Subtractive hybridization and capture methods and kits for differential isolation of nucleic acids including disease-associated sequences

The invention disclosed here allows for the differential isolation of nucleic acid sequences that are present in one nucleic acid population and not in another. The method is based upon using restriction endonucleases to digest two populations of nucleic acid, preferably cDNAs, preferably ligating different sets of adaptors to each of the two nucleic acid populations, followed by hybridization, restriction digestion and isolation of the desired molecules. The unique aspects of this invention include the use of a restriction enzyme to isolate the target duplex DNA molecule from a hybridization mixture. Certain embodiments of the invention include the direct or indirect incorporation of a capture molecule or ligand (e.g., biotin, dioxigenin) within the amplified nucleic acid fragments, which allows for a system in which molecules can be rescued from both the captured population as well as the effluent or otherwise uncaptured population.

This application claims the benefit of U.S. Provisional Application No. 
60/010,207, filed Jan. 18, 1996, the disclosure of which is incorporated 
by reference. 
FIELD OF THE INVENTION 
This invention relates to the field of nucleic acid analysis and 
differentiation. 
BACKGROUND OF THE INVENTION 
The ability to identify and target nucleic acid sequences which appear in 
one nucleic acid sample and not in another is of intense interest in the 
field of molecular biology. The identification of novel nucleic acid 
sequences can provide valuable clues as to genetic bases for disease, 
inherited dominant and recessive traits, genetic alterations which give 
rise to diseases such as cancer, determining species similarities and 
differences, genotyping, and taxonomic classification. Such technology has 
applications thus in diagnostics, medicine and health, forensics, 
taxonomic classification and the like. 
Various comparative nucleic acid techniques are available to analyze 
differences in nucleic acid populations. One widely known technique is 
referred to as "representational difference analysis" (RDA); see, for 
example, U.S. Pat. No. 5,436,142 and Lisitsyn et al., Science 259:946 
(1993). RDA is a subtractive hybridization method that uses restriction 
digestion of genomic DNA, followed by amplification and selection methods 
to isolate molecules that are present in one population but are lacking in 
a second nucleic acid sample. This method however requires multiple steps, 
numerous costly reagents and several weeks of time in the laboratory to 
obtain results. 
Genomic analysis, particularly at the human level, is highly complex and 
involves the analysis of tremendous amounts of nucleic acid. Processes 
which can selectively, simply and quickly isolate disease-associated 
sequences from complex nucleic acid samples will enable the science of 
molecular biology to uncover the keys to the genome and to disease. 
SUMMARY OF THE INVENTION 
The invention disclosed here allows for the differential isolation of 
nucleic acid sequences that are present in one nucleic acid population and 
not in another. The method is based upon using restriction endonucleases 
to digest two populations of nucleic acid, preferably cDNAs, ligating 
different sets of adaptors to each of the two nucleic acid populations, 
followed by hybridization, restriction digestion and isolation of the 
desired molecules. The unique aspects of this invention include the use of 
a restriction enzyme to isolate the target duplex DNA molecule from a 
hybridization mixture. Certain embodiments of the invention include the 
direct or indirect incorporation of a capture molecule or ligand (e.g., 
biotin, digoxigenin) within the amplified nucleic acid fragments, which 
allows molecules to be rescued from both the captured population as well 
as the effluent or otherwise uncaptured population. 
The methods described here are applicable to a wide variety of situations. 
In determining the presence or absence of particular DNA sequences, 
particularly associated with recessive or dominant traits, one can compare 
two related sources of DNA to determine whether they share the particular 
sequence, where the sequence may be a coding or non-coding sequence, but 
will be inherited in association with the DNA sequence associated with the 
trait. One can use the subject methods in forensic medicine, to establish 
similarities between the DNA from two sources, where one is interested in 
the degree of relationship between the two sources. The subject methods 
can also be applied in the study of diseases, where one can investigate 
the presence of a sequence associated with infection or cause, such as a 
vital sequence which may or may not be integrated into the genome. One may 
also use samples from a non-infectious disease source and compare it with 
a source without the disease to determine if there is a genetic basis, to 
identify genetic rearrangements, and for the identification of 
polymorphisms. Further, differences can be elucidated in species of 
interest to aid in taxonomic classification or even to determine possible 
contamination in nucleic acid samples of interest. 
