FOR RELEASE UNDER SPECIFIED TEMPERATURE CONDITIONS

BACKGROUND OF THE INVENTION

This invention relates generally to methods for performing biological reactions and, more specifically, to methods for avoiding undesirable side reactions by incorporating one or more essential reaction components into a wax medium.

The polymerase chain reaction ("PCR") is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A repetitive series of cycles involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5' ends of the primers. Because the primer extension products synthesized in one cycle can serve as templates in the next, the number of synthesized DNA copies approximately doubles at every cycle. Thus, 20 cycles of PCR yields about a million-fold (2 ²⁰ ) amplification. PCR technology and reagents are discussed and claimed in U.S. Patent Nos. 4,800,159; 4,683,202; and 4,683,195.

Initially, PCR methods used the Klenow fragment of E. coli DNA polymerase I to extend the annealed primers. This enzyme is inactivated by the high temperature required to separate the two DNA strands at the outset of each PCR cycle. Consequently, fresh enzyme had to be added during every cycle. The introduction of thermostable DNA polymerase (e.g., Taq polymerase, U.S. Patent No. 5,079,352) isolated from Thermus aquaticus transformed PCR into a simple and robust reaction which could be automated by a thermal cycling device. The reaction components (template, primers, Taq polymerase.

dNTP's, and buffer) could all be assembled and the amplification reaction carried out by simply cycling the temperature within the reaction tube. Although for any given pair of oligonucleotide primers an optimal set of conditions can be established, there is no single set of conditions that are optimal for all possible reactions.

The specificity of the PCR is typically analyzed by evaluating the production of the target fragment relative to other products by gel electrophoresis. The initial PCR method based on DNA synthesis by the Klenow enzyme at 37°C was not highly specific. Although a specific target fragment could be amplified up to a million-fold, most of what was synthesized was not, in fact, this target fragment. Thus, subsequent analysis with a specific hybridization probe or, in some cases, with internal "nested primers" to detect and characterize the amplified target sequence was required. By cloning a 3-globin amplification reaction and screening the individual clones with a β- globin probe to detect the target sequence and with one of primers to detect any amplified sequence, the specificity of the PCR was estimated to be -1%. Other primer pairs were somewhat more or less specific.

The use of the Tag polymerase not only simplified the PCR procedure but significantly increased the specificity of the overall yield of the reaction. The higher temperature optimum for the Taq polymerase (~75°C) allowed the use of higher temperatures for primer annealing and extension, thereby increasing the overall stringency of the reaction and minimizing the extension of primers that were mismatched with the template. At 37°C, many of these mismatched primers are sufficiently stable to be extended by the Klenow enzyme, resulting in non-specific amplification products. The increase in the specificity of the Tag PCR results in an improved yield

of the amplified target fragment by reducing the competition by non-target products for enzyme and primers. In the later cycles, the amount of enzyme is no longer sufficient to extend all the annealed primer\template complexes in a single cycle period, resulting in a reduced efficiency and a "plateau" in the amplification reaction. This plateau is reached later (e.g., about 30 cycles rather than 20 starting with 1 μg of genomic DNA) in Taq PCR than in the reaction with the Klenow enzyme due to the increased specificity of the former reactions. In addition to the increase in the specificity and yield of PCR made possible by Tag polymerase, the use of this enzyme allows the amplification of much longer fragments, up to 10 kb, albeit with reduced efficiency, than does the Klenow enzyme, which generally is restricted to less than 400 base pairs.

Changes introduced into the sequence of the PCR products due to nucleotide misincorporation can create potential problems. In the analysis of the population of amplified products, as in oligonucleotide probe hybridization or in direct sequencing, the rare errors in individual products are not detectable. However, in the sequence analysis of individual clones derived from a PCR, sequences must be determined from multiple clones to distinguish misincorporated nucleotides from the faithful copies of the template sequence.

The major obstacle to simple and routine single-copy detection by PCR now appears to be competing side reactions such as the amplification of nontarget sequences in background DNA ("mis-priming") and primer oligomerization. Furthermore, the success of "hot start" amplification methods of boosting low-copy-number specific amplification at the expense of side reactions implies that the most damaging, troublesome primer

oligomerization and mis-priming occur pre-PCR, that poorly defined interval of at least a few minutes when all reactants have been mixed, usually at room temperature, before thermal cycling is started.

In hot start PCR, reagent addition to the reaction tube is designed so that all reactants do not mix until reaching a temperature high enough to suppress primer annealing non-target sequences. Typically in the manual hot start method, all reactants except Taq polymerase are mixed at room temperature below a conventional mineral oil vapor barrier. Then, after all tubes have been loaded in a thermal cycler and the temperature has been raised to hold at 60-80°C, enzyme is added separately to each tube, changing the pipette tip after each transfer to prevent cross-contamination. After all tubes have been sealed, thermal cycling is started. Though such a manual procedure can increase amplification specificity and yield, it is inconvenient, error- and contamination-prone, and inherently imprecise, as each tube in a given experiment has a unique heating/mixing history.

One method which has attempted to eliminate the weaknesses of the hot start method is the use of wax beads to form a seal over the PCR reaction mixture. An omitted reagent was then added on top of the wax seal, being mixed into the reaction upon reaching the melting temperature of the wax.

Although the above described method allowed for more precise temperature control of PCR, it necessarily complicated PCR through the addition of steps which increased the risk of error and decreased efficiency both of time and expense. Thus, there exists a need for a method of controlling undesirable side reactions which is both time and cost effective. The present invention fulfills these needs.

