA dependant RNA polymerase and cause the initiation of transcription at that site in a particular direction (Green et al., 1983). RNA copies made by in vitro transcription have a defined starting point. If template DNA is cleaved with a restriction endonuclease in a strategic position, RNA transcripts can also have a defined endpoint. Finally, since RNA transcription is asymmetric, RNA probes are single stranded and cannot reanneal with themselves during hybridization in solution. Hybridization can be scored by meauring resistance of the label-probe-target hybrids to RNase. TARGET AMPLIFICATION

The sensitivity of solution hybridization formats is in the tens or hundreds of thousands of target molecules. One reversible affinity capture system was reported to measure 50 000 targets with a signal to noise ratio of 5 and to detect as few as 15 000 targets (Thompson el al., 1989 ). Affinity capture techniques which are not reversible ordinarily have detection limits in the hundreds of thousands of target molecules. This presents a serious challenge for using DNA probes in clinical situation, because earlier we established our needed sensitivity at around 1000 target molecules. The only known solutions to this dilemma is to amplify assay signal by repetitively copying intended targets or repetitively copying hybridized label-probe. Other theoretical so-

DNA PROBES AS DIAGNOSTIC REAGENTS

229

lutions exist which have not worked in practice. The search for a "superlabel" which would give sensitivity greater than 32p is so far a failure. Several fluorescent and luminescent probes have been reported to have a greater sensitivity than 32p, but these isolated reports have never translated into general use. Another theoretical approach is to form probe "networks" such that several label-probes become associated with each other, then with target (Urdea et al., 1987). Since the number of probes per target is increased, signal increases. In practice, while probe networking probably explains the exquisite and unexpected sensitivity of in situ hybridization (Harper and Saunders, 1981 ), virtually all other attempts to use probe networking to increase signal and not increase noise have failed. DNA target amplification (Saiki et al., 1988 ) is the repetitive copying of a 200 to 500 nucleotide region of an intended target by thermostable, DNA dependent DNA polymerase. DNA target amplification has been called polymerase chain reaction (PCR), because the number of copies of target increases exponentially as target copies serve as templates for further copying. The PCR mixture includes targets, primers, deoxynucleoside triphosphates and polymerase. Technically the following steps are involved: cycle 1 (A) heat-denature DNA at 95°C; (B) cool to 55°C to anneal primers for DNA synthesis; (C) heat to 70°C for DNA synthesis; cycle 2, (A) heat-denature DNA at 95 ° C; (B) cool to 55 ° C to anneal primers to target; (C) heat to 70 ° C for DNA synthesis. Theoretically, each "cycle" of denaturation, primer annealing and target copying doubles the number of target sequences. The length of PCR products is determined by the spacing between the two primer sites on the target. The great advantages of PCR are that target sequences are amplified millions or billions of fold starting from as few as one target molecule and that what is copied is a sequence which is characteristic of the target, not of a probe. For applying PCR to clinical diagnostics there are still challenges to overcome. PCR is not absolutely specific. In order to be amplified, a target must possess sequences complementary to each of two oligonucleotide primers. If absolutely specific primer-annealing condition can be established, then this requirement would essentially preclude amplification of any target but the intended target. However, primer annealing is conducted at 50°C to 55 °C and DNA polymerization occurs as the sample is being heated to 70 ° C, so during PCR the specificity-setting conditions are largely indeterminate. In practice, some reports show exclusive amplification of the intended target while others show major misamplification of unintended targets. Conditions are not optimized, so PCR operates at less than theoretical efficiency. This is important because at theoretical efficiency true amplification products are highly selected for. They are the only products which are amplified logarithmically from start to finish. Misamplification products will be amplified at a slower rate during initial cycles. Therefore, in PCR, efficiency is linked to specificity.

230

D. GILLESPIE

Contamination takes on a new meaning in PCR. Contamination of a negative sample by a single molecule of intended target can lead to a false positive result. Worse, this increased problem of contamination is in the context of synthesizing huge quantities of intended target by amplification. Contamination during PCR experiments has proven to be difficult to cope with in a research setting: in a clinical setting, contamination presents a nightmare. One major step to controlling contamination is to aliquot reagents in a single-use sizes and only use rigorously quality-controlled reagents. A second ingredient is isolating samples from each other; in the long run, sample isolation will be accomplished by automation. However, the best way to limit contamination may be to limit amplification process to the lowest acceptable levels. This will minimize quantities of contaminants generated and will also minimize the chance that contamination will result in a false positive result. Even so, some presumptive false positive results will need to be confirmed to rule out contamination. This can be done by amplifying from the same specimen a larger region containing the original sequence, by amplifying a different region of the same target or by determining the sequence of the PCR product. RNA target amplification can also be accomplished by PCR, providing the first step in the process is copying of the RNA target into DNA by reverse transcriptase. While there is good evidence that a single DNA molecule in a specimen can be amplified to detectable limits, this sensitivity has not been shown for RNA targets. If RNA, rather than DNA has been amplified, omission of reverse transcriptase will eliminate signal. A target amplification technique different from PCR and using T7 RNA polymerase promotors has been described (Kwoh et al., 1989 ) and is referred to as transcription amplification system (TAS). This system uses primers with T7 promotor sequences attached and requires two rounds of target copying to create a double-stranded target copy adjacent to the promotor. This DNA then serves as a template for transcription by DNA dependent RNA polymerase, creating 50 to 1000 single-stranded RNA copies of the target sequence. This amplification, too, can be carried through several cycles by copying RNA amplification products back into DNA then retranscribing them back into a 50 to 1000-fold excess of new RNA copies. Each TAS cycle of amplification increases amplification products about 50-fold. TAS has the advantage over PCR of a larger fold amplification/cycle and the disadvantage of requiring the action of three different enzymes. They differ fundamentally in the fact that PCR products are dsDNA of defined length while TAS products are ssRNA of more or less uniform length. PROBE AMPLIFICATION

