Boomerang DNA Amplification (BDA) according to the present invention provides a way to amplify a DNA sequence of interest when only one primer region associated with the sequence of interest is known. BDA also permits amplification of sequences that would otherwise reside outside a region suitable for PCR. As used herein, to "amplify" a DNA sequence of interest means to increase the amount of said sequence relative to other DNA sequences that may also be present.

BDA employs "adapters." Adapters are single-stranded or double-stranded polynucleotides that have internal sequences complementary to each other that are capable of annealing to each other to form a duplex under appropriate conditions. Such sequences are termed "self-complementary sequences" or "SCSs."

One type of adapter 50, termed a single-stranded (or "panhandled") adapter, is shown schematically in FIG. 2A. The adapter 50 has a first SCS 51 and a second SCS 52 that may anneal to each other to form a duplex structure 53 resembling a "panhandle." A "spacer" region 54 is situated between the first and second SCSs 51, 52. Because the spacer 54 is not complementary to other sequences of the adapter 50, it forms a single-stranded loop on one end of the panhandle 53. The opposing end of the panhandle 53 is "ligatable" (i.e., able to be enzymatically coupled) to a similar end on either another adapter or a fragment generated by cleavage of DNA. Preferably, the ligatable end is a "sticky" end 56, meaning that said end is capable of annealing and being ligated to complementary sticky-ended DNA fragments generated by cleavage of DNA using a restriction endonuclease.

Another type of adapter, termed a "double-stranded" adapter 58, is shown schematically in FIG. 2B, wherein features that are identical to features shown in FIG. 2A have the same reference designators (with suffixes "a" or "b" to distinguish complementary strands). Double-stranded adapters have SCSs 51a, 52a, and 51b, 52b, wherein one of said sequences is preferably located at each end of the adapter 58. It is also possible for the SCSs 51a, 52a, 51b, 52b to not be located on the ends of the double-stranded adapter 58, but such a configuration is not preferred for BDA because it is less efficient. The double-stranded adapter 58 also comprises a "spacer" region 54 comprising strands 54a and 54b, wherein the spacer strand 54a is situated between the self- complementary sequences 51a and 52a, and the spacer strand 54b is situated between the self complementary sequences 51b and 52b. Double-stranded adapters are generally fully duplex except for a single-stranded overhang 56a, 56b on at least one end as may be desirable for making the end "sticky" for ease of ligation. In any event, at least one end of the double-stranded adapter 58 is "ligatable."

As shown in Figure 2C,.double-stranded adapters 58 are convertible into panhandled adapters 50a, 50b by denaturing the double-stranded adapters 58, isolating the resulting single strands, and self-annealing the single strands.

In either panhandled or double-stranded adapters, the size of the spacer 54 is not critical and is preferably kept somewhat small for reasons of economy. Preferably, the spacer 54 is at least 10-15 bases long, up to about 200 bases long. Larger spacers will work but may require BDA production of excess unneeded DNA (which can either prematurely exhaust the supply of primers, dNTPs, and DNA polymerase in the BDA reaction or necessitate the addition of extra amounts of expensive primers, dNTPs, and DNA polymerase). Spacers smaller than 10-15 bases, including hairpin loops (0-3 bases) formed by contiguous self-complementary ends, should also work, but their use may result in unpredictable products due to the interaction of the DNA polymerase with the unusual structure posed by single-stranded nucleic acids tightly bent in this manner.

In the following description of adapters, the suffixes "a" and "b" are not used with reference designators 50, 51, 52, 54, and 56 for the sake of brevity. However, it will be understood that, where applicable and unless otherwise indicated, said discussion applies to both panhandled and double-stranded adapters.

The SCSs 51, 52 may be comprised of one or more polylinker DNA sequences. As used herein, a "polylinker" is a region of DNA composed of tightly clustered multiple restriction sites. A polylinker is also referred to in the art as a "polylinker cloning site." The SCSs 51, 52 can have any of a wide variety of different polylinker DNA sequences, thereby enabling one to have great latitude in selecting an appropriate sticky end for annealing the adapters to ends of DNA fragments generated using different restriction endonucleases. Also, multiple restriction sites in the polylinkers facilitate downstream cloning of DNA amplified by BDA. However, it is also possible for the SCSs 51, 52 to have only one restriction site or only an end compatible for ligation to other restricted DNAs and still be useful for BDA.

SCSs 51, 52 usually have a length of at least about 15-20 base pairs (bp). This length is the minimal length normally required for SCSs to form a duplex at room temperature. While SCSs can have lengths as long as 100 to 200 bases, or longer, it is preferred for reasons of economy to not use SCSs that are longer than necessary.