DETAILED DESCRIPTION 
The methods and kits of this invention provide for simple and relatively 
inexpensive means to determine similarities or differences between two 
nucleic acid populations. Basically, the methods provide for: 
(1) Fragmentation of sample nucleic acid by restriction digestion. The 
fragmentation of the nucleic acid in which unique sequences are suspected 
(the "first nucleic acid sample") and the restriction digestion of the 
nucleic acid to which the first nucleic acid is to be compared (the 
"second nucleic acid sample"). Preferably the nucleic acid samples are 
cDNA samples derived from RNA. The first and second nucleic acid samples 
are subjected to a restriction endonuclease to produce fragments ("first 
and second nucleic acid sample fragments", respectively). 
(2) Ligation of Adaptors. Adaptors with a restriction site are ligated to 
the first nucleic acid sample fragments and adaptors with a second and 
preferably a different restriction site are ligated to the second nucleic 
acid sample fragments. The adaptors may optionally contain a ligand 
binding end (defined below). 
(3) Optional Fragment Amplification. If the first and second nucleic acid 
sample fragments are amplified, they are amplified with primers containing 
a ligand binding end and a sequence complementary to the adaptors. 
(4) Hybridization of First and Second Nucleic Acid Fragments. The first and 
second nucleic acid fragments are combined under hybridization conditions. 
(5) Isolation of Target Nucleic Acid. Double-stranded nucleic acid 
fragments in which both strands are first nucleic acid fragments are 
isolated. 
The last step is preferably done by first removing the adaptors by 
restriction digestion and then capturing the molecules which still contain 
the ligand binding end. The molecules which are not captured may then be 
isolated and amplified. The method will be described in more detail below. 
A. Fragmentation of Sample Nucleic Acid. 
For the purposes of this invention, two or more sources of nucleic acid are 
used from which the test samples are to be derived for comparison 
purposes. The sources of nucleic acid may be any sources of nucleic acid 
in which one is interested in comparing for differences. The sources may 
be eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, 
non-mammalian, plant and others. 
The methods described here are particularly well-suited to the use of cDNA, 
which is preferred when complex genomes are of interest. RNA may be 
isolated by any known means as a subset of the genomic nucleic acid and 
subsequent synthesis of cDNA. The use of RNA provides a unique source as 
it represents an initial fragmentation of the genome. 
It is also desirable to use cDNA as the first nucleic acid sample in assays 
in which cDNA or RNA is used as the second nucleic acid sample to prevent 
the isolation of products that are derived from intronic genomic 
sequences. For any of the analysis methods described here, it will be 
understood that RNA viruses, novel mRNAs expressed in cancers and other 
RNA of interest such as RNA used as a representation of a genome of 
interest can be detected by first obtaining corresponding cDNA by any 
method known in the art such as by using reverse transcriptase. See, for 
example, Innis et al., PCR Protocols, infra and Ehrlich, ed., PCR 
Technology, W. H. Freeman and Company, N.Y. (1991), incorporated by 
reference herein. 
If genomic DNA is to be the source, it is isolated, freed of protein, and 
then substantially completely digested with at least one restriction 
nuclease. Not all restriction endonucleases will be equivalent in the ease 
with which target DNA may be identified. Therefore, in each case it may be 
desirable to use a plurality of restriction endonucleases in separate 
determinations, not only to ensure that one obtains target DNA within a 
reasonable number of cycles, but also to increase the number of target DNA 
sequences that may be obtained. Alternatively and conveniently, though, as 
described above, RNA may be the source of nucleic acid, and cDNA is 
synthesized for testing, representing a subset of a genomic sample. 
Normally the first and second nucleic acid samples will be those which are 
expected to have substantially similar nucleic acid sequences. 
Whatever the source, the first and second nucleic acid samples are 
separately subjected to at least one restriction endonuclease. The 
restriction endonuclease may provide for blunt ends or staggered (sticky) 
ends, usually staggered ends. It is preferred that both first and second 
nucleic acid samples are subjected to the same restriction endonuclease 
and that such endonuclease is one which recognizes and cuts at a four base 
site. For the subsequent steps it is further preferred that such 
restriction endonuclease be one which recognizes a four base sequence 
found within a longer six base or eight base sequence recognized by a 
restriction endonuclease. Almost 1500 restriction endonucleases are now 
known and at least 150 are commercially available. Complete lists plus 
details of restriction sites and reaction conditions are published, for 
example in Brown, T. A. Molecular Biology Labfax, BIOS, Oxford (1991). 