SUMMARY OF THE INVENTION

The present invention provides a method of selectively adding a reagent to a temperature dependent reaction. One or more reagents are incorporated into a wax medium. The other non-incorporated reagents of the reaction are combined. The wax medium containing the incorporated reagents are then added to the reaction mixture. The temperature of the reaction mixture is then raised to at least the melting temperature of the wax medium thereby releasing the reagents into the reaction mixture.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of temperature controlled release of reagents into a reaction mixture. The present invention further provides a method of minimizing undesirable side reactions which may occur in a chemical or biochemical reaction. The described method includes the utilization of a wax medium which incorporates one or more reagents of the desired reaction. The present invention further provides a method of controlling the release of reagents into a reaction mixture by increasing the mixture temperature to the melting point of the wax medium. These undesirable side reactions may occur in both temperature and non- temperature dependent reactions. According to the present invention, the wax medium may be added to the reaction mixture at any time. Where it is desirable to delay release of the incorporated reactants, the wax medium is added to the mixture at a temperature below the melting point of the wax medium.

The term "reagent" is defined as any substance that participates in a chemical reaction. Thus, "reagent" includes any reactant or catalyst. A reactant

is defined as any atom, ion or molecule that enters into a chemical reaction. The reactant may be a substance which is essential or non-essential to the reaction. The catalyst may be any organic (e.g., enzyme) or inorganic substance that changes the rate of the chemical reaction.

The term "reagent" also includes any substance which may be used for the detection or determination of another substance. Thus, reagent includes, for example, antibodies, enzymatic and radioactive labels, flourochromes and the like.

The term "wax medium" includes any inert substance into which a reagent may be incorporated which while in its solid phase does not release the reagent upon contact with the reaction mixture. Further, the reagent is released into the reaction mixture only where the temperature of the reaction mixture has reached the melting point of the wax medium. In one embodiment, the waxes used in the methods of the invention are neutral lipids consisting of esters formed from fatty acids and long-chain alcohols other than glycerol. Such waxes can include those having the physical characteristics of being malleable. Further, where the invention is to be practiced as a means to minimize undesirable side reactions, these waxes have a melting point above the optimal temperature at which the undesirable side reactions take place and below the boiling point of the reaction mixture.

The term "wax medium" also includes synthetic polymers which can be designed for the release of reaction components at different temperatures. For example, a medium having a melting temperature of 42°C could be used to release reverse transcriptase primers and buffer components for first strand synthesis. A medium having a melting temperature of 50°C could be used

to release DNA polymerase and buffer components for second strand synthesis. A medium having a melting temperature of 75°C could be used to release ligase, linkers and cut plasmids for ligation. For general methods which can be modified by this invention, see Sambrook et al.. Molecular Cloning: a Laboratory Manual Cold Spring Harbor Lab. Press., Cold Spring Harbor, NY (1989), incorporated herein by reference.

The invention further contemplates diagnostic kits where specific primers are incorporated into the medium optimizing for dNTP and enzyme ratios. This technique not only simplifies diagnostic testing, but also increases the reliability of the assay by decreasing the degree of variability. Such diagnostic kits would include the wax having the primer, suitable reaction buffer, thermostable nuclease and instructions for performing the procedure. Primer kits for the detection of pathogens such as HIV and for the diagnosis of disease such as sickle cell anemia are included in this invention.

Generally, the reaction mixture will be in a liquid state. However, the addition of the wax medium to a gaseous or solid reaction mixture is also provided for by the method of the present invention.

In one embodiment, the method of the present invention is used in a modification of the Polymerase Chain Reaction (PCR) described in U.S. Patent Nos. 4,800,159, 4,683,202, and 4,683,195, incorporated herein by reference. In a further embodiment, the wax medium can be used as an alternative to mineral oil and to automate the "hot start" technique, described in Bassam and Caetano-Anolles, BioTechniques, Vol. 14:31-33 (1993), incorporated herein by reference. This technique, an improvement of PCR, eliminates false priming at low

temperatures and reduces nonspecific PCR products. The hot start technique works by removing one or more essential components of the PCR reaction until the mixture has been heated above the melting temperature of the primers.

The present invention provides a method of minimizing the non-specific priming and generation of spurious results during the amplification of DNA by polymerase chain reaction using a thermostable polymerase by permitting the reaction temperature to increase to greater than or equal to 60°C allowing primers to properly and specifically anneal before the addition of a critical reactant to the reaction mixture.

The present invention further describes a method of creating wax beads or pellets which contain reaction components. Once the wax melts, the reactants are released to admix with other reactants. For example, where the present invention is used in a modified version of PCR, dNTPs (dinucleotide triphosphates) or Taq polymerase may be incorporated. dNTPs are particularly good reagents for wax incorporation as they are relatively stable and because the concentration of dNTPs in the reaction mixture is not as critical as are other components such as magnesium.

The wax medium can be formed into a ball or pellet shape enabling it to float freely on top of the reaction components. This ensures that the wax medium will rise more smoothly and uniformly to the top upon melting. The volume of the wax ball is important, the optimal being the minimum volume necessary to just cover the surface of the lower reaction components to prevent evaporation and to allow rapid mixing of upper and lower components. After rising, the wax provides a seal which serves as an overlay preventing evaporation during the

reaction. This effectively eliminates the conventional use of mineral oil as used, for example, in PCR. The size of the wax medium can vary with the volume amplified as well as the vessel used for amplification, but commonly 30-40 μl is used. Different kinds of wax medium with various melting temperatures can be used depending on the optimal reaction conditions. In general, the wax medium must liquefy above the melting temperature of the reagents.

For example, where the method is to be applied to PCR, the melting temperature of the wax must be above the melting temperature of the primers or target nucleotides and must release the dNTPs quickly enough to prevent evaporation and uneven mixing. The optimal melting temperature would thus be between about 50-60°C.