Both panhandled adapters 50 and double-stranded adapters 58 can be generated from circular or linear DNAs by any of several possible methods. FIG. 2C is a schematic diagram showing a way in which adapters can be generated from a circular duplex DNA 60. (An example of another possible method for generating adapters is described in Example 1.) The circular DNA 60 comprises a first SCS duplex 51a, 52b, and a second SCS duplex 51b, 52a. The SCS duplex 51a, 52b has a sequence substantially identical to the SCS duplex 51b, 52a but in the opposite orientation. Thus, the SCSs 51a, 52a are complementary to each other in the same strand. Likewise, the SCSs 51b, 52b are complementary to each other in the same strand. It will also be appreciated that SCS 51a is substantially identical to SCS 51b and SCS 52a is substantially identical to SCS 52b. A spacer region 54 is situated between the SCS duplexes. The spacer 54 is preferably at least 10 to 15 bp long. The duplex circular DNA 60 is cleaved using a restriction endonuclease that preferably cuts at a locus adjacent each SCS duplex 51a, 52b and 51b, 52a. Subsequent gel purification yields the double-stranded adapter 58, as described above. Subsequent separation of the single strands on a denaturing gel and self-annealing yields the corresponding panhandled adapters 50a, 50b. Each double-stranded adapter has a sticky end 56a, 56b on each end thereof, and each panhandled adapter 50a, 50b has a sticky end 56a, 56b, respectively.

It is also possible for the adapters to have blunt, rather than sticky, ligatable ends. However, attachment of adapters having blunt ends to other pieces of DNA so as to practice BDA is inefficient compared to attachment of sticky ended adapters.

A general sequence of events in BDA, using only one primer and panhandled adapters 50, is illustrated schematically in FIGS. 3A-3D. Referring first to Figure 3A, BDA begins with a sample DNA 80 usually comprising a number of DNA sequences but also containing a sequence of interest (SOI) 82 to be amplified. For example, the sample DNA 80 can comprise genomic DNA. The SOI 82 can have any of a variety of lengths. Although, in principle, there is no limit to the length of the SOI 82, BDA (like PCR) is more difficult to perform if the SOI 82 is longer than about 4 kilobases (kb). Longer SOI sequences can cause premature exhaustion of BDA reaction constituents (such as DNA polymerase dNTPs, or primers), or untimely drop-off of DNA polymerase enzyme molecules from the DNA being amplified.

The SOI 82 can either span a primer target site (comprising sequence 84) or be situated adjacent the primer target site 84, as shown in Figure 3A. For best BDA results, the primer target site 84 should be uniquely associated with the SOI 82. The primer target site 84 is selected using criteria similar to criteria used in selecting primer target sites for PCR. For example, if the SOI 82 belongs to a group of related genes in other organisms or to a family of related genes in the same organism, wherein the sequence of another gene in the group or family is entirely or partially known, a possible primer target site 84 can be ascertained from said sequence. Alternatively, if the sequence of a polypeptide encoded by the SOI 82 is at least partially known, possible primer target sites can be ascertained using the genetic code (and allowing for the degeneracy of the code). Another way in which a primer target site 84 can be selected is by selecting a known conserved or "consensus" region upstream of a particular gene, such as a promoter.

As can be surmised from its name, the primer target site 84 serves as a region to which a complementary single-stranded primer of the same length will bind (anneal). The primer target site 84 should have a length sufficient to form a stable duplex with a complementary primer at the annealing temperature of the BDA reaction. In general, a minimal length satisfying this criterion is about 15 to 20 bases long. The primer target site should also have sufficient length to be "discriminating," i.e., to ensure that the primer binds substantially only to it and not to other sequences as well. As is known in the art, longer primers are more discriminating than short primers in their binding of target sequences when appropriate conditions for annealing are employed. I have found that primer target sites of about 30 bases long exhibit satisfactory discriminatory binding to complementary primers. Longer primer target sites can be used, but other reasons, such as economy or simply lack of sequence information about any more of the SOI 82 usually preclude such use.

It will be appreciated that primer length is ideally equal to the length of the corresponding primer target site 84, but in some cases can (and may) vary slightly. Primers can be conveniently made using a conventional DNA oligonucleotide synthesizer.

The sample DNA 80 is cleaved preferably using either the same restriction endonuclease used to create the adapter 50 or any other restriction endonuclease that creates sticky ends 86 homologous to the sticky ends 56 on the adapters 50. Such cleavage yields a number of DNA fragments each terminating with a sticky end 86. Some fragments 87 contain the primer target site 84 and the SOI 82. A relatively large number of other fragments 88 lack the SOI 82 and, consequently, the primer target site 84.