B. Ligation of Adaptors. 
Once the first and second nucleic acid samples have been separately 
fragmented to produce first and second nucleic acid sample fragments, 
double-stranded oligonucleotide adaptors are ligated onto the ends of each 
of the strands of the fragments. The adaptor will usually be staggered at 
both ends, with one strand being longer than the other. The adaptors will 
generally serve to provide the sequence complementary to a primer to be 
used when a subsequent amplification step is employed. Thus, typically one 
end of the adaptor will be double-stranded and have one end complementary 
to the ends of the double-stranded nucleic acid fragments from the 
digestion, sometimes referred to herein as the proximal end of the 
adaptor. Each adaptor will preferably further contain a restriction site 
located distal to the proximal end. 
The restriction site in the adaptor is preferably one which has a six or 
eight base consensus sequence, and most preferably is such a one that 
further contains a 3' sequence that ends in a four base consensus sequence 
that has ends that are complementary to the same ends that are created by 
the six or eight base cutter that is adjacent and external to it. Examples 
of such restriction endonucleases include, but are not limited to, DpnII 
('GATC); BglII (A'GATCT); BamHI (G'GATCC); Tsp509I ('AATT), EcoRI 
(G'AATTC) and PacI (TT'AATTAA). 
It is preferred for the purposes of the methods of this invention that the 
adaptors used for the first nucleic acid sample fragments contain a 
restriction site which is different than the one used in the adaptors for 
the second nucleic acid sample fragments. The adaptor may further 
optionally contain a ligand binding end. A ligand binding end is 
particularly important if the fragments will not be amplified. It is 
preferred that the adaptor have one strand longer than the other to serve 
as a complement to primers if the fragments are to be amplified. 
In one embodiment, only the first nucleic acid sample fragments will 
contain adaptors having a restriction site. The second nucleic acid sample 
fragments do not necessarily need to have adaptors or a primer used for 
amplification with a restriction site. If this embodiment is employed, the 
adaptors and/or primers for the second nucleic acid sample fragments will 
have a ligand binding moiety to enable capture of the second nucleic acid 
sample fragments. 
Further, in another embodiment, it is possible to ligate the same adaptors 
onto nucleic acid of the first nucleic acid sample and the second nucleic 
acid sample if different primers are subsequently used to amplify the two 
sample populations so long as a restriction endonuclease site is encoded 
within the primers used to amplify the first nucleic acid sample 
fragments. 
Additionally, the adaptor ligated onto the first nucleic acid sample 
fragments may have "non-nested" restriction endonuclease sites; e.g. 
5'EcoRI--GATC3', where the EcoRI site is external to an initial DpnII 
digestion site. This protocol is less preferred, however, because when the 
EcoRI site is subsequently targeted by the restriction endonuclease in 
order to release the homoduplex from its biotinylated adaptors, 
approximately 1/16 of the cDNA molecules may contain an internal EcoRI 
site. 
C. Optional Amplification of First and Second Nucleic Acid Fragments. 
The first and second nucleic acid fragments may be separately amplified to 
enhance the assay, preferably by the polymerase chain reaction or other 
methods discussed in general below, using primers containing a sequence 
complementary to the respective adaptors and a ligand binding end. 
Thus, the second nucleic acid sample fragments and the first nucleic acid 
sample fragments may be amplified separately by adding appropriate primers 
complementary to the adaptors using the polymerase chain reaction (PCR), 
typically for about 10-35 cycles, more typically about 20 cycles, 
depending upon the initial concentration of second or first nucleic acid 
sample fragments being amplified. For a general overview of PCR, see PCR 
Protocols: A Guide to Methods and Applications (Innis, M.; Gelfand, D.; 
Sninsky, J. and White, T.; eds.), Academic Press, San Diego (1990), and 
U.S. Pat. Nos. 4,683,195 and 4,683,202, all incorporated herein by 
reference. In the methods described, here, the adaptors do not need to be 
removed. 
D. Hybridization of First and Second Nucleic Acid Fragments. 
The amplified first and second nucleic acid sample fragments are combined 
under hybridization conditions such that the fragments hybridize together 
creating essentially several possible complexes: first nucleic acid/second 
nucleic acid matches, second nucleic acid/second nucleic acid matches, and 
first nucleic acid/first nucleic acid matches. It is preferred that the 
second nucleic acid fragments are present in excess of the first nucleic 
acid sample fragments to increase the probability that the first nucleic 
acid/first nucleic acid complexes are representative of nucleic acid not 
found in the second nucleic acid sample. 
