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WO1989001526A1 - Librairie et procede de clonage par coincidence - Google Patents

Librairie et procede de clonage par coincidence Download PDF

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Publication number
WO1989001526A1
WO1989001526A1 PCT/US1988/002631 US8802631W WO8901526A1 WO 1989001526 A1 WO1989001526 A1 WO 1989001526A1 US 8802631 W US8802631 W US 8802631W WO 8901526 A1 WO8901526 A1 WO 8901526A1
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Prior art keywords
fragments
dna
mixture
fragment
sequences
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PCT/US1988/002631
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English (en)
Inventor
Francis Collins
Sherman Weissman
Charles Cantor
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Genelabs Incorporated
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Publication of WO1989001526A1 publication Critical patent/WO1989001526A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the present invention relates to methods for obtaining DNA sequences which are common to two DNA frag ⁇ ment mixtures derived from different sources, and to uses of the method for gene mapping and cloning.
  • Sorted chromosomes isolated by physical methods from various cell types, and cloned sequence libraries prepared from sorted chromosomes, many of which are commercially available (American Type Culture Collection, Rockville, MD) contain genetic material from a selected chromosome, and are available for most, although not all, human chromosomes. While such sorted chromosomes have been valuable in providing genetic sequences for regions of interest in many cases, they do have some important limitations. One is a relatively high level of contamina ⁇ tion with nonspecific genetic material, which decreases the utility of sorted chromosome material in isolating sequences of interest.
  • Subtractive hybridization techniques have proven to be very valuable in isolating target genetic sequences present in only one of two sources. This is useful, for example, in isolating mRNAs (or the corresponding cDNAs) which are expressed in various cell types after activation 'or other stimuli. These methods rely on the use of two cell sources which are largely identical, only one of which contains the sequence of interest. Most often these are mRNAs or the corresponding cDNAs, although genomic DNA may also be used. Furthermore, these methods rely on the use of an excess of sequences from the source which does not contain the sequence of interest, in order to drive the hybridization reaction towards the formation of heteroduplexes.
  • Another object of the invention is to provide a variety of techniques which can be used to obtain such common sequences, according to the method of the inven ⁇ tion.
  • Still another object of the invention is to use
  • fl sequences in a mixture of genomic fragments is yet another object of the invention.
  • the method of the invention is designed for obtaining from a first mixture of DNA duplex fragments derived from one source, those fragments which are
  • the frag ⁇ ments are generated in a manner which allows heteroduplex, end-hybridized fragments formed by the hybridization of homologous DNA strands from the two DNA fragment mixtures to be isolated from homoduplex fragments produced by hybridization between opposite strands of the fragments in the first or second mixture only, and from heteroduplex fragments which are not end-hybridized. Denatured strands from the fragments of the first and
  • hybridization reaction may be carried out with a molar ratio of the two fragment mixtore or with a molar excess of one of the mixtures.
  • the fragments are generated in such a way that when the paired strands form-
  • the ligatable fragment ends in the end- hybridized heteroduplex fragments may be generated either
  • linkers may additionally contain methylated sites which allow generation of unique end pairs in heteroduplex fragments
  • one of the fragment mixtures is modified with a label which allows physical separation of heteroduplex fragments from homoduplexes.
  • the label may be an affinity label, such as biotin, which allows separation of heteroduplex species based on (a) initial binding to an affinity column and (b) subsequent release of the unlabeled strand of the heteroduplex by duplex denaturation.
  • the label may be a density label which permits physical separation of heteroduplex from homoduplex strands based on density
  • the fragments in the two mixtures are cloned in a vector which allows expression of one fragment strand or its transcript from one mixture, and the opposite fragment strand or its ⁇ g transcript from the other mixture. Separation of the heteroduplexes in this procedure is based on duplex forma ⁇ tion and separation, for example, on a hydroxylapatite column.
  • one method of use is for cloning ahd/or analyz ⁇ ing the gene sequences, and preferably the single-copy sequences, which are carried on defined chromosomes or chromosome regions.
  • the sources of the two DNA fragment mixtures may be a two-species cell hybrid
  • Another important application of the method is for obtaining clones derived from a DNA fragment contained in a mixture of fragments, such as are typically obtained when DNA fragments are subfractionated, as by gel
  • partial digest fragments of genomic DNA when fractionated by pulse field gel electrophoresis, will yield several band regions contain ⁇ ing a gene region of interest, as evidenced by the binding of a selected probe to each of the regions of interest. After eluting the fragments from each of two such gel regions, these are then hybridized, according to the method of the invention, to produce common-sequence heteroduplex fragments derived from the desired probe- binding fragments on the gel.
  • the method may also be used for identifying and
  • Another application of the method is for identifying and cloning specific chromosomal regions, such as the telomere regions at the end of chromosomes which Q appear to be required for chromosome stability.
  • the method here involves cloning the coincident gene sequences from hybrid cells, each of which contains the chromosomal region of interest.
  • the method can also be used to enrich a mixture g of genomic DNA fragments for single-copy sequences, either applied to a single DNA fragment mixture, such as total genomic fragments from a given source, or in conjunction with other applications mentioned above, in which the co ⁇ incident fragments isolated by the method are enriched for
  • the invention includes a library of cloned DNA sequences produced by treating two DNA fragment mixtures according to the method of the invention, where the end-hybridized heterologous fragments
  • Figure 1 is illustrates the method of the inven ⁇ tion, wherein heterologous duplex fragments are isolated from homologous fragments on the basis of different frag ⁇ 0 ment ends present in the heteroduplexes;
  • Figure 2 illustrates the method in another embodiment wherein fragments in the two fragments are equipped with different linkers, and heteroduplex frag ⁇ ments are selected on the basis of different restriction c sites formed by the two linkers at the opposite fragment ends;
  • Figure 3 illustrates the method in another .embodiment, in which the original homoduplex fragments are methylated at one of two different restriction sites, and Q heteroduplex fragments are isolated on the basis of unique opposite-end restriction sites after digestion with endonucleases corresponding to the two methylase sites;
  • Figure 4 shows the method in a related embodiment, in which linkers attached to each of the 5 mixtures of fragments contain two common internal restric ⁇ tion sites, one of which is methylated, and different end sites, and heteroduplexes are distinguished from homoduplexes on the basis of different end sites which result after digestion with endonucleases specific for the internal linker sites;
  • Figure 5 illustrates another general embodiment of the method, in which heteroduplex molecules are isolated on the basis of binding to an affinity column and release of one strand of the heteroduplex on denaturation, where the released single-strand is contained in a cloning vector which can be readily converted to-- double-strand form;
  • Figure 6 illustrates an embodiment which is similar to that in Figure 5, but where the released single-strand material is annealed to form duplex frag ⁇ ments which can be cloned into a suitable cloning vector;
  • Figure 7 illustrates the method of the invention as it can be practiced using density-gradient centrifuga ⁇ tion to separate heteroduplex from homoduplex fragments;
  • Figure 8 illustrates another method for carrying
  • Figure 9 shows the steps in the application of the invention to isolating clones from single fragments obtained from a gel band region; and 15 Figure 10 illustrates the application of the method to isolating a human chromosome telomere.