In a separate embodiment, the incorporated reagents are soluble and are released quickly once the wax medium liquefies. The wax medium can be in the form of a moderately unstable wax/aqueous emulsion. Although an emulsion can be achieved with sonication [VibraCell Sonics and Materials, Danbury, CT] using varying ratios of wax to water (at 25W from 1-60 seconds), the emulsion is relatively stable and the reagents are not released rapidly enough to permit the reaction to proceed in an optimal fashion. To facilitate the reagents release, various detergents can be employed to emulsify the wax in a reversible manner. It is important that a detergent is selected which does not interfere with or inhibit the incorporated reagents.

As applied to the PCR, one cationic detergent, dimethyldioctadecyl ammonium bromide (DDAB) , in combination with Aristowax 125 and sufficient dNTPs to achieve a final concentration of -200-500 μM of each

nucleotide, achieved the best results when formulated as described below.

Special attention must be paid in handling and storing the wax medium both before and after the desired reactants have been incorporated. Although the following description involves dNTPs, it provides relevant guidelines to one skilled in the art as to the emulsification, handling and storage of other combinations of wax mediums and reagent components.

In formulating the wax medium/ dNTPs, it is recommended that a minimum amount of liquid per pellet (2 μl or less) be used in order to maximize dispersal of dNTPs. A minimum amount (50 μg/ml) of detergent is recommended to form a workable unstable emulsion. The volume of the pellets should be kept as small as possible (-30-40 μl) . Furthermore, high ionic strength buffer (5 mM MgCl ₂ and greater) should be avoided. In order to minimize the separation of the emulsion, the wax should be vortexed well just prior to cooling and solidification of the wax.

The forming process must be completed rapidly (less than 1 minute) during production since water can evaporate or sublimate quickly if the wax emulsion is heated for too long, or if it is exposed to air for long periods.

Due to difficulties and expense in the manufacture of these pellets, a novel method for scaled up production was developed. The wax emulsion is formed and then is hardened by quickly pouring the wax into a chilled mold at -20°C. The cold, solidified emulsion is ground into powder using a spinning metal blade and is then quickly formed into pellets using a pharmaceutical pelleting machine. Alternatively, the reagent is

encapsulated into wax beads using a variation of the method described in U.S. Patent Nos. 3,015,128, 3,310,612, 3,389,194 and 4,764,317, incorporated herein by reference. Method may include centrifugal extrusion, submerged extrusion, stationary extrusion, rotating disk, or vibrating nozzle (Dietert, R., Application of Vibration to Encapsulation Process, American Laboratory 19-25 (1979)).

The pellets should be stored immediately at -20°C to prevent degradation of the dNTPs. The wax medium/dNTPs described above were found to be unstable if stored at room temperature. Pellets stored for more than seven days at room temperature (22°C) generated a smear and little or no specific PCR product.

In one embodiment, the method of the present invention involves a modification of PCR. An important property of PCR, particularly in diagnostic applications, is the capacity to amplify a target sequence from crude DNA preparations (Saiki et al.. Nature 324:163 (1986)) as well as from degraded DNA templates (Bugawan et al..

Bio /Technology 6:943 (1988)). The DNA in a sample need not be chemically pure to serve as a template provided that the sample does not contain inhibitors of thermostable polymerase. The ability to amplify specific sequences from crude DNA samples has important implications for research applications, (e.g., sperm lysates, Li et al.. Nature 335:414 (1988)), for medical diagnostic applications (e.g., mouthwash, Lench et al.. Lancet ii:1356-1358 (1987) or archival paraffin-embedded tissue samples, Shibata et al.. Cancer Res . 48:4564-4566 (1988)) and for forensics (e.g., individual hairs, Higuchi et al.. Nature 332:543 (1988)).

Standard PCR is typically done in a 50- or 100- μl volume which, in addition to the sample DΝA, contains

50 mM KC1, 10 mM Tris.HCl (pH 8.3 at 42°C), 2.5 mM MgCl ₂ , 0.001% μg/ml gelatin, 0.25 μM of each primer, 200-500 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), and 2.5 units of Taq polymerase (PCR Technology, Erlich, ed., p8 (1989)). Either dNTP or Taq polymerase may be incorporated into the wax medium essentially as described in Examples I and II infra. The type of the DNA sample will vary, but will usually have between 10 ² to 10 ⁵ copies of template (e.g., 0.1 μg human genomic DNA) .

The amplification can be conveniently performed in a DNA Thermal Cycler (Perkin-Elmer Cetus Instruments, Norwalk, CT) using the "Step-Cycle" program according to the manufacturer's instructions set to denature at 94°C for 20 seconds, and extend at 72°C for 30 seconds, for a total of 30 cycles. A pre-denaturation step is required at the initiation of PCR to allow the reagents time to escape the wax medium and mix with the lower components. Otherwise, the first 2-3 cycles will be incomplete and the yield of the product DNA will be lower than a standard PCR reaction. The "Step-Cycle" program causes the instrument to heat and cool to the target temperatures as quickly as possible. In the current instrument, this results in a heating rate of about 0.3°C per second and a cooling rate of about 1°C per second, for an overall single cycle time of approximately 3.75 minutes.

The deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) are usually present at 50 to 200 μM of each. Higher concentrations may tend to promote misincorporations by the polymerase (i.e., "thermodynamic infidelity") and should be avoided (Petruska et al., Proc . Nat 'l . Acad. Sex . USA 85:6252-6256 (1988)). At 50 and 200 μM, there is sufficient precursor to synthesize approximately 6.5 and 25 μg of DNA, respectively.