Second nucleic acid sample fragments will then be combined with the 
adaptor-ligated first nucleic acid fragments, with the second nucleic acid 
sample fragments present in excess, usually at least 5-fold excess and 
less than 500-fold excess, preferably about 100-fold excess for the first 
cycle of hybridization. Hybridization will be allowed to proceed at high 
stringency temperatures, usually about 60.degree.-70.degree. C. Various 
buffers and salt concentrations may be used to adjust for the desired 
stringency as will be appreciated by those in the art. 
E. Isolation of First Nucleic Acid/First Nucleic Acid Complexes. 
The first nucleic acid/first nucleic acid complexes present in the combined 
first and second nucleic acid solution can be readily separated from the 
other complexes depending upon the ligand used. Most conveniently, all of 
the combined fragments will be subjected to a restriction enzyme which 
recognizes the site in the first nucleic acid sample adaptors which will 
effectively remove the ligand binding end from the first nucleic 
acid/first nucleic acid molecules and not from the others. Thus, by 
capture technology which will attract the ligand binding end of the second 
nucleic acid/first nucleic acid complexes, one can readily separate out 
the first nucleic acid/first nucleic acid complexes. The first nucleic 
acid/first nucleic acid complexes may be further amplified and isolated, 
by, for example, ligating new adaptors onto the ends of the molecules and 
amplifying by PCR. 
It may be of interest to carry out the process more than once, where 
different restriction endonucleases are used. Different fragments may be 
obtained and result in additional information. 
Any resulting unique first nucleic acid sequences (i.e. those not found in 
the second nucleic acid sample) can be used as probes to identify sites in 
the first nucleic acid sample which differ from the second nucleic acid 
source. For this purpose they may be labeled in a variety of ways. 
Desirably in order to obtain substantially homogeneous compositions of 
each of the first nucleic acid sample sequences, the first nucleic acid 
sample sequences may be cloned by inserting into an appropriate cloning 
vector for cloning in a prokaryotic host. If desired, the cloned DNA may 
be sequenced to determine the nature of the target DNA. Alternatively, the 
cloned DNA may be labeled and used as probes to identify fragments in 
libraries carrying the target DNA. The target DNA may be used to identify 
the differences which may be present between the two sources of nucleic 
acid. 
The resulting target DNA will be greatly enriched. It may be used as probes 
to identify sites on the first nucleic acid sample sequences which differ 
from the second nucleic acid. The target nucleic acid may be sequenced 
directly by PCR or it may be cloned by inserting it in a cloning vector 
for cloning into a host cell. The cloned DNA can be sequenced to determine 
the nature of the target DNA through the use of dot blotting or other 
procedure. It may also be labeled and used as probes to identify fragments 
in libraries carrying the target DNA. Sequences can be identified and 
cloned for sequencing. Comparative searches with sequences described in 
accessible libraries such as Genbank (National Center for Biotechnology 
Information, Natl. Library of Medicine, National Institutes of Health, 
8600 Rockville Pike, Bethesda, Md. 20894); Protein Identification Resource 
(PIR, Natl. Biomedical Research Foundation, 3900 Reservoir Road NW, 
Washington, D.C. 20007; EMBL, European Molecular Biology Laboratory, 
Heidelberg, Germany) can aid in identifying the sequence. 
Other Definitions and General Techniques 
The term "ligand" or "ligand binding end" refers to a component which may 
directly or indirectly be detected or captured by another component, the 
"anti-ligand" which permits the physical or chemical separation of 
compositions bearing the ligand from those which do not. The ligand will 
be attracted to an anti-ligand molecule such that molecules which do not 
bear the ligand will not be captured or otherwise attracted to the 
anti-ligand. The ligand will need to be one which may be attached directly 
or indirectly to nucleic acid sequences. Examples of direct ligand binding 
include the use of biotin labeled nucleotides or the use of digoxigenin. 
These molecules can be used as the ligand binding component. They can be 
readily captured by their anti-ligand, e.g. avidin or streptavidin in the 
case of biotin and an anti-digoxigenin antibody, bound on a suitable 
substrate. (These reagents are all readily available, see Clontech 
Laboratories, Inc., Palo Alto, Calif. for digoxigenin reagents, for 
example.) Molecules which do not bind the anti-ligand can be collected and 
captured, by for example passing them through a streptavidin column. This 
direct capture method is preferred as it is likely to be the simplest, 
least costly and most efficient of the capture technologies available. 