  • End-hybridized A fragment is end- hybridized if it is formed from end-hybridizable frag ⁇ ments. Typically, the strands forming an end-hybrized fragment will be hybridized along their entire lengths.
  • Ligatable ends The ends of a duplex frag ⁇ ments are ligatable if the fragment can be selectively incorporated into a cloning vector having defined ligation ends, in the presence of suitable ligation enzymes in vitro or in vivo.
  • Ligatable ends include sticky ends, i.e., ends with short orverhang sequences capable of hybridizing with complementary overhang sequences, and blunt ends. Typically end-hybridized fragments will have ligatable ends.
  • Homoduplex fragments are those formed by hybridization between homologous- fragment strands derived from the the same DNA fragment mixture.
  • Heteroduplex frag ⁇ ments are those formed by hybridization between homologous-fragment strands derived from different DNA fragment mixtures.
  • the method of the invention is aimed at obtain ⁇ ing gene sequences which are coincident in, i.e., common to the DNA fragments in two different mixtures of gene fragments. More particularly, the method is designed to obtain from the first mixture of duplex fragments, those fragments which are homologous to and end-hybridizable with the duplex fragments in the second fragment mixture.
  • At least one of the mixtures is prepared in a manner such that when a strand from one mixture is hybridized with a homologous, end-hybridizable strand from the second mixture, the resulting end-hybridized heteroduplex fragment has proper ⁇ ties which allow its separation from homoduplex fragments formed by hybridization between opposite strands of the 0 fragments in the first or second mixtures only, and from duplex heteroduplex fragments which are not end- hypridized.
  • Part D below describes embodiments of the method in which end-hybridized heteroduplex separation is based on unique fragment ends which allow cloning into a 5 vector with selected insertion sites.
  • Part C the methods of separating heteroduplex from homoduplex fragments involve physical separation of labeled fragments.
  • Part C describes another general ap ⁇ proach to the invention, in which heteroduplex separation 0 is based on duplex formation from cloned, single-strand species.
  • the mixture(s) of duplex DNA fragments used in 5 the invention can be derived from a variety of multi-gene DNA source(s) , such as the genomic DNA from eukaryotic cells or tissue samples, isolated chromosomes, mitochondrial DNA, and subfractions of DNA obtained by various DNA fragment separation procedures, such as gel Q electrophoresis or centrifugation methods.
  • the actual source material used for DNA isolation may be whole cells, or subfractionations thereof, such as cell nuclei, or isolated chromosomes from cells.
  • the cell line used as the _ DNA source for at least one of the fragment mixtures is a hybrid cell line containing at least one- chromosome or chromosome region from one species, and the balance of the chromosome material from one or more other species.
  • hybrids may be obtained from known sources, or produced according to published methods.
  • Example 1 utilizes as one source of DNA material, the genomic DNA obtained from the somatic cell hybrid HHW661, a hamster- human hybrid containing a translocation of human chromo ⁇ some region 4p onto hamster chromosome 5 (Wasmuth) .
  • the two sources of DNA are both hybrid cells, one containing a human chromosome 8, and another a human chromosome 4 with a translocated portion of human chromosome 8.
  • the DNA can be isolated by standard procedures, which typically include successive phenol and phenol/chloroform extractions (Maniatis, p. 280).
  • Example 1 describes the isolation of genomic DNA from two cell lines.
  • the DNA mixtures are derived from subfractionated DNA fragments, such as from the agarose gels, conventional methods of DNA extraction, such as electroelution, gel maceration, or the like are used. The elution of DNA fragments from agarose gel regions is described in Example 9.
  • the isolated DNA is obtained in relatively intact form, and is fragmented by digestion with one or more selected restriction endonucleases, to form the desired mixture of DNA fragments.
  • the DNA fragments in the mixture are formed by complete digestion with one or more endonucleases, to final fragment sizes of preferably between about 200 to 10,000 basepairs. Since in most applications, the heteroduplex fragments formed in the method are cloned, the upper size limit of fragments in the two mixtures is limited to clonable fragment sizes, generally less than 40 kilobases, and preferably no more than 10-20 kilobases.
  • restriction endonucleases used in forming the DNA fragments in the two mixtures will depend on the specific approach used for isolating heteroduplex fragments, as will be clear from the various approaches described in Parts B-D below. In particular, the approach used will dictate optimal fragment size and nature of the cut ends.
  • the fragments may be further modified by fill ⁇ ing recessed ends, ligation of end linkers and/or restriction-site methylation (Part B), by nucleotide 0 labeling (Part C) , and/or by cloning into a single-strand vector (Part D) . Methods for performing such modifica ⁇ tions are detailed in Examples 1-8 below.
  • the method is effective to isolate heteroduplexes consisting of end- Q hybridizable, homologous strands from homoduplex fragments and from duplex fragments which are not end-hybridized, i.e., which have one or more extended, non-hybridized end regions. Since the preponderance of duplex fragments which are not end-hybridized are formed by hybridization 5 between repeat sequences, the method is therefore effec ⁇ tive in enriching for single-copy sequences which are co ⁇ incident to the two fragment mixtures.
  • DNA-I and DNA-II are each prepared by digesting the corresponding DNA material to completion with a restriction endonuclease, such as Mbol, which produces sticky fragment ends.
  • a restriction endonuclease such as Mbol
  • One of these fragment g mixtures, e.g., the DNA-1 mixture is further treated with Klenow fragment in the presence of the required nucleotides, to fill in both recessed ends of the frag ⁇ ments, forming blunt end fragments, as indicated.
  • the two fragment mixtures are now denatured and reacted under hybridization conditions which yield homoduplex and heteroduplex fragments.