Neutralized dNTP solutions can be obtained from Pharmacia (Piscataway, NJ) . _T

As deoxynucleotide triphosphates appear to quantitatively bind Mg ²⁺ , the amount of dNTPs present in a reaction will determine the amount of free magnesium available. In the standard reaction, all four triphosphates are added to a final concentration of 0.8 mM; this leaves 0.7 mM of the original 1.5 mM MgCl ₂ not complexed with dNTP. Consequently, if the dNTP concentration is changed significantly, a compensatory change in MgCl ₂ may be necessary.

Taq polymerase is available from Perkin-Elmer Cetus Instruments, Norwalk, CT. The concentration of enzyme typically used in PCR is about 2.5 units per 100 μl reaction. For amplification reactions involving DNA samples with high sequence complexity, such as genomic DNA, there is an optimum concentration of Tag polymerase, usually 1 to 4 units per 100 μl. Increasing the amount of enzyme beyond this level can result in greater production of non-specific PCR products and reduced yield of the desired target fragment.

PCR is performed by incubating the samples at three temperatures corresponding to the three steps in a cycle of amplification-denaturation, annealing, and extension. This cycling can be accomplished either manually with pre-set water baths, or automatically with the DNA Thermal Cycler.

In a typical reaction, the double-stranded DNA is denatured by briefly heating the sample to 90-95°C, the primers are allowed to anneal to their complementary sequences by briefly cooling to 40-60°C, followed by heating to 70-75°C which allows extension of the annealed primers with the Taq polymerase. In addition, at the

initiation of PCR, a 2-3 minute pre-denaturation step at 94°C is required to allow the incorporated reagents to escape the wax pellets.

Insufficient heating during the denaturation step is a common cause of failure in a PCR reaction. It is very important that the reaction reaches a temperature at which complete strand separation occurs. A temperature of about 94°C should be adequate in most cases. After the initial pre-denaturation step, as soon as the sample reaches 94°C, it can be cooled to the annealing temperature.

The temperature at which annealing is done depends on the length and GC content of the primers. A temperature of 55°C is a good starting point for typical 20-base oligonucleotide primers with about 50% GC content; even higher temperatures may be necessary to increase primer specificity.

As observed for several DNA polymerase activities isolated from thermophilic microorganisms, 94 kDa Taq DNA Polymerase has a relatively high temperature optimum (T _opt ) for DNA synthesis. Depending on the nature of the DNA template, an apparent T _opt of 75-80°C has been found with a K _oat approaching 150 nucleotides/sec/enzyme molecule. Even at lower temperatures. Tag DNA Polymerase has extension activities of -0.25 and 1.5 nt/sec at 22° and 37°C, respectively.

Although Tag DNA polymerase has a very limited ability to synthesize DNA above 90°C, the enzyme is relatively stable to and is not denatured irreversibly by exposure to high temperature. In a PCR mix. Tag DNA Polymerase retains about 50% of its activity after 130 min, 40 min, and 5-6 min at 92.5°, 95°, 97.5 ^β C, respectively. Preliminary results indicate retention of

65% activity after a 50-cycle PCR when the upper limit temperature (in tube) is 95°C for 20 seconds in each cycle.

Low, balanced concentrations of dNTPs have been observed to give satisfactory yields of PCR product, to result frequently in improved specificity, to facilitate labeling of PCR products with radioactive or biotinylated precursors, and to contribute to increased fidelity of Tag polymerase. In a 100-μl PCR with 40 μM each dNTP, there are sufficient nucleotide triphosphates to yield 2.6 μg of DNA when only half of the available dNTPs are incorporated into DNA. It is likely that very low dNTP concentrations may adversely affect the processivity of Tag DNA polymerase.

In further embodiments, it is contemplated that the methods of the present invention can be used in conjunction with various existing methods for sequencing DNA. For example, DNA may be sequenced using the dideoxy or enzymatic method as originally described by Sager, Proc. Natl . Acad. Sci . USA 74:5463-5467 (1977), incorporated herein by reference.

DNA may also be sequenced using the chemical approach described by Maxam-Gilbert in Proc. Natl . Acad. Sci . USA 74:560-564 (1977) and Meth . Enzymol . 65:499-559 (1980) each incorporated herein by reference.

In further embodiments, polymerase chain reaction products may be sequenced according to this invention by modification of existing techniques. For example, dideoxy sequencing of single-stranded products generated by asymmetric PCR may be performed using the methods described herein. The dideoxy sequencing method is described in "Current Protocols in Molecular Biology"

Green Publishing Assoc. and Wiley-Interscience 15.2.1 (1991) and is incorporated herein by reference.

cDNA amplification may also be performed by modifying existing anchored PCR protocols. Such protocols are described in "Current Protocols in

Molecular Biology" Green Publishing Assoc. and Wiley- Interscience 15.6.1 (1991), incorporated herein by reference.

It is contemplated that other thermostable enzymes can be incorporated into the wax medium according to this invention. These include, but are not limited to, polymerases, ligases, exonucleases and endonucleases. For example, thermostable ligases may be incorporated into the wax medium as used in genetic disease detection and DNA amplification by modification of the procedure described in Barany, Proc . Natl . Acad. Sci . USA 88(l):189-93 (1991), incorporated herein by reference.

Examples of specific enzymes having varying temperature optima include BstXI 55C, BstNI 60C and BsrI 65C.

As described above, the method of the present invention may be applied to any chemical or biochemical reaction where the temperature controlled release of a reagent is desired. The present method may also be applied to any reaction where it is further desired that an effective seal is formed over the reaction mixture to prevent the evaporation of initial reagents, intermediates, final products and the like.