Probe amplification is increasing hybridization probe molecules in number by repetitive copying or by some other means. The first probe amplification

DNA PROBES AS DIAGNOSTIC REAGENTS

231

to be introduced used probes containing RNA bacteriophage replication sequences and the RNA dependent RNA polymerase, called "replicase" (Chu et al., 1986). The model bacteriophage for this technique is QB and the replicating RNA, MDV, is a 300 nucleotide "midivariant" RNA containing only that portion of the QB 5000 nucleotide genome required for replication. QB probes contain MDV RNA plus a sequence complementary to target RNA or target DNA. QB probes are hybridized with target and probe-target hybrids are purified away from unhybridized probe. QB replicase is added and probe replication occurs at a rate of one generation per 20 seconds. After some fixed period of time the quantitiy of replicated probe is measured. QB amplification results in a billion-fold probe increase in 20 minutes, without temperature cycling. The same contamination issues raised for target amplification also apply to probe amplification. In addition, all unhybridized probe must be removed prior to probe amplification to eliminate false-positive results. Since probe is amplified, there is no direct way to know that target was involved, so confirmation of positive results will be important. A reasonable strategy for analyzing future clinical specimens may be to screen using QB amplification and confirm positives with PCR. Another strategy for probe amplification is ligation amplification. Two oligonucleotides complementary to adjacent target sequences are brought into juxtaposition by hybridization, then ligated enzymatically as described by Landergen et al. (1988 ). One oligonucleotide carries an affinity ligand and the other a label, hybridization products can be scored by affinity capture. If the hybridization mixture also contains oligonucleotides complementary to those described above, then amplification can be accomplished as follows: cycle 1 (A) hybridize at 65°C; (B) add ligase and ligate at 37°C; (C) denature at 95°C; cycle 2 (A) hybridize at 65°C; (B) add ligase and ligate at 37°C; (C) denature at 95 °C. Like in PCR, products formed during ligation amplification serve as substrate for further amplification, so ligation amplification products accumulate logarithmically. Temperature cycling is necessary for ligation amplification. Since all available ligases are thermolabile, the present scenario require that ligase be added at each cycle. Many other kinds of amplification are possible and remain to be developed. At present, amplification is the only known way to achieve the sensitivity needed for clinical diagnostics. However, amplification adds to the cost burden of diagnostic assays and, especially in veterinary medicine, reduces their utility from only high value tests to only very high value tests. Since amplification is enzymatic, relatively pure nucleic acids are needed. Amplification from crude extracts is possible, especially with small samples, but such a strategy may lack the flexibility and reliability needed for routine diagnostics. One approach is to use reversible target capture to achieve purification of targets or probe-target hybrids, then perform amplification on relatively sequence-pure material.

232

D. GILLESPIE

CONCLUSION T h e p r o d u c t i v e f u t u r e o f D N A p r o b e s in clinical m e d i c i n e s e e m s assured. S o l u t i o n s to p r o b l e m s o f s e n s i t i v i t y a n d n o i s e are in h a n d . A s s a y s c a n b e p e r f o r m e d in 24 h o u r s o r less, y i e l d i n g i n f o r m a t i o n n o t o b t a i n a b l e a n y o t h e r way. T h e p r o d u c t i v e f u t u r e o f D N A p r o b e s in v e t e r i n a r y m e d i c i n e a n d agric u l t u r e is m u c h less a s s u r e d d u e to h i g h cost a n d m a y l i m i t the use o f D N A p r o b e s in v e t e r i n a r y m e d i c i n e a n d a g r i c u l t u r e to selected p r o b l e m s . ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d in p a r t b y g r a n t s f r o m t h e N I H ( C A 2 9 5 4 5 ) a n d T h e A d v a n c e d T e c h n o l o g y C e n t e r o f S o u t h e a s t e r n P e n n s y l v a n i a . I w a s enl i g h t e n e d b y h e l p f u l d i s c u s s i o n s w i t h J. R i c h a r d s , J. T h o m p s o n a n d I. B r o d sky. I a m i n d e b t e d to J. L e v i n f o r p r e p a r i n g t h e m a n u s c r i p t .