An excess amount of adapters 50 is added to the cleaved sample DNA under conditions wherein the sticky ends 56 of the adapters 50 anneal to the sticky ends 86 of the cleaved sample DNA. "Excess" in this context refers to a number of adapter sticky ends 56 greater than the total number of sticky ends 86 represented by the population of cleaved sample DNA. The number of sticky ends represented by the cleaved sample DNA can be approximated by persons skilled in the art by knowing the approximate molecular weight of uncleaved sample DNA, the particular site at which the restriction enzyme cleaves the sample DNA, the AT/( C composition of the sample DNA, and the actual amount of sample DNA to be cleaved. Preferably, "excess" means at least about an 8:1 ratio of adapter sticky ends 56 to cleaved sample- DNA sticky ends 86, to greater than about 100:1. Too low a ratio can result in cleaved sample DNA pieces annealing to themselves rather than to adapters, decreasing the efficiency of BDA. The annealing of excess adapters to themselves causes no problem in BDA (other than a decrease in efficiency) because such annealed molecules are not primable when proper annealing conditions and primer sequences are employed.

Annealing of adapters 50 to cleaved sample DNA is performed at a relatively low temperature, such as within a range of about 0° C. to about 25° C., typically about 16° C.

A DNA ligase (such as T4 DNA ligase obtainable from New England BioLabs, Inc., Beverly, Mass., and used according to the manufacturer's directions) is used to covalently bond the adapters 50 to the fragments of sample DNA to form a population 89 of various "closed-loop structures." The closed- loop structures, as can be seen, comprise a length of duplex DNA terminated on each end by a loop 54 contributed by the adapter 50. Although potentially all the restriction fragments of the sample DNA can form closed-loop structures 89, at least one group 90 of the closed-loop structures includes the primer target site 84 and the SOI 82 and can therefore serve as a BDA template. It will be appreciated that T4 DNA ligase can ligate blunt ends. Thus, again, blunt ended adapters can be used to perform BDA. Another possible DNA ligase is E. cold DNA ligase (also obtainable from New England BioLabs). However, this ligase cannot ligate blunt ends.

Once the closed-loop structures 89 are prepared, other ingredients (reactants) are added thereto to prepare a reaction mixture. The reaction mixture preferably contains sufficient amounts of reactants so that an entire "round" of BDA comprising multiple cycles of amplification of the SOI 82 can occur without having to replenish the reactants part way through the round. Reaction conditions for BDA should closely approximate buffer requirements of the DNA polymerase employed. A representative BDA reaction mixture comprises the population 89 of closed-loop structures, primers 92, buffer (such as 10 to 50 mM Tris-HCI buffer, pH about 8.3 to about 8.8 at 25° C.) all four dNTPs (in equal concentrations to minimize misincorporation errors, wherein the concentration of each dNTP ranges between 20 to 200 JIM), a DNA polymerizing agent such as a DNA polymerase, and a magnesium salt such as magnesium chloride or magnesium sulfate (depending on the DNA polymerase used).

The DNA polymerase employed is preferably a "thermostable" DNA polymerase capable of withstanding denaturation temperatures. Examples of thermostable polymerases include the well-known Taq DNA polymerase and Vent (trademark) DNA polymerase (available from New England BioLabs, Beverly, Mass.) which can withstand nearly boiling temperatures such as 95° C. (or higher for Vent (trademark) DNA polymerase). Less thermostable DNA polymerases can also be used, but may require replenishment after each cycle. An example of the latter is the Klenow fragment of E cold DNA polymerase.

As a first approximation, the amount of DNA polymerase added to the reaction mixture can be according to the enzyme manufacturer's suggestions. However, the amount actually required in a particular BDA reaction mixture will depend upon several factors, including the type of polymerase, the temperature at which DNA replication will occur, the intended number of replication cycles in the BDA "round," and the combined length of the SOI, polylinkers, and loops. Skilled artisans are familiar with altering the concentration of DNA polymerase in a DNA replication reaction and how to ascertain optimal concentrations of the enzyme.

Depending upon the DNA polymerase used in the reaction, addition of other ingredients (or omission of some of the ingredients described above) may be useful for optimal polymerization. Optional ingredients may include KCl (typically 50 mM or lower), DMSO, gelatin or bovine serum albumin (generally at about 100 µg/mL to stabilize the DNA polymerase), ammonium sulfate (10 mM), magnesium sulfate (5 mM), Triton X-100 (0.1%), or other ingredients depending on the particular DNA polymerase employed.

The amount of primer 92 present in the BDA reaction mixture is a concentration in large excess relative to the concentration of the primer target sequence 84. Preferably, as in PCR, enough primer 92 is added to the reaction mixture to last through the intended number of cycles in the BDA "round." Calculating the amount of primer 92 to add is within the purview of persons skilled in the art having a knowledge of the amount of sample DNA in the BDA reaction mixture and the number of copies of the SOI in the sample DNA. To illustrate the magnitude of the "excess," the molar ratio of primer to primer target sites can be about 30 million or more. Such an excess also helps prevent reannealing of the denatured closed-loop structures 90 with themselves in the region of the priming sequence.