The methods of the invention are illustrated but in no way limited by the following examples.

EXAMPLE I Preparation of Wax Pellets Incorporating dNTP

Trial wax pellets were formulated with the following components:

1 ml molten wax (Aristowax 125, from Calwax,

Azusa, CA)

25 μl of 25 mM dNTPs in 2 mM Tris pH7 (2-10 mM effective range),

1 μM dithiothreitol (DTT) (not required for emulsion)

0.5-1 μl of red or yellow food coloring (to visualize the emulsion)

0.5 μl of DDAB, 100 mg/ml in chloroform.

These components were mixed and heated to approximately 70-80°C to liquefy the wax. The mixture was then vortexed to form a wax/aqueous emulsion. The pellets were formed by pipetting 40 μl of molten mixture onto a glass surface. A moving belt apparatus can be used if automation of the pellet-forming process is desired. This step should be done quickly as the emulsion begins to separate after 20 seconds. The wax pellets solidified at 52°C. This process ultimately yielded approximately 25 pellets. This yield will, of course, vary depending upon the size of the pellets. This in turn is dependent upon the volume needed to cover the surface area of the reaction mixture.

EXAMPLE II Preparation of Wax Pellets Incorporating Taq Polymerase

Wax pellets incorporating Tag polymerase were formulated essentially as described in Example I. Trial wax pellets were formulated with the following components:

1 ml molten wax (Aristowax 125, from Calwax, Azusa, CA) ,

1 unit of Tag polymerase,

1 μM dithiothreitol (DTT) (not required for emulsion) ,

0.5-1 μl of red or yellow food coloring (to visualize the emulsion) ,

0.5 μl of DDAB, 100 mg/ml in chloroform.

These components were mixed and heated to approximately 70-80°C to liquefy the wax. The mixture was then vortexed to form a wax/aqueous emulsion. The pellets were formed by pipetting 40 μl of molten mixture onto a glass surface. A moving belt apparatus can be used if automation of the pellet-forming process is desired.

This step should be done quickly as the emulsion begins to separate after 20 seconds. The wax pellets solidified at 52°C. This process ultimately yielded approximately 25 pellets. This yield will, of course, vary depending upon the size of the pellets. This in turn is dependent upon the volume needed to cover the surface area of the reaction components.

EXAMPLE III PCR Using Pellets

Materials

1. 10X PCR Buffer: Prepare a solution containing 500 mM KCl, 200 mM Tris-HCl, pH

8.4, 25 mM MgCl ₂ , 1 mg/ml nuclease-free BSA (Pharmacia, cat. # 27-8914-01).

2. 10 mM dNTP stock: Prepare a solution containing 10 mM dATP, 10 mM dCTP, 10 mM dGTP, and 10 mM dTTP. Prepare from 100 mM stocks (Pharmacia, cat. # 27-2050-01, 27- 2060-01, 27-2070-01, and 27-2080-01, respectively) , diluting with 10 mM Tris- HCl, pH 7.5. 3. PCR primers: 30-50 pmoles/μl, containing both the 5'- and S'-amplimers.

4. Tag polymerase (5 U/μl, Perkin-Elmer Cetus) .

5. Microfuge tubes: Use tubes certified for use in the thermal cycler.

6. 3% NuSievel/1% SeaKe agarose (FMC Corporation, cat. 3 50092).

7. 6X loading buffer (6X LB): 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in water.

8. TEA electrophoresis buffer: 40 mM Tris- HCl, pH 7.5, 1 mM EDTA, 5 mM sodium acetate.

Method

1. Prepare this PCR:

1 μl cell lysate 10 μl 10X PCR buffer 1 unit of Tag polymerase

1 μl upstream primer (30-50 pmoles/μl) 1 μl downstream primer (30-50 pmoles/μl) 87 μl H ₂ 0 100 μl Total

2. PCR amplification a. Add 1 unit of 25 mM of each dNTP wax pellets [original: 10 μl - 10 mM dNTPs]. b. Incubate the reaction at 95°C for 2-3 minutes. c. Perform 25-35- cycles:

• 95°C, 30 seconds (denaturing step) .

• 50-72°C, 30 seconds (annealing step) .

• 72°C, 30-120 seconds (extension step) .

3. Add 2 μl of 6X LB to 10 μl of PCR reaction and load into a composite 3% NuSievel/1% SeaKem agarose gel in TEA buffer.

4. Electrophorese, stain with 10 μg/ml ethidium bromide for 10 minutes, destain with H ₂ 0 for 20 minutes, and visualize on a UV light box. Blot the DNA, if desired.

EXAMPLE IV RNA Amplification

Materials

10X PCR buffer: 500 mM KCl, 200 mM Tris.HCl (pH 8.4 at room temp), 25 mM MgCl ₂ and 1 mg/ml nuclease free BSA. Deoxynucleotide Triphosphates: Neutralized, 100 mM solutions (Pharmacia

or PL-Biochemicals) . The dNTPs are combined to make a 10 mM stock solution of each dNTP using 10 mM Tris.HCl (pH 7.5) as diluent. 3. RNasin (Promega Corporation at 20-40 units/μl) . 4. Random hexamers: 100 pm/μl solution in TE

(10 Mm Tris.HCl, 1 mM EDTA, pH 8.0)

(Pharmacia) . 5. PCR Primers: The primers are usually 18-

22 bases in length and dissolved in TE at

10-100 pm/μl. 6. Reverse transcriptase: Mo-MuLV (Bethesda

Resear h Labs) at 200 units/μl. 7. 1 unit wax pellets containing Tag polymerase.