  • the hybridization reaction may be carried out by traditional hybridization methods, involving slow hybrid formation in a single-phase aqueous or aqueous/formamide medium, at a reaction temperature slightly below the melting temperature of the duplex material, according to published methods (Britten, 1968; Britten, 1985; Hames).
  • the hybridization reaction is performed according to a more recent phenol emulsion reaction technique (PERT), or formamide-phenol emulsion reaction technique (F-PERT), which greatly accelerates the hybridization reaction (Kohne, Casna) .
  • PERT phenol emulsion reaction technique
  • F-PERT formamide-phenol emulsion reaction technique
  • the hybridization reaction produces three general classes of duplex fragments.
  • the first of these include original homoduplex fragments formed by hybridization between end-hybridizable homologous strands of the fragments in the first or second mixtures only. These homologous duplex fragments have either- opposite blunt ends or opposite sticky ends.
  • the second class of fragments are homoduplex or heteroduplex fragments formed from opposite strands which are not end- hybridizable. Typically such fragments are formed from imperfect copies of themselves, as is expected of repeat sequences contained in a variety of different-size digest fragments. At least one of the ends of these non- hybridized fragments is irregular in that it has a relatively long end-region of non-hybridized single-strand DNA.
  • the third class of fragments are end-hybridized heteroduplex fragments. As seen in the mid portion of Figure 1, these fragments have opposite sticky and blunt ends.
  • the fragment mixture formed by reacting the op ⁇ posite strands from the first and second DNA mixture are now cloned into a cloning vector which is designed to in ⁇ corporate selectively only those duplex fragments having
  • the vector is a plasmid, such as the pUC18 plasmid illustrated in Figure 1, which is cut at a polylinker site to expose ends which are compatible with the sticky and blunt ends-of the desired heteroduplex
  • reaction fragments are ligated into the vector after removal of the small polylinker segment.
  • Selection of successful recombinants, on a suitable host, is carried out by conventional methods. Since some of the
  • __ end-hybridized heteroduplex fragments may be formed from end-hybridizable homologous strands, the successful re ⁇ combinants may be further screened with labeled repeat sequence to eliminate the small percentage of repeat sequences.
  • Example 1 This method is detailed in Example 1, which generally follows the reaction scheme shown in Figure 1. Three out of five clones which were screened were heterologous fragment inserts, i.e., derived from sequences common to both genomic DNA sources. Only one of the 48 clones which were screened by repeat-sequence
  • the two DNA fragment mixtures are prepared by (a) digesting the first and second DNA with different endonucleases, such that the first and second fragment mixtures have different sticky
  • the nucleases used are selected such that the hybrid sticky ends formed by hybridization between first- and second-mixture equal-size strands are different from either of the homoduplex sticky ends in the original frag ⁇ ment mixtures.
  • Figure 2 illustrates another approach for generating fragment mixtures in which homoduplex and end- hybridized heteroduplex fragments can be separated by an appropriate cloning vector.
  • characteristic sticky ends used to distinguish homoduplex from end-
  • the first fragment, mixture is then mixed with one linker, designated linker I in the figure, which is designed for attachment to the fragment sticky end and provides an internal, preferably infrequent restriction 2 Q site, such as the Xhol site indicated. Treatment of this fragment mixture with the linker-site endonuclease now yields relatively small fragments with opposite rare- cutter site ends.
  • a second linker similarly designed for attachment to the original digest mixture and carrying a 25 second internal and preferably infrequent endonuclease site, such as NotI (linker II in Figure 2) is similarly attached to the second fragment mixture, which is then treated with the linker-site endonuclease, to generate a second fragment mixture composed of small fragments with 30 infrequent-site sticky ends.
  • linker sequences are also designed for hybridization with one another, as illustrated in linkers I and II in Figure 2.
  • the two fragment mixtures are now mixed, de- _ natured, and reannealed, as above, to produce hybridized fragments consisting of end-hybridized and non-end- hybridized homoduplex and heteroduplex fragments.
  • end-hybridized homoduplex fragments have opposite sticky ends which are either both linker I or both linker II ends; non-end-hybridized homoduplex and heteroduplex frag ⁇ ments have at least one irregular end; and end-hybridized heteroduplex fragments have one linker I end and an op ⁇ posite linker II end, as indicated. These fragments are mixed into a cloning vector which selectively incorporates the linkerl/linker II ends, and successful recombinants 0 are selected as above.
  • the two fragment mixtures are prepared with Xhol and NotI sticky ends, and the hybrid ⁇ ized fragments are cloned into the Xhol/NotI site of a 5 Bluescripts plasmid.
  • Figure 3 illustrates another procedure for preparing the fragment mixtures for selection of heteroduplex fragments on the basis of hybridized-end characteristics. This procedure utilizes methylation at Q internal restriction sites, followed by endonuclease treatment of the hybridization products, to generate unique fragment ends in equal-size heteroduplexes.
  • the DNA fragment mixtures are initially prepared by complete digestion with a one or more selected 5 endonucleases, where the endonuclease(s) used is selected to produce preferred fragment sizes of at least about 1,000-2,000 kilobases, to insure that most of the fragmenst contain internal frequent-cutting sequences, such as Alul and Haelll sequences.
  • the fragments shown in Figure 3 which are produced by BamHI (B) digestion, contain a single internal Alul (A) and two Haelll (H) restriction sites.
  • the first fragment mixture designated DNA-I in the figure, is treated with a selected methylase, such as Alul methylase, to methylate both fragment strands at one frequent-cutting site, as indicated by the "*" symbols in- the figure.
  • a selected methylase such as Alul methylase
  • the second fragment mixture is treated with a second methylase, such as Haelll methylase, to methylate both fragment strands at a second frequent-cutting site in the strands.
  • the two fragment mixtures are mixed, denatured, and reannealed, as above, to produce hybridized fragments consisting of both homoduplex and heteroduplex fragments.
  • hybridized fragments consisting of both homoduplex and heteroduplex fragments.
  • a fourth method of heteroduplex selection by cloning employs elements of both the end-linker and site- methylation approaches just described.
  • this method which is illustrated in Figure 4, fragment digestion and attachment of different linkers (linkers I and II in the
  • the linkers contain, in addition to the "proximal" sticky end used for ligation to the fragments, such as an Mbol sticky end, and a rare-cutting sequence near the "distal" linker end, such ⁇ g as a NotI sequence, two "internal" restriction sequences, in the present example, Alul, and Haelll sites.