Nevertheless other methods may be used as well so long as they accomplish a 
similar purpose. The ligand could alternatively be a specific nucleic acid 
sequence with the anti-ligand being the complement of the sequence or an 
antibody specific for the sequence. The ligand could include labeled 
molecules which may be manipulated on a substrate so that they are 
physically or chemically separated from non-ligand bearing molecules. 
Alternatively, the ligand molecule can have affinity for an anti-ligand 
molecule which is labeled or inherently detectable. These compositions can 
be further detectable by spectroscopic, photochemical, biochemical, 
immunochemical, or chemical means. For example, useful nucleic acid labels 
may include enzymes (e.g., LacZ, CAT, horse radish peroxidase, alkaline 
phosphatase and others, commonly used as detectable enzymes, either as 
marker gene products or in an ELISA), nucleic acid intercalators (e.g., 
ethidium bromide) and colorimetric labels such as colloidal gold or 
colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) 
beads, substrates, cofactors, inhibitors, fluorescent moieties (e.g., 
fluorescein and its derivatives, Texas red, rhodamine and its derivatives, 
dansyl, umbelliferone and the like), chemiluminescent moieties (e.g. 
luciferin and 2,3-dihydrophthalazinediones), magnetic particles, and the 
like. Labeling agents optionally include e.g., monoclonal antibodies, 
polyclonal antibodies, proteins, or other polymers such as affinity 
matrices, carbohydrates or lipids, fluorescent dyes, electron-dense 
reagents, enzymes (e.g., as commonly used in an ELISA), or haptens and 
proteins for which antisera or monoclonal antibodies are available. A wide 
variety of labels suitable for labeling nucleic acids and conjugation 
techniques are known and are reported extensively in both the scientific 
and patent literature, and are generally applicable to the present 
invention for the labeling of nucleic acids, or amplified nucleic acids 
for detection and isolation by the methods of the invention. The choice of 
label depending on the sensitivity required, ease of conjugation of the 
compound, stability requirements, available instrumentation, and disposal 
provisions. Separation and detection of nucleic acids proceeds by any 
known method, including immunoblotting, tracking of radioactive or 
bioluminescent markers, Southern blotting, northern blotting, southwestern 
blotting, northwestern blotting, or other methods which track a molecule 
based upon size, charge or affinity. 
Means of detecting labels are well known to those of skill in the art. 
Thus, for example, where the label is a radioactive label, means for 
detection include a scintillation counter or photographic film as in 
autoradiography. Where the label is a fluorescent label, it may be 
detected by exciting the fluorochrome with the appropriate wavelength of 
light and detecting the resulting fluorescence, e.g., by microscopy, 
visual inspection, via photographic film, by the use of electronic 
detectors such as charge coupled devices (CCDs) or photomultipliers and 
the like. 
Similarly, enzymatic labels may be detected by providing appropriate 
substrates for the enzyme and detecting the resulting reaction product. 
Finally, simple colorimetric labels are often detected simply by observing 
the color associated with the label. Thus, in various dipstick assays, 
conjugated gold often appears pink, while various conjugated beads appear 
the color of the bead. 
Substrates to be used as an environment for the capture and separation of 
the ligand bound molecules from those without ligand depend on the ligand 
being used and the desired format. For instance, the solid surface is 
optionally paper, or a membrane (e.g.,. nitrocellulose), a microtiter dish 
(e.g., PVC, polypropylene, or polystyrene), a test tube (glass or 
plastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, 
and the like), a microcentrifuge tube, or a glass, silica, plastic, 
metallic or polymer bead or other substrate as described herein. The 
desired anti-ligand may be covalently bound, or noncovalently attached to 
the substrate through nonspecific bonding. 
A wide variety of organic and inorganic polymers, both natural and 
synthetic may be employed as the material for the solid surface. 
Illustrative polymers include polyethylene, polypropylene, 
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene 
terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene 
difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose 
acetate, nitrocellulose, and the like. Other materials which are 
appropriate depending on the assay include paper, glasses, ceramics, 
metals, metalloids, semiconductive materials, cements and the like. In 
addition, substances that form gels, such as proteins (e.g., gelatins), 
lipopolysaccharides, silicates, agarose and polyacrylamides can be used. 
Polymers which form several aqueous phases, such as dextrans, polyalkylene 
glycols or surfactants, such as phospholipids, long chain (12-24 carbon 
atoms) alkyl ammonium salts and the like are also suitable. Where the 
solid surface is porous, various pore sizes may be employed depending upon 
the nature of the system. 