In a final vol of 20 μl IX PCR buffer assemble the following: 1 mM of each dNTP, 1 unit/μl RNasin, 100 pmoles of random hexamer, 1 μg of total or cytoplasmic RNA and 100-200 units of BRL MuLV reverse transcriptase. Incubate 10 min at room temp, then 30-60 min at 42°C. To stop reaction, heat tube in 95°C water bath for 5-10 min, then quick chill on ice. Note that other manufacturers of reverse transcriptase recommend different amounts of enzyme than BRL. Follow the manufacturer's prescribed amounts. It is sometimes helpful to heat treat the RNA sample at 90°C for 5 min and quick chilling before adding an aliquot to the reaction mix. Presumably the heat treatment breaks up RNA aggregates and some secondary structure which may inhibit the priming step.

PCR Reaction

To the heat treated 20 μl reverse transcriptase reaction add 80 μl of IX PCR buffer containing 10-50 pmoles each of upstream and downstream primer and 1-2

units of wax pellets containing the Tag polymerase. After pre-denaturing the mixture at 94°C for 2-3 minutes, run 20-50 cycles depending on the abundance of the target. A "typical" PCR cycle which works well for amplifying lengths of 500 or more bases is: 95°C denaturation for 30 seconds, 1 min cooling to 55°C, annealing of primers at 55°C for 30 seconds, 30 seconds heating to 72°C, extension of primers at 72°C for 30 seconds, and 1 min heating to 95°C, etc. Other PCR cycle profiles work just as well, but be careful about times allotted for heating and cooling. If heating and/or cooling is too fast, the reaction solutions will not have time to equilibrate to the correct temperatures and amplifications will be inefficient or nil.

EXAMPLE V

Inverse Polymerase Chain Reaction

A procedure is described which extends the utility of the polymerase chain reaction by allowing the geometric amplification of an unknown DNA sequence that flanks a core region of known sequence. DNA containing the core region is digested with appropriate restriction enzymes to produce a fragment of suitable size for PCR amplification. The ends of the fragment are then ligated to form a circular molecule. Primers for PCR are homologous to the ends of the core region included within the circle, but oriented such that chain elongation proceeds across the uncharacterized region of the circle rather than across the core region separating the primers. This "inverse PCR" procedure can be used to amplify the sequences that originally flanked the core sequence. Inverse PCR has applications in producing probes of anonymous sequences or in determining the sequences of upstream and downstream flanking regions themselves.

The basis for this procedure ("inverse PCR") is to convert flanking DNA to interior region by cutting the molecule outside of the core region using an appropriate restriction enzyme, and forming circular molecules by self-ligation of the restriction fragments. Progressive PCR amplification of the unknown region in the circles is possible using primers homologous to the ends of the core region, but oriented with their 3' ends toward the unknown region.

DNA digestions are carried out using conventional buffers and other conditions recommended by the suppliers. Fragments of suitable size for inverse PCR are determined by the size of fragment that can be amplified by PCR, which at present has a practical upper limit of 3-4 kb. In many cases, preliminary Southern hybridizations will be needed to identify restriction enzymes that produce end fragments of suitable size for circularization and amplification of inverse PCR. Enzymes that cleave within the core region allow inverse PCR amplification of either the upstream or downstream segment of DNA that serves as a template for PCR primers (depending on choice of primers), whereas enzymes that do not cleave within the core region allow amplification of both flanking sequences with their junction determined by the restriction enzyme(s) and the type of circularization (e.g., ligation of complementary overhanging ends versus blunt ends) . For amplification of left or right hand sequences, good choices for initial trials include enzymes with four-base recognition sites known to have conveniently located cleavage sites within the core region. If inverse PCR is to be carried out to probe hybridization probes from a large number of different sequences cloned into the same vector, it may be advisable to introduce convenient restriction sites into the vector in advance.

Circularization is performed with T4 DNA ligase in a dilute DNA concentration that favors the formation of monomeric circles (Collins et al., Proc . Nat 'l Acad. Sci USA 81:6812-6816 (1984)). Generation of suitably sized fragments for inverse PCR may require the use of two restriction enzymes with ends that be incompatible for ligation, in which case the ends of the fragments should be repaired (made flush) using Klenow or bacteriophage T4 DNA polymerase prior to the circularization step. Prior to ligation, it is necessary to inactivate restriction enzymes from the previous step by phenol or heat denaturation. It is not normally necessary to cleave the circular molecules within the core region in order to obtain efficient PCR amplifications.

Polymerase chain reaction conditions are those conventionally used (e.g., Saiki et al.. Science 230:1250-1354 (1985); Ochman et al.. Genetics 120:621-623 (1988); Triglia et al., Nucl . Acids Res . 16:8186 (1988)). For example, a pre-denaturation step of 2-3 minutes at

94°C followed by 30 cycles of denaturation at 94°C for 30 seconds, primer annealing at 58°C for 30 seconds, and extension with Tag polymerase at 70°C for 3 min. Either dNTP or Tag polymerase containing wax pellets may be utilized. The PCR conditions can be altered for specific products. When inverse PCR is used in sequencing applications, it is often useful to use amplification primer set back from the ends of the core sequence, allowing the sequencing primers to be close to the junction between the amplified part of the core sequence and the unknown flanking sequence to minimize interference from the amplification primers.

EXAMPLE VI

Production of First-Strand cDNA,

High- or Low-Specific-Activity

Materials

1. poly(A) ⁺ RNA,(+) 20-120 μg.

2. MoMLV-RT reverse transcriptase (BRL, Grand Island, NY., cat. # 8025SA) .

3. RNasin (Promega, Madison, WI., cat. # N2111). 4. p(dT) (Pharmacia, cat. # 27-7858-01).

5. p(d ) , random primers (Pharmacia, cat. # 27-2166-01) .