REFERENCES Bonner, J., Kung, G. and Bekor, I., 1967. A method for the hybridization of nucleic acid molecules at low temperature. Biochemistry, 6: 3650-3653. Brahic, M., Haase, A. and Cash, F., 1984. Simultaneous in situ detection of viral RNA and antigens. Proc. Nat. Acad. Sci. USA, 81: 5445-5448. Chu, B. C. F., Kramer, F. R. and Orgel, L. E., 1986. Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res., 14:5591-5603. Gall, J. and Pardue, M. L., 1969. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Nat. Acad. Sci. U.S.A., 63: 378-383. Gillespie, D. and Bresser, J., 1983. mRNA immobilization in NaI. Quick Blot. Biotech., 2:184192. Gillespie, D. and Gillespie, S., 1971. Ribonucleic acid-deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide. Biochem J., 125:481-487. Gillespie. D. and Spiegelman, S., 1965. A quantative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol., 12: 829-842. Green, M.R., Maniatis, T. and Melton, D.A., 1983. Human B-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell, 32:681-694. Hamaguchi, K. and Geiduschek, E. P., 1962. The effect of electrolytes on the stability of the deoxyribonucleic helix. J. Am Chem. Soc., 84:1329-1338. Harper, M. E. and Saunders, G. F., 1981. Localization of single copy DNA sequences on G-branded human chromosomes by in situ hybridization. Chromosoma, 83:431-439. Kafatos, F. C., Jones, W. C. and Efstratiadis, A., 1979. Determination of nucleic acid sequence homologies and relative concentration by a dot hybridization procedure. Nucleic Acids Res., 7: 1541-1551. Kwoh. D. Y., David G.R., Whitfield, K. H., Chappelle, H.L,. DiMichelle, L. J. and Gingeras, T. R., 1989. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc. Nat. Acad. Sci. U.S.A., 86:1173-1177. Landergen, U., Kaiser, R., Sandres, J. and Hood, L., 1988. A ligase-mediated gene detection technique. Science, 241: 1077-1080.

DNAPROBESASDIAGNOSTICREAGENTS

233

Lawerence, J.B. and Singer, R.H., 1985. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res., 13: 1777-1799. Maniatis, T., Sambrook, J. and Fritsch, E.F., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Meinkoth, J. and Wahl. G., 1984. Review: hybridization of nucleic acids immobilized on solid supports. Anal. Biochem., 138: 267-284. Rigby, W. J., Dieckman, M., Rhodes, C. and Berg, P., 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by translation with DNA polymerase I. J. Mol. Biol., 113: 237251. Saiki, R. K., gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich, H. A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239:487-491. Southern, E., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98:503-517. Thomas, P. S., 1980. Hybridization ofrenature RNA and small DNA fragments transferred to nitrocellulose. Proc. Nat. Acad. Sci. U.S.A., 77: 5201-5205. Thompson, J. and Gillespie, D., 1987. Molecular hybridization with RNA probes in concentrated solutions of guamide thiocyanate. Anal. Biochem., 163:281-291. Thompson, J. Solomon, R., Pellegrino, M., Sakai, K., Lewin, M., Field, M., Castrovinci, M. Sacramone, 1. and Gillespie, D., 1989. A noise free molecular hybridization procedure for measuring RNA in cell lysates. Anal. Biochem., 181: 371-378. Urdea, M. S., Running, J. A., Horn, T., Clyne, J., Ku, L. and Warner, B., 1987 a novel method for the rapid detection of specific nucleotide sequences in crude biological samples without blotting or radioactivity; application to the analysis of hepatitis B virus in human serum. Gene, 61: 253-264. Virtanen, M., Laaksonen, M., Soderlund, H., Palva, A., Halonen, P. and Rankis, M., 1983. Novel test for rapid viral diagnosis: detection of adenovirus in nasopharyngeal mucus aspirates by means of nucleic-acid sandwich hybridisation. Lancet, i: 381-383. Wallace, R. B., Johnson, M. J., Hirose, T., Miyake, T., Kamashima, E. H. and Itakura, K., 1981. The use of synthetic oligonucleotide as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit B-globin DNA. Nucleic Acids Res., 9: 879-894. Wetmur, J. and Davidson, N., 1968. Kinetics of renaturation of DNA. J. Mol. Biol., 31: 349370. White, B.A. and Bancroft, F. C., 1982. Cytoplasmic dot hybridization : simple analysis of relative mRNA levels in multiple small cell or tissue samples. J. Biol. Chem., 257: 8569-8572.