  • the two internal-site sequences are referred to more generally as A and B sequences, and the distal-site sequences, such as NotI and Xhol sequences, as C and D sequences.
  • linker I in the figure has the sequences A/B/C and linker II, the sequences A/B/D.
  • the DNA-I fragment mixture is treated with a methylase which is specific for the A linker sequence, and
  • the DNA-II fragment mixture having the linker-II ends, is treated with a methylase specific for the B linker sequence.
  • the resulting fragment mixtures are methylated at both linker strands, at either the A or B sequence and at any " A or B internal sequences in the fragments, as indicated in the figure.
  • the two fragment mixtures prepared as above are now mixed, denatured and annealed, as above, to produce (a) end-hybridized homoduplex fragments which are protected at one or the other but not both of the A or B linker sequences, (b) non-end-hybridized homoduplex and heteroduplex fragments having at least one irregular end and (c) end-hybridized heteroduplex fragments which are protected at both A and B linker sequences, by virtue of different-strand methylation in the linker region, and having opposite-end C and D sequences.
  • Digestion of the reaction fragments with endonucleases specific for both A and B sequences cuts the homoduplexes at all A or B sequences, producing fragments with either A-sequence or B-sequence opposite sticky ends.
  • Heteroduplexes by contrast, are not cut by either endonuclease, and thus 0 retain their opposite C and D sequences.
  • Further diges ⁇ tion with endonucleases specific for C and D sequences now produce C and D sticky ends in the opposite ends of end- hybridized heteroduplex fragments. It can be appreciated that a small percentage of fragments containing internal C 5 or D sequences may have opposite C or opposite D sticky ends.
  • the digest fragments are now cloned into a suit ⁇ able vector containing C and D sticky end sites, and the successful recombinants selected as above.
  • the fragments ⁇ may also be cloned into vectors containing opposed C- sequence sticky ends, or D-sequence sticky ends, to clone those heteroduplex fragments containing internal C or D sequences.
  • Example 4 details a procedure which follows the general scheme shown in Figure 4. 5 As can be appreciated from the above, all of the procedures presented above share a number of common features and advantages:
  • the two fragment mixtures are generated from the associated DNA source in such a way that the hybridization products produced by reacting the two fragment mixtures under hybridization conditions can be separated on the basis of selective incorporation into a suitable cloning vector.
  • the method for isolating the desired heteroduplex fragments also yields a fragment library which is enriched for end-hybridizable, coincident sequences.
  • heteroduplex fragments are separated from homoduplex fragments on the basis of a physical property related to a nucleotide label.
  • the label may be either a density label, such as an 15N- 5 labeled nucleotide, or an affinity label, such as biotin, which is incorporated into both strands of one fragment mixture.
  • Heteroduplex fragment separation then involves isolating fragments containing one labeled and one unlabeled strand from completely labeled or completely 0 unlabeled homoduplex fragments.
  • DNA-II is cloned into a vector, such as M13, which can be grown in single strand form. Because the cloning vector is used as a source of one strand only (either the sense or anti-sense strand) , the original fragments are prepared by digestion with two
  • the DNA-I fragments are prepared by digestion with the same pair of enzymes.
  • one of the two mixtures, and preferably the c DNA-I fragment mixture is initially treated to remove repeat sequences. This can be done by conventional slow hybridization techniques carried out in a single-phase reaction system, as referenced above.
  • the de ⁇ natured fragments in the mixture are hybridized to an ⁇ J ⁇ Q initial C t value at which most of the repeated sequences are hybridized, and most of the single-copy sequences are still in single-strand form.
  • the sing 3 le-strand material is carried to a second Cot value at
  • biotin is the preferred affinity label
  • any label which can be incorporated into polynucleotides and which has a binding partner capable of binding the label specifically and with high affinity may be used.
  • the affinity label is also referred to herein as Q an epitopic molecule, and the binding partner, as a bind ⁇ ing molecule.
  • Exemplary binding pairs of epitopic molecule/binding molecule include biotin/avidin, biotin/ streptavidin, antigen/antibody, and carbohydrate/lectin.
  • the labeled, single-copy strands are now mixed with the cloning vector containing the DNA-II fragment inserts and grown under conditions which yield one vector strand (sense or anti-sense) only.
  • the mixture is de ⁇ natured and allowed to reanneal, as above.
  • the annealing reaction produces homoduplex fragments, heteroduplex fragments consisting of a labeled fragment strand from the DNA-I mixture, and the homologous DNA strand from the cloned DNA-II mixture, and single- strand species from both mixtures (not shown) .
  • a related method which does not require removing repeat sequences from one of the fragment mixtures is il ⁇ lustrated in Figure 6.
  • both fragment mixtures are generated by digestion with the same endonuclease, and one of the fragment mixtures is labeled, as indicated.
  • the labeled and unlabeled mixtures are now mixed, denatured, and reannealed, as above, producing homoduplex fragments with both or neither fragments labeled, and heteroduplex fragments with one strand only labeled.
  • the hybridization products are passed through an avidin or streptavidin column, binding labeled homoduplex and heteroduplex fragments to the column, with elution of the unlabeled homoduplex fragments.
  • the bound fragments are now denatured, as above, and the unlabeled single- strand species are eluted.
  • the eluted DNA strands are (a) all derived from the unlabeled fragment mixture, (b) represent both end- hybridizable and non-end-hybridizable strands, and (c) include both sense and anti-sense strands.
  • These single strand species are ethanol precipitated, and reannealed, forming homoduplex fragments which are derived from heteroduplex fragments only, i.e., are all coincident with fragments in the labeled fragment • mixture.
  • the reannealed end-hybridized duplex fragments (representing predominantly single-copy fragments), contain the same sticky ends as the original unlabeled fragments, whereas the duplex fragments which are not end-hybridized contain at least one irregular end.
  • the total fragments are mixed with a suitable cloning vector which selectively in ⁇ corporates the regular sticky end fragments, with selec ⁇ tion for successful recombinants as above.
  • the method is detailed in Example 6, where the fragment mixtures are formed with Mbol digestion, and the reannealed unlabeled fragments are cloned into the Mbol site of a p ⁇ C18 vector.
  • Figure 7 illustrates a method of density gradi ⁇ ent separation of heterologous and homologous fragments.