6. [α- ³ P]dATP (6000 Ci/mmole, New England Nuclear, Boston, MA) . 7. dATP, dCTP, dGTP, and dTTP, at 100 mM each

(Pharmacia, cat. # 27-2050-01, 27-2060-01, 27-2070-01, and 27-080-01, respectively).

8. Siliconized micro tubes.

9. 5X reverse transcriptase buffer (5X RT) : Prepare a solution containing 250 mM Tris- HCl, pH 8.3, 375 mM KCl, 15 mM MgCl ₂ , 10 mM DTT. Make this buffer fresh.

10. tRNA (yeast, Sigma, St. Louis, MO., cat. # R0128). 11. Sephadex G-50 spoin columns (Worthington

Biochemicals, cat. #LS4404).

12. 7.5 M ammonium acetate.

13. DEPC-treated H ₂ 0.

14. Bromophenol blue (Sigma, cat. # B5525). 15. Dimethyldichlorosilane (silane, Sigma, cat. # D3879) .

16. Dithiothreitol (DTT, Sigma, cat. # D9779).

17. 8-hydroxyquinoline (Sigma, cat. # H6878).

18. Xylene cyanole FF (Sigma, cat. # X0377).

Method cDNA Synthesis

1. Synthesize high- or low-specific activity cDNA a. Synthesis of high-specific-activity cDNA: Prepare this reaction on ice: 20.0 μl mRNA(+) (1.0 mg/ml; heat denatured: 68°C, 5 minutes, 0°C, 5 minutes) 40.0 μl 5X RT buffer

5.0 μl RNasin (40 U/μl) 1 unit of 25 mM of each dNTP wax pellets; [10.0 μl dNTPs (10 mM dCTP, dGTP, and dTTP; 0.4 mM dATP) 30.0 μl [ - ³² P]dATP (6000 Ci/mmole)

20.0 μl p(dT) (100 pmole/μl) 20.0 μl MoMLV-RT (200 U/μl) 55.0 μl H ₂ 0, DEPC-treated 200.0 μl Total Note: Random primers (2.5 mg/ml) can be substituted for oligo dT. Use 50 μl of random primers and 25 μl of H ₂ 0. b. Synthesize of low-specific-activity cDNA: Prepare this reaction on ice: 20.0 μl mRNA(+) (1.0 mg/ml; heat- denatured: 68°C, 5 minutes, 0°C, 5 minutes)

40.0 μl 5X RT buffer 5.0 μl RNasin (40 U/μl) 1 unit of 25 mM of each dNTP wax pellets; [10.0 μl dNTPs (10 mM each of dATP, dCTP, cGTP, and dTTP)] 10.0 μl [α- ³² P]dATP (6000 Ci/mmole) 20.0 μl p(dT) (100 pmole/μl) 20.0 μl MoMLV-RT (200 U/μl)

75.0 μl H ₂ 0, DEPC-treated 200.0 μl Total

2. Incubate the reaction at 37°C for 1 hour to synthesize first strand.

3. Add 1 μl of the cDNA-synthesis reaction to 39 μl of water. Remove 5 μl aliquots for TCA-precipitation analysis.

4. Add 20 μl of 1 N NaOH to the 199 μl of the cDNA-synthesis reaction remaining. Incubate for 20 minutes at 68°C to hydrolyze the mRNA 5. Add 20 μl of 1 M Tris, pH 7.4 to the cDNA- synthesis reaction 6. Add 6 μl of the cDNA-synthesis reaction to 20 μg of tRNA for an unsubtracted-probe control. Store at -20°C. 7. Perform a TCA-precipitation analysis on 5 μl aliquots from step 3 to assess incorporation. (Remember that the concentration of dATP in the high- specific-activity-cDNA-synthesis reaction is 20 μM, and remember to set the counter to the ³ H channel for Cerenkov analysis. ) 8. Removal of unincorporated dNTPs. a. Add 117 μl of 7.5 M ammonium acetate to the cDNA-synthesis reaction. b. Add 700 μl of 95% ethanol to the cDNA-synthesis reaction. c. Centrifuge at 4 ^β C and 14 Krpm in a microfuge for 20 minutes. d. Resuspend the pellets in a total volume of 300 μl of TE. Add 150 μl of 7.5 M ammonium acetate and 1.0 ml of 95% ethanol. e. Centrifuge at 4°C and 14 Krpm in a microfuge for 20 minutes. f. Repeat steps (d) and (e) twice. g. Resuspend the pellet in 100 μl of H ₂ 0.

EXAMPLE VII dCTP-Tailing of ds cDNA with Terminal Transferase

Materials

1. Obtain 0.1 μg of ds cDNA for the production of the library and 0.1 μg of ds cDNA to optimize the terminal-transferase- tailing reaction.

2. Optimizing the tailing of cDNA a. Prepare this reaction: 6.5 μl 10 X TdT

1.0 μl 1 mM dCTP

X μl ds cDNA (0.1 μg) Y μl H ₂ 0 64.5 μl Total b. Prewarm the tube at 37°C for 15 minutes. c. Prepare tubes labeled 45", 60", 75", 90", 105", and 120", and add 78 μl of 1 mM EDTA stop solution to each tube. d. Add 60 units of terminal transferase

(=0.5 μl) to the reaction cocktail. Incubate at 37°C. Remove 10 μl at each time point and pipette directly into the stop solution. e. For each time point, mix:

44.0 μl C-tailed cDNA time point 1.0 μl G-tailed vector 5.0 μl 10X AB 50.0 μl Total f. Anneal and transform DH5α. The optimal time point for dCTP-tailing of cDNA is that time point that results in the highest number of bacterial transformants.

g. Prepare the same cocktail as in step (a) . Prewarm the reaction as before, then add 60 units of terminal transferase. Incubate at 37°C for the determined optimal time period, and immediately terminate the reaction by adding 500 μl of 1 mM EDTA. h. Perform a large-scale transformation and solid-state amplification.