To begin a cycle of DNA replication in a BDA process, the population 89 of closed-loop structures is heated (Figure 3A), generally at a temperature of about 93° to about 100° C., for a time sufficient for full denaturation (up to about five minutes). The temperature is then lowered to a point where the primers 92 anneal efficiently to the primer target sites 84.

Proper annealing of primers in BDA, like PCR, is regulated by the annealing temperature and the concentration of primers. (As noted above, the concentration of primers may require alteration to effect efficient and accurate priming.) Several factors affect the annealing temperature. These include (but are not necessarily limited to) the primer length, the salt concentration, and the primer base composition (i.e., number of A and T bases relative to number of G and C bases). Persons skilled in the art are familiar with methods for calculating a temperature Tm at which half the primer target sites will have primer molecules annealed thereto. Generally, an applicable annealing temperature is about 5° C. below the Tm of the primers. Thus, annealing temperatures are generally within a range of about 50° C. to about 70° C. The annealing temperature should be high enough to prevent non-specific binding of the primers to the sample DNA. Increasing the annealing temperature generally enhances discrimination against incorrectly annealed primers. As in PCR, some empirical experimentation may be required to ascertain the optimal annealing temperature.

Annealing of primers at a temperature within the stated range occurs rapidly (less than one minute). However, at this beginning stage of BDA wherein primers are annealed to the BDA templates 90 for the first time, more time is usually allowed, generally about two minutes, to ensure that primers bind to the BDA templates 90 as efficiently as possible.

Tetramethylammonium chloride may be added to the reaction mixture to enable one to more accurately predict an effective annealing temperature. As known in the art, this compound equalizes the binding strength of G:C base pairs relative to A:T base pairs, thereby allowing one to more precisely determine and use an annealing temperature that prevents mismatched sequences from hybridizing to form a duplex.

In BDA, it is preferred that only the templates 90 that include the primer target site 84 experience replication. Thus, annealing is performed under conditions wherein the primers 92 anneal substantially only to the primer target sites 84. (For simplicity, closed-loop structures lacking the primer site 84 are not shown further in FIGS. 3A-3D.)

Referring now to Figure 3B, the primed template 91 is shown having a first region 94 containing a primer target site 84 to which a primer 92 is annealed. The opposing second region 96 (complementary to the first region 94) includes a sequence 98 substantially identical to the primer 92. 5' and 3' ends of the primer target site 84 and primer 92 are denoted to indicate orientation. The primed template 91 also has a "spacer" region 100, 101 located on each end. Each spacer 100, 101 was contributed by a panhandled adapter 50 (Figure 3A) and was originally the looped spacer 54 of said adapter.

The oligonucleotide primers 92 annealed to the primer target sites 84 serve as initiation sites of DNA replication (replication is also termed "primer extension") during subsequent steps in each cycle. As shown in Figure 3B, primer extension begins on the 3'-ends of the primers 92 and proceeds in a 5' to 3' direction. Since the primed templates 91 are closed loops, replication can proceed down the first region 94, around a spacer 100, and down the second region 96, thereby generating a primer extension product 102. (The procession of replication around the spacer 100 suggested the motion of a boomerang, hence the coined name of the present method.) It will be appreciated that primer extension continuing past the region 98 generates a new primer target site 104 as part of the primer extension product 102.

Primer extension as described above is allowed to proceed only for a short time during each cycle. The exact point on the primed template 91 where DNA replication stops is not critical, so long as replication has proceeded from the primer 92 down the first region 94, around the spacer 100, and into the second region 96 toward the region 98 a sufficient distance to produce a primer extension product 102 that can form a duplex with itself between a segment thereof complementary to the first region 94 and a segment thereof complementary to the second region 96. Although, under certain conditions, a primer extension product 102 could form such a duplex with the segment complementary to the second region 96 being as small as one base, a stable duplex between a segment complementary to the first region 94 and a segment complementary to the second region 96 would generally require that the segment complementary to the second region be at least about 10 bases long. Preferably, primer extension is allowed to proceed on through the opposing complementary region 98, thereby generating a primer target site 104 on the primer extension product 102.

It will be appreciated by persons skilled in the art that primer extension products that do not include a complementary region 98 can be extended in subsequent BDA cycles. This is because the duplex formable by the primer extension product can, in fact, function as a primed template for DNA replication.