In preparing the surface, a plurality of different materials are optionally 
employed, e.g., as laminates, to obtain various properties. For example, 
protein coatings, such as gelatin can be used to avoid non specific 
binding, simplify covalent conjugation, enhance signal detection or the 
like. If covalent bonding between a compound and the surface is desired, 
the surface will usually be polyfunctional or be capable of being 
polyfunctionalized. Functional groups which may be present on the surface 
and used for linking can include carboxylic acids, aldehydes, amino 
groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups 
and the like. In addition to covalent bonding, various methods for 
noncovalently binding an anti-ligand component can be used. For additional 
information regarding suitable ligand-anti-ligand and labeling technology 
as it relates to nucleic acids, see, for example, Essential Molecular 
Biology, ed. T. A. Brown IRL Press (1993); In Situ Hybridization 
Protocols, ed. K. H. Andy Choo, Humana Press (1994). 
By a nucleic acid sequence "homologous to" or "complementary to", it is 
meant a nucleic acid that hybridizes, duplexes or binds only to DNA 
sequences encoding one protein or portions thereof. A DNA sequence which 
is homologous to a target sequence can include sequences which are shorter 
or longer than the target sequence so long as they meet the functional 
test set forth. Hybridization conditions are specified along with the 
source of the DNA. Typically the hybridization is done in a Southern blot 
protocol using a 0.2XSSC, 0.1% SDS, 65.degree. C. wash. The term "SSC" 
refers to a citrate-saline solution of 0.15M sodium chloride and 20 mM 
sodium citrate. Solutions are often expressed as multiples or fractions of 
this concentration. For example, 6XSSC refers to a solution having a 
sodium chloride and sodium citrate concentration of 6 times this amount or 
0.9M sodium chloride and 120 mM sodium citrate. 0.2XSSC refers to a 
solution 0.2 times the SSC concentration or 0.03M sodium chloride and 4 mM 
sodium citrate. 
Accepted means for conducting hybridization assays are known and general 
overviews of the technology can be had from a review of: Nucleic Acid 
Hybridization: A Practical Approach, Ed. Hames, B. D. and Higgins, S. J., 
IRL Press, 1985; Hybridization of Nucleic Acids Immobilized on Solid 
Supports, Meinkoth, J. and Wahl, G.; Analytical Biochemistry, Bol 238, 
267-284, 1984 and Innis et al., PCR Protocols, supra, all of which are 
incorporated by reference herein. 
Nucleic acids of interest in the present invention may be cloned or 
amplified, or any of the nucleic acid fragments may be amplified by in 
vitro methods, such as the polymerase chain reaction (PCR), the ligase 
chain reaction (LCR), the transcription-based amplification system (TAS), 
the self-sustained sequence replication system (3SR) and the Q.beta. 
replicase amplification system (QB). A wide variety of cloning and in 
vitro amplification methodologies are well-known to persons of skill. 
Examples of these techniques and instructions sufficient to direct persons 
of skill through many cloning exercises are found in Berger and Kimmel, 
Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic 
Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) Molecular 
Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor 
Laboratory, Cold Spring Harbor Press, N.Y., (Sambrook et al.); Current 
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current 
Protocols, a joint venture between Greene Publishing Associates, Inc. and 
John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion et al., U.S. 
Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864. Examples of 
techniques sufficient to direct persons of skill through in vitro 
amplification methods are found in Berger, Sambrook, and Ausubel, as well 
as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to 
Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, 
Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The 
Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. 
Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 
87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., 
(1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; 
Wu and Wallace, (1989) Gene 4, 560; and Barringer et al. (1990) Gene 89, 
117. 
The term "identical" in the context of two nucleic acid sequences refers to 
the residues in the two sequences which are the same when aligned for 
maximum correspondence. Sequences which are not identical are "different." 
F. Kits 
Further contemplated are kits for the assays described here. Combinations 
of reagents useful in the methods set out above can be packaged together 
with instructions for using them in the described method. In particular, 
kits containing a separate container for first nucleic acid sample 
adaptors and for second nucleic acid sample adaptors can be prepared. 
Preferably such kits will also contain instructions for the subtractive 
capture methods. Alternatively the kits can contain separate containers 
for primers to amplify the first nucleic acid sample fragments and primers 
to amplify the second nucleic acid sample fragments as described above. 
Further, the kits could contain separate containers of first nucleic acid 
sample primers, second nucleic acid sample primers, first nucleic acid 
sample adaptors and second nucleic acid sample adaptors, all as described 
above. 
All of the literature and patent references cited herein provide additional 
background and general guidance and are incorporated by reference herein.