EXAMPLE VIII

Synthesis of Radiolabeled Oligonucleotide Probe with Polynucleotide Kinase

The availability of synthetic oligonucleotides has changed the complexion of radiolabeled-probe synthesis. By this procedure (Carter et al., (1985)), you can kinase an oligonucleotide to very high specific activity with a minimum of work. We use such probes routinely for screening cDNA libraries for plasmid clones of known sequences. You should keep in mind that the efficiency of kination can vary from oligonucleotide to oligonucleotide.

Materials

1. 10X kinase buffer (10X KB): Prepare a solution containing 500 mM Tris-HCl, pH

8.0, 100 mM MgCl ₂ , 50 mM DTT, 1 mM EDTA.

2. Oligonucleotide, unphosphorylated.

3. [γ- ³² P]ATP (>3000 Ci/mmole, in aqueous solution; NEN, cat. # NEG-002H or Amersham, cat. # PB.10218).

4. T4 polynucleotide kinase (New England Biolabs, cat. # 201).

5. Stratagene push column (Stratagene, cat. # 400701).

Method

1. Prepare this reaction:

1.0 μl oligonucleotide (10 pmoles/μl)

2.0 μl 10X KB 16.0 μl [γ- ³² P]ATP

1.0 μl polynucleotide kinase (1-5 U/μl)

20.0 μl Total

2. Incubate at 37°C for 45 minutes, then at 68°C for 10 minutes. 3. Purify the probe by passing through a

Stratagene push column.

Examples VI to VIII can be modified to use a wax component having a melting temperature at 37°C or 68°C.

EXAMPLE IX

Labeling of DNA with Biotinylated dUTPtPCR

With this method, you can prepare very large quantities of a sequence-specific probe containing biotinylated dUTP using PCR.

Materials

1. 10X PCR buffer: Prepare a solution containing 500 mM KCl, 200 mM Tris-HCl, pH 8.4, 25 mM MgCl ₂ , 1 mg/ml BSA (DNase and RNase-free, Pharmacia, cat. # 27-8914-01). 2. 10X dNTP stock: 10 mM dATP, 10 mM dCTP, 10 mM dGTP, and 7.2 mM dTTP. (Prepare from 100 mM dNTP stock solutions from Pharmacia cat. # 27-2050-01, 27-2060-01, 27-2070-01, and 27-2080-01, respectively, diluting with 10 mM Tris-HCl, pH 7.5.)

3. 0.3 mM biotinylated dUTP: Prepare a solution of 0.3 mM Biotin-11-dUTP (Sigma,

cat. # B7645; Enzo Diagnostics, cat. # NU- 806) in 10 mM Tris-HCl, pH 7.5.

4. PCR primers (50 pmoles/μl).

5. Tag polymerase (5 U/μl, Perkin-Elmer Cetus) .

6. Microfuge tubes: Use tubes certified for use in thermal cycler.

7. Glass beads (GENECLEAN, BIO-101).

8. 3% NuSieve/1% SeaKem agarose (FMC Corporation, cat. # 50092).

9. 6X loading buffer (6X LB): 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in H ₂ 0.

10. TEA electrophoresis buffer: 40 mM Tris- HC1, pH 7.5, 1 mM EDTA, and 5 mM sodium acetate.

Method

1. Prepare this PCR:

1 μl target DNA (plasmid DNA, 1-5 ng/μl)

10 μl 10X PCR buffer

1 unit of 25 mM of each dNTP wax pellets [10 μl 10X dNTP stock] 17 μl 0.3 mM biotinylated dUTP 1 μl upstream primer (50 pmoles/μl)

1 μl downstream primer (50 pmoles/μl) 1 μl Tag polymerase (5 U/μl) 59 μl H ₂ 0 100 μl Total 2. PCR Amplification a. Denature the target DNA by incubating at 95°C for 8 minutes. b. Perform 25 cycles of • 95°C, 2 minutes • cooling 1 minute to 55°C

• 55 °C , 2 minutes

• heating 1 minute to 72°C

• 72°C, 3 minutes

• heating 1 minute to 95°C c. Perform a 26th cycle, but stop the cycle after reaching the 72°C extension step. Incubate for 7 minutes.

3. Purification of PCR Fragment a. Extract the mineral oil by adding 300 μl of TE-saturated chloroform, b. To the aqueous layer, add 50 μl of

7.5 M ammonium acetate and 300 μl of ethanol. c. Incubate in an ethanol/dry-ice bath for 20 minutes, or at -70°C for 1 hour. Steps b and c will remove oil and chloroform as well as precipitate the DNA. d. Spin in microfuge at 4°C and 14 Krpm for 30 minutes. e. Resuspend the pellet in 10 μl of TE and 2 μl of 6X LB. Load a 3% NuSieve/1% SeaKem agarose gel in TEA buffer. Electrophorese the PCR products in TEA buffer. f. Stain the gel with ethidium bromide. g. Glass-bead purify the PCR fragment. Elute in 50 μl of H ₂ 0.. h. Store the DNA probe at -20°C for up to one year.

4. Boil probe for 10 minutes, then quick- chill on ice for 5 minutes, before adding to filter-hybridization solution.

Note: PCR conditions should be optimized for the production of a single PCR product. The above amplification procedure is a suggested starting point.

Although the invention has been described with reference to specific examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.