  • the two fragment mixtures are prepared by digestion with a frequent-cutting endonuclease, such as Mbol, and one of the fragment mixtures is labeled, as above, by in ⁇ corporation of a heavy isotopic nucleotide, such as N- labeled nucleotides, where the label may be carried in one or more of the nucleotide species.
  • a heavy isotopic nucleotide such as N- labeled nucleotides
  • Incorporation of the labeled nucleotides is by one of the methods detailed in Example 5B for incorporation of biotinylated nucleotides into duplex DNA.
  • the labeled and unlabeled fragments are mixed, denatured and reannealed as above, yielding homoduplexes with both or neither unlabeled strands and coincident heteroduplex fragments with one labeled and one unlabeled strand.
  • These three species of duplex fragments are then fractionated by equilibrium density centrifugation, ac ⁇ cording to classical procedures, such as on a CsCl gradi ⁇ ent.
  • the heteroduplex fragments fractionate between the lighter unlabeled homoduplexes and the heavier, fully labeled homoduplexes.
  • the heteroduplex fraction is recovered by aspiration.
  • This fraction contains both end-hybridized and non-end- hybridized fragments, and the former are isolated by clon ⁇ ing into an appropriate cloning site in a plasmid vector, as in the method immediately above. Details of this method are given in Example 7.
  • each of the two fragment mixtures is initially prepared by digestion with two selected endonucleases, such as EcoRI and Hindlll, producing fragments which can be inserted in an oriented fashion in a cloning vector which can be grown in either a single-strand or double- strand form.
  • endonucleases such as EcoRI and Hindlll
  • the two fragments mixtures are cloned into a pair of cloning vectors which are designed to receive fragments in one or two defined orientations, in a double-strand form, and which therefore produce opposite insert strands, in a single-strand form.
  • One such vector pair includes the vectors M13/mpl8 and M13/mpl9 which have polylinkers arranged in opposite orientations, for accepting inserts cut with a pair of selected endonucleases, such as EcoRI and Hindlll, in op- posite orientations.
  • the cloning step is shown in Figure 8, where the first EcoRI/Hindlll fragment mixture is cloned into an mpl9 plasmid in one orientation, and the second EcoRI/HindiII fragment mixture is cloned into an mpl8 plasmid in the opposite orientation.
  • the mpl9 vector produces the sense (+) strand of the insert, and the mpl8 vector, the anti-sense (-) strand.
  • phage complexes formed from end- hybridizable inserts allow end region annealing of the opposite-strand polylinker sequences present in the two cloning vectors, so that the duplex inserts are bounded by defined duplex restriction sequences, and in particular, the sequences used for inserting the original fragments into the double-strand vectors.
  • these sequences are those recognized by EcoRI and Hindlll.
  • opposite strand complexes formed from non-end- hybridizable fragments have at least one irregular mismatch at the insert end which prevents annealing at the vector polylinker sequences.
  • the annealed fragments are now digested with endonucleases which cut at opposite vector polylinker sites, and preferably at the sites used to introduce the original fragments into the two vectors, to avoid cutting the inserts themselves at internal sites.
  • endonucleases which cut at opposite vector polylinker sites, and preferably at the sites used to introduce the original fragments into the two vectors, to avoid cutting the inserts themselves at internal sites.
  • the- ⁇ coRI and Hindlll sites used to generate the original fragment mixtures, and to introduce the fragment mixtures into the two cloning vectors are also used to digest the duplex phage species.
  • the resulting digest fragments are then cloned into a suitable cloning vector, such as pUC18 opened at its EcoRI and Hindlll sites, which selectively incorporates the equal-size duplex fragments. This method is illustrated in Example 8.
  • This approach has the potential for greater discrimination between coincident and non-coincident sequences, since only coincident sequences form hybrid duplexes, and therefore could be introduced into a duplex cloning vector.
  • the method also has the potential for good discrimination between end-hybridizable and non-end- hybridizable duplexes, since only equal-size duplexes are released from the hybridized products in a clonable form.
  • the limitation of the method is the need for two cloning steps, one in forming the single-strand fragment mixtures, and the second in selecting single-copy annealed hybridization products.
  • This section discusses applications of the co ⁇ incident cloning method to gene mapping, gene isolation, chromosome construction, cloning of conserved genomic sequences, and removing repeat sequences from genomic DNA.
  • C4 human chromosome 4
  • the problem is to clone all of the single-copy sequences in human chromosome 4 (C4), for purposes of constructing a library of probes for C4.
  • C4 human chromosome 4
  • the hybrid would be a mouse/human or hamster/human hybrid containing a single C4 chromosome.
  • restriction fragments from this source (DNA-I) and from the entire human genome (DNA-II) are mixed, and reacted under hybridization conditions, according to one of the methods from Section II above, to produce hybridization products representing coincident sequences, i.e., sequences associated with C4.
  • the hybridization products are further treated and cloned, as
  • Example 1 which demonstrates the cloning of single-copy species associated with human chromosome 5
  • PFGE pulsed-field gel electrophoresis
  • PFGE can be used to fractionate large genomic fractions, and the fragments of interest, i.e., those associated with one or more identified probes, can be identified on the gel by probe binding techniques, such
  • duplex DNA shown at the top in the figure represents a segment of DNA containing a probe-binding region P which is adjacent a
  • the restric ⁇ tion sites S. are preferably at least about 100 kilobases from one another.
  • the objective is to clone fragments in the S ⁇ S 4 fragment segment only, for purposes of further
  • the DNA is partially digested with the endonuclease which cuts at the rare S sequences.
  • Methods for forming partial DNA digests which are suitable in the present method are given in Example 9.
  • the partial digest produces a numoer of different-size fragments which contain the desired S.,/ S. segment, including the S ⁇ /S. fragment.
  • the partial digest fragments are now fractionated by PFGE, substantially according to methods described and referenced in Example 9, and the gel is examined for probe-binding regions (containing the S ⁇ /S. fragment) by Southern blotting, using the previously selected probe. Two of the probe binding regions are now removed and the digest fragments are eluted.
  • the probe-binding regions identi ⁇ fied as S./S 4 and S 3 /S. are so identified and eluted.
  • the two eluted gel fractions are used as the two DNA sources from which coincident sequences can be cloned, according to the method of the invention.
  • Each of these fragment mixtures is digested to completion with one or two selected endonucleases and prepared for hybridization, according to one of the methods detailed in Section II.
  • Hybridization and cloning of heteroduplex fragments formed from end-hybridized strands yield cloned subfragments which are common to both el ' utate mixtures.