The amount of time required to achieve the requisite amount of DNA replication per cycle is dependent upon several factors of which artisans skilled in PCR are familiar. These factors include the type of DNA polymerase being used, the length and concentration of the primed template 91, and upon the temperature. The temperature, of course, must be suitable for the particular DNA polymerase used. For example, the Vent (trademark) polymerase is typically used at a temperature of about 70° C. to about 76° C. (Higher temperatures may cause the primer to become denatured from the template.) At 72° C., rates at which this enzyme replicates DNA can vary two to three fold, depending upon the buffer, the pH, concentration of salts, and the nature of the template. As a starting point, one minute at 72° C. is often sufficient to produce primer extension products up to about 2 kb in length. Cycling times may be varied to insure the most efficient production of full-length primer extension products. As in PCR, it may be necessary for the skilled artisan to perform preliminary experiments to ascertain minimal amounts of time required to achieve the requisite amount of DNA replication.

The principal disadvantages of allowing DNA replication to proceed for too long is loss of process economy and possible premature exhaustion of the supplies of DNA polymerase and dNTPs in the reaction mixture. Aside from possibly necessitating an unplanned replenishment of DNA polymerase and dNTPs, allowing too much time does no real harm to the outcome of BDA because DNA polymerases, upon progressing once entirely around the primed template 91, will generally drop off the template.

Referring further to Figure 3B, after the primer extension products 102 are produced, the resulting duplex DNAs 106 are heat-denatured, thereby allowing the template 108 to separate from the primer extension product 109. For maximal BDA efficiency, each of these denatured DNAs 108, 109 has a primer target site 84. Thus, each also has a region 98 complementary to the primer target site 84. (It will be appreciated that the primer target sites 84, 104 are identical. Therefore, on the denatured primer extension product 109, the primer target site 84 now has the same reference designator as the primer target site 84 on the denatured template 108.)

It is desirable that denaturation result in complete separation of the primer extension product 109 from the template 108. Incomplete separation can allow these denatured DNAs to rapidly reanneal together, thereby reducing the ultimate yield of amplified DNA. Typical denaturation conditions comprise a temperature from about 93° C. to about 96° C. (preferably about 95° C.) for one minute or less. Too high a temperature or too long a denaturation time can cause premature exhaustion of the enzyme activity and/or dNTPs in the reaction mixture.

Referring now to Figure 3C, a subsequent cycle of DNA replication is initiated by annealing primers 92 to the denatured DNAs 108, 109 to form the primed templates 110, 111. The annealing temperature is usually the same as employed previously (about 50° to about 70° C.). However, the annealing time is usually shorter than before, typically about 30 seconds or less. The DNA polymerase in the reaction mixture effects primer extension from the 3'-ends of the primers 92, thereby generating duplex structures 112, 114. The duplex structure 112 comprises the template 108 and a new primer extension product 116. As in the preceding cycle, primer extension is allowed to proceed at least for a time sufficient to produce a primer extension product 116, 118 that can form a duplex with itself between a segment thereof complementary to the first region 94 and a segment thereof complementary to the second region 96. Each primer extension product 116, 118 preferably includes a region 104 having a sequence complementary to the primer 92 and identical to the primer target site 84. Thus, the region 104 can serve as a primer target site in a subsequent BDA cycle.

Subsequent heat-denaturation of the duplex structures 112, 114 would signify the beginning of yet another BDA cycle. As shown in Figure 3D, cycles as described above are repeated a sufficient number (n) of times until the desired amount of amplified DNA 120 is obtained. Generally, n is about 30 to 60 so as to achieve a satisfactory degree of amplification.

It will be appreciated by persons skilled in the art that at least one BDA cycle, such as a final one or more cycles, can be performed using one or more labeled dNTPs, thereby producing labeled BDA products.

When all cycles have been completed, DNA polymerase activity can be stopped by chilling the reaction mixture to about 4° C. or by adding EDTA to the reaction mixture to a concentration of about 10 mM.

After all the cycles of BDA are completed, samples of the amplified DNA can be loaded onto gels for analysis, using techniques well-known in the art. When working with new DNAs or when optimizing the BDA process for a particular application, cleaving the amplified DNA using one or more restriction endonucleases and looking for expected sizes of cleavage fragments in gels is a good way to confirm that the BDA was successful. Alternatively, one may choose to excise the amplified sequence of interest from the adapter using a restriction endonuclease and ligate the amplified sequence of interest into a cloning vector for sequence analysis.

Performing BDA using only one primer can result in amplification of either a portion of the SOI or the entire SOI, depending upon the location of the primer target site on the SOI.

To this point, the descriptions of BDA have pertained to amplification of SOIs situated adjacent the primer target site. BDA also permits one to amplify an SOI that has a primer target site located completely within the SOI, such as when one wishes to amplify an entire genomic restrictions fragment. Figure 4 schematically depicts how a BDA template 140, made using panhandled adapters, can be used to amplify an SOI 142, starting from a primer target site 148 located within the SOI 142. A similar reaction could be performed using double-stranded adapters, but the intermediate would not be a closed-loop structure.