  • the method yields clones containing only sequences present in the S ⁇ /S. fragment, and enriches single-copy sequences. With this limited library, mapping of and gene identification in the S,/S. fragment is greatly simplified.
  • the method is similarly applicable with other recombinants for generat ⁇ ing fragments that fractionate in different parts of the gel, or in more than one gel, which contain coincident sequences. This may be accomplished, for example, by using a source or sources containing a restriction fragment-linked polymorphism for the rare cutter enzyme S in the region of interest, or by cutting with two differ ⁇ ent rare cutter enzymes.
  • ⁇ j e are likely to represent the most important functional genes in an organism, it would be advantageous to obtain all of the conserved sequences of an organism, particularly in humans, in cloned form.
  • 2Q from related species are selected.
  • a primate species such as lemur would be preferred, since a more closely related species, such as chimpanzee, may give too much general gene homology.
  • the two DNA sources are fragmented, denatured, reannealed and
  • telomere sequences may be important, for example, for constructing stable chromosomes which can be used for gene therapy.
  • One approach to cloning telomere sequences, ac ⁇ cording to the present invention, is outlined in Figure 10. The upper portion of the figure shows a known translocation in the end regions of human chromosomes 4 and 8 in which end portions of the two chromosomes, including the telomere region, are exchanged.
  • the objec ⁇ tive of the method is to clone those sequences, presumably including telomeric sequences, which are present in the C8 translocation on the C4 chromosome.
  • hybrid cells containing in one case chromosome 8, and in the other case, chromosome 4 with the chromosome 8 translocation are produced.
  • one hybrid cell is a Chinese hamster ovary (CHO) cell containing the C4/C8 translocation chromosome
  • the other hybrid is a mouse cell containing a normal human C8 chromosome, as indicated in the figure.
  • DNA from the two cell types is isolated, and fragmented as above, to form the two DNA fragment mixtures used in the method.
  • the coincidence sequences which include those single-copy sequences derived from the translocated portion of C8, as well as those sequences conserved between Chinese hamsters and mice, are obtained by one of the coincidence methods discussed in Section II. Those clones containing rodent conserved sequences are then identified and removed by screening with total DNA from either rodent cell.
  • This application is aimed at cloning DNA sequences derived from infectious microorganisms which (a) have not yet been identified and isolated, and (b) are infectious toward disparate hosts, such as humans and rodents.
  • the two infected cell types from the disparate hosts are used to produce the two DNA fragment mixtures from which coincident sequences will be derived, according to the method of the invention.
  • the library of cloned sequences may be further screened with the sequences derived from the two host sequences, such as human and hamster genomic sequences, to remove host sequences from the library.
  • the remaining cloned fragments now represent sequences derived from the infectious agent.
  • These clones in turn, can be used as probes for identifying the infection in cells, or for determining sequences in the genome in the infectious agent, for purposes of preparing diagnostic or vaccine reagents.
  • the method of the invention may also be used for removing repeat sequences from genomic DNA, to enrich a genomic fragment mixture for single-copy sequences. This application is based on the 5 ability of the method to discriminate against heterologous fragments formed from non-end-hybridizable strands, as ⁇ sociated predominantly with repeat sequence hybrids.
  • the genomic material of interest is divided into two portions, and each of Q these is then used in generating the two fragment mixtures which are to be hybridized.
  • the two mixtures are reacted under hybridization conditions which yield heteroduplex fragments, as discussed above, and these are further cloned to selectively remove fragments formed from non- 5 end-hybridized fragments.
  • the resulting genomic library can be further screened with known repeat sequences to further enrich the library for single-copy sequences.
  • the method of Q the invention provides a simple, practical approach for selecting out of two large mixtures of genomic fragments, those coincident sequences which are common to both mixtures.
  • the method typically yields a library of cloned coincident sequences which are enriched 5 for single-copy sequences.
  • the method may be performed by a variety of procedures which rely on fragment end characteristics, physical properties, and/or duplex forma ⁇ tion in cloned single-strand form.
  • the method can be applied usefully to a number of significant problems in genetic mapping and gene clon ⁇ ing, including the specific applications described in this section.
  • M13/mpl8 and M13/mpl9 are obtained from New England Biolabs (Beverly, MA).
  • Cloning plasmid pUC18 and its host E. coli strain JM103 are obtained from Pharmacia. Bluescripts cloning vector containing NotI and Xhol clon ⁇ ing site is supplied by Stratagene (San Diego, CA) .
  • Terminal transferase (calf thymus), alkaline phosphatase (calf intestine), polynucleotide kinase, Klenow reagent, and SI nuclease are all obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN); SP6 and T7 polymerase, from Promega Biotech (Madison, WI); and proteinase K r RNase and DNase, from Sigma (St. Louis, MO);
  • Synthetic oligonucleotides for vector modifica ⁇ tions to introduce NotI and Sfil linkers are prepared by conventional phosphotriester methods (Duckworth) or the phosphoramidite method as reported (Beaucage; Matteucci) , and can be prepared using commercially available automated oligonucleotide synthesizers. Alternatively, custom
  • 10 designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) . Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 1 nmole substrate in
  • Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes)
  • cleaved frag ents After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid re ⁇ covered from aqueous fractions by precipitation with ethanol (70%). If desired, size separation of the cleaved frag ents may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzy ology (1980) 65:499-560.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow reagent) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20° to 25°C in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 0.1-1.0 mM
  • dNTPs 10 dNTPs.
  • the Klenow fragment fills in at 5' single-stranded overhangs in the presence of the four nucleotides.
  • selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the overhang.
  • Ligations are performed in 15-50 ul volumes under the following standard conditions and temperatures: for example, 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 mg/ml BSA, 10 mM-50 mM NaCl, and either 40 mM ATP,
  • vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal
  • CIP alkaline phosphatase
  • Diges ⁇ tions are conducted at pH 8 in approximately 10 mM Tris- HCl, 1 mM EDTA using about 1 unit per mg of BAP at 60°C for one hour or 1 unit or CIP per mg of vector at 37°C for about one hour.
  • the preparation is extracted with phenol/ chloroform and ethanol precipitated.
  • religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion and separation of the unwanted fragments.
  • This example describes the use of coincidence cloning to identify common genomic sequences in LAZ342, a human lymphoblastoid cell line, and the somatic cell hybrid HHW661, a hamster-human hybrid cell line containing only a single human chromosome: a translocation of human chromosome region 4p onto hamster chromosome 5.
  • the HHW661 cell line was prepared according to published methods (Wasmuth) .
  • Genomic DNA from the two cell lines was obtained by conventional methods (Maniatis), and both DNAs were cut to completion with Mbol, which generates fragments pre ⁇ dominantly less than 1 kb in length.
  • Mbol which generates fragments pre ⁇ dominantly less than 1 kb in length.
  • the HHW661 DNA fragments were further blunt- ended with Klenow fragment in the presence of all four nucleotides, so that the final HHW661 fragments are blunt- ended homoduplexes (DNA-I fragments in the figure) and the lymphoblastoid cell fragments are sticky-ended (DNA-II in the figure) .
  • Both mixtures of DNA fragments were mixed in a 1:1 ratio, alkaline denatured at pH 13, and then reannealed by a phenol emulsion reassociation technique (F-PERT) (Kohne; Casna) .
  • F-PERT phenol emulsion reassociation technique
  • the denatured DNA fragments were mixed, and phenol and formamide were added to final volume concentrations of 27 and 8 percent, respectively.
  • a two-phase emulsion was formed by vigorous shaking with a vibratory shaker run at 1/2 to 3/4 maximum speed. Total reannealing time was about 20-24 hours at 22° C.
  • the annealed DNA was recovered by phenol extrac ⁇ tion and ethanol precipitation, according to known methods.
  • the reannealed fragments include original blunt-ended homoduplexes from the DNA-I fragments, DNA-II homoduplexes with opposite Mbol sticky ends, and heteroduplexes with opposite Mbol (or BamHI) and blunt ends, as indicated. Reassociation of repeat sequences in the fragment mixtures would not be expected to yield clonable ends, since the repeats are likely to hybridize with imperfect copies of themselves.
  • pUC18 plasmids were treated with BamHI and Smal restriction endonucleases, to cut the plasmid in its polylinker region, and the small linker fragment was removed by PEG precipitation. The reassociated fragments from above were mixed and ligated with the cut plasmids under standard conditions.
  • the recombinant plasmids are used to transform JM103 host cells, and successful transformants are selected by plat ⁇ ing in the presence of isopropylthiogalactoside (IPTG) and 5-bromo-4-chloro-3-methyl-indolyl-beta-D-galactoside (Xgal).
  • IPTG isopropylthiogalactoside
  • Xgal 5-bromo-4-chloro-3-methyl-indolyl-beta-D-galactoside
  • Minipreps of the plasmid DNA designated pUC/HD in Figure 1, revealed detectable inserts in the 200-1,000 bp range in 60% of the clones.
  • Genomic DNA from the lymphoblastoid and HHW661 cell lines above is cut to completion with Mbol, as described, yielding predominantly 200-1,000 bp fragments with Mbol sticky ends, as illustrated in Figure 2, where again the HHW661 fragments are indicated DNA-I and the 0 lymphoblastoid-cell fragments, as DNA-II.
  • Synthetic linkers having an Mbol sticky end and an internal Xhol site (linker I in the figure) or an internal NotI site (linker II) are prepared by conventional oligonucleotide methods, as described above. 5
  • the Xhol linker is ligated to the DNA-I fragments, and the fragments are cut to completion with Xhol endonuclease, yielding DNA-I fragments with Xhol sticky ends, as indicated.
  • the DNA-II fragments are ligated with the NotI linker, and the resulting fragments are cut _ to completion with NotI endonuclease, yielding DNA-II fragments with NotI sticky ends as shown.
  • a Bluescripts plasmid containing NotI and Xhol sites in the plasmid's polylinker region is cut with Xhol and NotI endonucleases, and the small linker fragment is removed by polyethyleneglycol (PEG) precipitation.
  • PEG polyethyleneglycol
  • the reassociated homoduplex and heteroduplex fragments from _ above are mixed and ligated with the cut plasmids under standard conditions.
  • only the end-hybridizable heteroduplexes, with their opposite NotI and Xhol sticky ends are compatible with the cut ends, of the plasmid, and n therefore only these heteroduplexes are expected to form successful recombinants.
  • Genomic DNA from the lymphoblastoid and HHW661 cell lines above is cut to completion with BamHI, yielding fragments predominantly in the 2-10 kilobase size region and having BamHI (B) sticky ends, as seen in Figure 3, where the fragments derived from the HHW661 and lymphoblastoid cell lines are designated DNA-I and DNA-II, respectively.
  • BamHI BamHI
  • the reannealed fragments include the original homoduplexes from the DNA-I and DNA-II fragments, having opposite BamHI ends, same-size heteroduplex fragments also having opposite BamHI ends, and unequal-strand homoduplex and heteroduplex fragments (predominantly different-size
  • the reannealed fragments are now digested to completion with both Alul and Haelll under standard digest conditions to cut those fragments at internal, non-
  • the digest fragments from above are now ligated into a pUC18 plasmid which has been linearized by BamHI digestion.
  • the digested fragments are mixed and ligated with the cut plasmids under standard conditions, and the plasmids are selected for successful recombinants, which should contain only the matched heteroduplex frag ⁇ ments.
  • Non-repeat clones are further purified and labeled as above, for screening genomic fragments from hamster, human and HHW661 cells, to identify those clones which are specific for both human and HHW661 genomic fragments, as determined, for example, by probe binding to southern blots of the genomic fragments.
  • Genomic DNA from the lymphoblastoid and HHW661 cell lines above is cut to completion with Mbol or BamHI, as above yielding predominantly 200-1,000 bp fragments with Mbol sticky ends, or 200-20,000 bp fragments with BamHI sticky ends as illustrated in Figure 4.
  • the HHW661 fragments are indicated as DNA-I and the lymphoblastoid-cell fragments, as DNA-II.
  • Synthetic linkers having an Mbol (M) sticky end and internal Haelll (H) and Alul (A) sites and either an Xhol (X) or a NotI (N) site adjacent the opposite linker end are prepared by conventional methods, as detailed in the Materials and Methods section above.
  • the nucleotide sequence of the two linkers is shown in Figure 4.
  • the Xhol linkers (linker I) are ligated to the DNA-I frag ⁇ ments, yielding fragments having groups of H/A/X sites at each end region.