Referring further to Figure 4, the BDA template 140 is comprised of a first SOI region 144 and a second SOI region 146 complementary to the first SOI region 144. The first SOI region 144 includes a first primer target site 148; the second SOI region 146 includes a second primer target site 150 complementary to the first primer target site 148. BDA of such a template 140 can be performed using a first primer 152 complementary to the first primer target site 148 and a second primer 154 complementary to the second primer target site 150. Thus, the first and second primers 152, 154, respectively, are complementary to each other and are identical to primer target sites 150, 148, respectively.

Although Figure 4 indicates that BDA involving the first primer 152 and the second primer 154 are performed in separate reactions, it is to be understood that BDA according to Figure 4 can also be performed using both primers in a single reaction. However, such single-reaction BDA is not ideal because the primers 152, 154, being complementary to each other, can anneal to each other, thereby decreasing the efficiency of the BDA reaction.

Referring further to Figure 4, a first BDA reaction mixture is prepared comprising the closed-loop structure 140, buffer, dNTPs, DNA polymerase, and required salts, as described above, and molecules of the first primer 152. A second BDA reaction mixture is prepared that is identical to the first reaction mixture except that the second primer 154 is added thereto instead of the first primer 152. The primers 152, 154 are allowed to anneal to the closed-loop structures 140. Subsequent primer extension (DNA synthesis) as described above yields the duplex structures 156, 158 comprising primer extension products 160, 162, respectively. Repeated(n) cycles of heat denaturation, annealing of primers, and primer extension yield a large population of the structure 164 from the first BDA reaction and the structure 166 from the second BDA reaction. As can be seen, the BDA product 164 overlaps the BDA product 166 at the primer target sites. This overlap region can be effectively employed, should one desire, to unite the two products 164, 166 together into a single two-loop structure using molecular biological techniques known to skilled practitioners in the relevant art.

It is also possible to perform BDA using double-stranded adapters. Referring to Figure 5A, a polynucleotide 300 contains an SOI 302 (comprised of segment 302a and complementary segment 302b) and a primer target site 304. Cleavage of the polynucleotide 300, such as by using a restriction endonuclease, generates a number of discrete fragments 306 that lack the primer target site, and fragments 308 that include the primer target site 304 and at least a portion of the SOI 302. Although each fragment 306, 308 preferably has "sticky" ends 310, the ends are in any event ligatable to adapters. (For clarity and simplicity of illustration, only the fragment 308 containing the primer target site 304 is shown and discussed further hereinbelow.)

The double-stranded adapters 312 have first self- complementary sequences 314a, 316a and second self-complementary sequences 314b, 316b. Thus, sequence 314b is identical to sequence 314a and sequence 316b is identical to sequence 316a. For optimal coupling to the fragments 306, 308, the adapters have ligatable ends 318. As shown in FIG. 5A, the ends 318 are preferably sticky to the sticky ends 310 of the fragments 306, 308. A spacer region 315a is situated between self-complementary sequences 314a and 316a, and a spacer region 315b, complementary to spacer region 315a, is situated between self complementary sequences 314b and 316b.

It may be desirable, although not necessary in principle, to chemically alter the sticky ends 318 of the adapters 312 so that they are ligatable only to the ends of the fragments 306, 308 and not to each other. Thus, use of adapters with altered sticky ends can improve the efficiency of BDA performed using double stranded adapters. One way to perform this alteration, as shown in Figure 5A, is to treat the 5' ends of the adapters 312 with a phosphatase enzyme (such as calf- intestinal phosphatase or alkaline phosphatase) so as to remove the phosphate 320 normally present on said 5' ends (leaving a hydroxyl group 322 on treated adapters 323). Duplex DNAs treated in this manner are not ligatable to each other but can be ligated to untreated duplex DNAs such as the fragments 306, 308 having S'-phosphates 324. That is, the hydroxyl group normally present on each 3' end 326 of either an untreated adapter 312 or a treated adapter 323 can be covalently coupled to a 5' phosphate 324. Thus, ligation of treated adapters 323 to fragments 308 can be achieved (albeit of only one strand because a hydroxyl normally present on each 3' end 328 of the fragments 308 cannot be ligated to a 5' hydroxyl 322 on a treated adapter 323) Treated adapters 323 cannot be ligated together because 5' hydroxyls 322 cannot be ligated to hydroxyls normally present on the 3' ends 326 of the adapters.

Ligation of a treated adapter 323 to each end of a fragment 308 produces a duplex structure 330 having unligated gaps 332. The gap 332 is formed because a 5' hydroxyl 322 does not couple to the hydroxyl normally present on each 3' end 328 of the fragment 308.

Turning now to Figure 5B, heat-denaturation of the duplex structures 330 followed by addition of oligonucleotide primers 334 substantially homologous to the primer target site 304 yields primed linear templates 336 and unprimed single stranded molecules 338,340. (The unprimed single-stranded molecules 338, 340 are not discussed further hereinbelow even though it will be understood that the molecule 340 can form a duplex with itself which is extendable in a subsequent BDA cycle.) Primer extension of the linear template 336 produces a duplex molecule 342 that includes a primer extension product 344 which comprises a regenerated segment 302a of the SOI 302. The primer extension product 344 also includes the self-complementary sequence 314a, the spacer segment 315a, and an amount of the self- complementary sequence 316a sufficient to enable the primer extension product 344 to form a duplex with itself (via Watson-Crick pairing of bases in the self-complementary sequences 314a and 316a). Ideally, primer extension continues through the self complementary sequence 316a to include a short segment 346 complementary to the sticky end 318. Of course, if the adapters 312 had blunt ends, the short segment 346 would not be formed.

Referring to Figure 5C, heat denaturation of the duplex molecule 342 and annealing of primers 334 reproduces the primed template 336 and produces a looped template 348 from the primer extension product 344. The looped template 348 includes a duplex region 350 formed by the annealing together of self-complementary sequences 314a and 316a. The spacer 315a thus becomes a loop. The short segment 346, if present in the duplex 342, is preserved in the looped template 348.

Continuing with Figure 5C, primer extension of the templates 348, 336 yields the duplexes 352, 354, respectively. Use of a DNA polymerase such as Vent (trademark) polymerase (which has a 3'-exonuclease function) removes the unpaired segment 346. Thus, as can be seen, in the looped template 348, the self complementary sequence 316a serves as a primer for the regeneration of segment 302b and the primer target site 304. As can be seen, the duplex 354 is structurally similar to the duplex 342 formed earlier (Figure 5B). The duplex 352 is structurally similar to the BDA product 120 of Figure 3D. Subsequent cycles of BDA result in amplification of the duplex 352, which comprises the SOI 302, as well as production of more molecules of the duplexes 352 and 354.

Thus, BDA allows researchers to clone entire genomic restriction fragments with a knowledge of the sequence of only one priming region in the restriction fragment. In addition, a portion of a DNA sequence amplified by BDA can be used as a primer for subsequent BDA reactions on templates made with adapters ligated to DNAs cut with a different restriction enzyme. This would effectively enable one to "walk" along genomic DNAs one restriction fragment at a time and determine the sequence of or clone each fragment.

BDA differs from existing PCR methods in two important aspects. First, BDA is not limited to amplifying the region located between two priming sites. Because of its inability to amplify sequences lying outside two priming sites, PCR is most commonly employed only as an aid in cloning gene sequences located between priming sites. For example, PCR can be used to amplify a section of a gene useful for making a radioactive probe. Such probes are useful for isolating clones containing an entire gene from genomic (or cDNA) libraries. But, making such libraries and probing them with PCR- generated probes can be exceedingly time-intensive, often taking months to obtain entire genes. BDA, on the other hand, which can produce amplified entire restriction fragments from a single priming region, can amplify an entire gene in a single overnight reaction after selection of the appropriate restriction fragment.

A second primary advantage of BDA is its ability to allow users to "walk" along a genomic DNA and determine its entire sequence by employing multiple "rounds" of BDA and DNA sequencing. In such a scheme, each preceding round yields information utilized in the subsequent round. Briefly, the BDA portion of each "round" is comprised of multiple BDA cycles as described above. A first round is begun by cutting the genomic DNA using a first restriction endonuclease. The resulting population of linear duplex DNA fragments includes a group of substantially identical fragments to be sequenced which include a first primer target site, a first region (to be sequenced) and an opposing second region complementary to the first region. To prepare the fragments for BDA, suitable adapter molecules as described generally above are ligated thereto. The adapters, of course, have ends ligatable to the fragments. The resulting templates are denatured. Primers homologous to the first primer target site are annealed to the denatured templates. The primers are then extended as described above, forming primer extension products. After denaturing the primer extension products from the templates, further BDA cycles are performed, as described above, until sufficient DNA is produced to permit conventional sequencing of the amplified DNA to be performed. After sequencing, a second round is initiated by selecting a new primer target site situated within the sequence obtained in the first round, but downstream of the first primer target site (thereby ensuring that DNA amplified in the second round will overlap DNA amplified in the first round). The genomic DNA is then cut using a second restriction endonuclease. Using new primers homologous to the second primer target site, multiple cycles of BDA are performed, as described above, yielding DNA amplified 3 sufficiently to permit sequencing. By registering the sequence information determined in the first round with sequence information determined in the second round, the combined sequence of the DNA amplified in both rounds is obtained. Further rounds could, in theory, be performed in 3 each direction until a complete genomic segment such as a chromosome has been sequenced.

This "walking" feature is extremely useful for sequencing and/or cloning very large genetic sequences spanning many restriction fragments. Very large genes, such as those extending across multiple restriction fragments, are oftentimes very difficult to clone using genomic libraries and conventional methods due to the limited capacity of most vectors to carry large sequences. In these cases, BDA may be the only means of obtaining complete large gene sequences, particularly without spending large amounts of time. This "walking" ability of BDA should prove extremely useful in projects involving genomic sequencing, such as the human genome effort or similar efforts proposed for commercially important crops or livestock.

BDA can also be used to selectively clone an SOI, thereby ultimately achieving a de facto amplification of the SOI without the need for performing multiple cycles of annealing, primer extension, and denaturation. An example of a BDA cloning method is as follows:

Referring to Figure 6, genomic or other DNA 180 containing one or more copies of an SOI 182 (including a primer target site 184) is cleaved using a restriction endonuclease. The particular restriction endonuclease used is chosen to ensure that the cleavage products therefrom have a desired "sticky end" 186. Of the population of cleavage products so generated, a first subpopulation 188 will include the SOI 182 and a second subpopulation 190 will not.

BDA adapters 192 are then added to the cleavage products. The BDA adapters have the same "panhandle" configuration as described above, including a single-stranded spacer 194, a duplex region 196, and a sticky end 198. The sticky end 198 is complementary to the sticky ends 186 on the cleavage products 188, 190. The duplex region 196 is an inverted repeat, as described above. The single-stranded spacer 194 includes a replication origin 200 expressible in a cloning host cell such as E. coli. The spacer 194 also includes a selectable marker 202, such as a gene conferring resistance to an antibiotic. (Figure 6 and the above description depict the replication origin 200 and selectable marker 202 as being located in the spacer 194. However, there is no reason per se why the replication origin 200 and selectable marker 202 could not be situated either partially or wholly within the duplex region 196.) Thus, the adapters 192 used for BDA cloning will typically be larger than adapters used for BDA alone.

The mixture of adapters 192 and cleavage products 188, 190 are ligated together as described above, thereby generating closed-loop structures 204, 206.

Primers 208 homologous to the primer target site 184 in the SOI 182 (and therefore specific for the SOI) are added to the closed-loop structures 204, 206. All four dNTPs and a suitable DNA polymerase, as described above, are also added. The amount of dNTPs and DNA polymerase added are sufficient for a single BDA cycle. The resulting mixture is heat- denatured, then cooled to allow the primers 208 to anneal to the primer target sites 184. Of course, closed loop structures 206 lacking the SOI (and therefore the primer target site) will not experience any significant binding of primers thereto.

Subsequent DNA replication (primer extension) is allowed to proceed for a time sufficient for the DNA polymerase molecules to fully circumnavigate the closed loop structure 204 containing the SOI 182. Such a time would normally be slightly longer than the time, as described above, that would allowed for a DNA polymerase to at least travel past the region on the SOI complementary to the primer target site 184. Thus, the closed-loop structure 204 containing the SOI 182 will become fully replicated, thereby generating circular duplexes 210 that include the SOI 182, the replication origin 200, and the selectable marker 202. The closed-loop structures 206 lacking the SOI 182 are not replicated. Thus, the closed- loop structures 206 lacking the SOI 182 (and therefore lacking the primer target site) retain their single-stranded loop portions after DNA replication.

After DNA replication, the reaction mixture is treated with a single- strand- specific nuclease (a nuclease that specifically degrades single-stranded DNA). Representative nucleases for this purpose include, but are not necessarily limited to, Mung Bean nuclease and S1 nuclease. If S1 nuclease is to be used, it is desirable to treat the replication mixture with a DNA ligase to convert the gapped structure 210 produced by primed DNA replication into a fully covalently closed circular duplex. This is because S1 will nick gapped duplexes. Mung Bean nuclease, however, will not nick gapped duplexes and no such ligase treatment is necessary. The single-stranded portions of all DNAs (in particular, closed-loop structures 206 lacking the SOI) are degraded, leaving only a linear duplex 212 lacking the replication origin and selectable marker. The circular duplex DNAs 210 are not degraded.

After nuclease treatment, the DNA mixture is transformed into cells of a susceptible host such as E. coli. The resulting transformed cells are cultured under conditions favoring survival of cells that received the fully duplex DNA, such as by culturing in the presence of the antibiotic corresponding to the antibiotic resistance marker 202. Transformed cells that receive only a linear duplex 212 lacking the replication origin and selectable marker do not survive. Transformed cells that receive a circular duplex 210 including the replication origin 200 and selectable marker 202 do survive, enabling large amounts of the SOI to be produced by continued selective culturing of the cells.

In order to further illustrate the invention, the following examples are given.




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