  • the frag ⁇ ments illustrated in the figure are also shown as having
  • the NotI (linker II) are ligated to Q the DNA-II fragments, yielding fragments having groups of H/A/N restriction sites at each fragment end. Methylation of these fragments with Haelll methylase gives the frag ⁇ ments indicated with methylated Haelll sites in both of the DNA-II sites.
  • the reannealed fragments include the original homoduplexes from the DNA-I and DNA-II fragments, having either H/A/X or H/A/N linkers, respectively, at their op ⁇ posite ends, repeat sequences with different-length ends, and heteroduplexes with an H/A/X linker sequence at one end and an H/A/N linker sequence at the other end.
  • the reannealed fragments are now digested to completion with both Alul and Haelll endonucleases, under standard digest conditions.
  • digestion of the Alul methylated homoduplexes (the DNA-I homoduplexes) with the combination of endonucleases cleaves the fragments at all Haelll sites, including the end linker sites, producing fragments whose opposite ends have Haelll blunt ends.
  • diges ⁇ tion of the Haelll methylated homoduplexes (the DNA-II homoduplexes) with the combination of endonucleases cleaves the fragments at all Alul sites, including the end linker sites, producing fragments whose opposite ends have Alul blunt ends.
  • heteroduplex frag ⁇ ments formed from same-length strands
  • all of the Alul and Haelll sites are methylated on one strand or the other, and so no endonuclease digestion occurs, yielding intact heteroduplex fragments with opposite Xhol and NotI ends.
  • Duplex fragments which are not end-hybridized sequences will be cleaved by the Alul or Haelll endonucleases only in duplexes where the homologous strands are derived from the same original DNA mixture, thus yielding fragments with irregular ends, or fragments where one or both ends are Alul or Haelll ends.
  • the digest fragments from above are now ligated into a Bluescript ⁇ vector having NotI and Xhol polylinker sites. Briefly, the vector is digested with the both NotI and Xhol, with removal of the small linker fragment. As above, the digested fragments are mixed and ligated with the cut plasmids under standard conditions, and the plasmids are selected for successful recombinants, which should contain only the matched heteroduplex fragments.
  • Non-repeat clones are further purified and labeled as above, for screening genomic fragments from hamster, human and HHW661 cells, to identify those clones which are specific for both human and HHW661 genomic fragments, as determined for example, by probe binding- to Southern blots of the genomic fragments.
  • Genomic DNA from the lymphoblastoid cell line above is cut to completion with Hindlll and EcoRI, substantially as described, yielding predominantly 200-
  • HHW661 cell line are dissolved in 0.12 M phosphate buffer
  • the T is between about 80 -90°C.
  • the ⁇ ° material is then cooled mslowly to about 25 C below the T , and allowed to anneal to a C t value (mole/liter x sec) of about ⁇ . 100, at which the repeat-sequence material is pre ⁇ dominantly in reannealed form, and the non-repetitive
  • HAP hydroxyapatite
  • the DNA material is loaded onto the column and the single-strand material eluted with several volumes of the buffer. This material is combined, and precipitated with cold ethanol, as above.
  • the precipitated single-strand material is redissolved in annealing buffer, and the entire separation procedure repeated, except that the reannealing is performed at a temperature about 10 below the above T value.
  • biotinylated nucleotides used are Bio-11- dUTP (Brigati) which has an 11-atom linker arm separating the biotin and the pyrimidine base, and Bio-19-SS-dUTP
  • a typical reaction carried out in 60ml final volume, contains 1 ug DNA in 50 mM Tris-Cl pH 7.5, lOmM
  • MgS04 0.1 mM DTT, 100 mM of each of the following nucleotides: dATP, dGTP, and Bio-11-dUTP or Bio-19-SS- dUTP, 5 uCi of [alpha-32P] dCTP (Amersham, specific activ ⁇ ity 3,000 Ci/mmole), 30 U DNA polymerase I, and 27 pg/ml DNAse ⁇ I.
  • the reaction mixture is incubated at 14°C for one hour, stopped by addition of EDTA to 10 mM and heated at 68 C for 5 min.
  • Labeled DNA is recovered by chromatography over Sephadex G50 equilibrated and eluted with 10 mM Tris-Cl, pH 7.5/1 mM EDTA (T.E.). When large amounts of DNA are required, two to three nick- translations are run in parallel and loaded onto one column to obtain a concentrated DNA solution. 2. Tailing by Terminal Transf rase
  • the reaction mixture consists of 1 ug DNA in 100 mM potas ⁇ sium cacodylate (pH 7.2), 2 mM CoCl j , 0.2 mM DTT, 100 mM Bio-11-dUTP, 50 mCi [alpha- 32 P] dCTP, and 20 U terminal transferase, added last. After incubation at 37°C for 45 min, an additional 20 ⁇ of enzyme is added and the incuba ⁇ tion repeated. The reaction is terminated by EDTA added to 10 mM, the DNA is recovered as described above,
  • the reaction contains 1 ug of DNA in 33 mM Tris- 5 OAc (pH 7.9), 66 mM NaOAc, 10 mM MgOAc, 0.5 mM DTT, 0.1 mg/ l BSA, and 0.5 U T4 DNA polymerase.
  • dATP, dGTP, and Bio-11-dUTP are added to a final concentration of 150 mM
  • dCTP is added to 10 mM
  • 50 mCi of [alpha- 32 P] dCTP (3000 Ci/m ole) and 0 TrisOAc, NaOAc, MgOAc, BSA, and DTT are added to maintain previous concentrations.
  • This reaction is incubated at 37 C for 30 min, then dCTP is added to a concentration of 150 mM, and the reaction incubated for an extra 60 min at 37 C.
  • the reaction is stopped by addition of EDTA to 10 5 mM, heated at 65 C for 10 min, chromatographed and processed as described before.
  • the reannealed material is passed over an avidin column, for binding of biotinylated DNA to the column material.
  • a 1 ml silanized syringe plugged with silanized glass wool is packed with 0.3 ml streptavidin-agarose and washed with 0.15 PB, 2 mM EDTA.
  • the hybridization mixture from above is loaded onto the column which is then washed with several volumes of the same buffer, to remove non- hybridized cDNA.
  • the material bound to the column is alkaline denatured at pH 13, and the released (non-biotinylated) DNA strands are eluted with the same high pH medium.
  • the non-biotinylated strand material which elutes is carried in the single-stranded phage.
  • This material which constitutes human lymphoblastoid DNA sequences which are homologous to single-copy sequences from the HHW661 sequences, is transfected into the JM103 host, and grown in either single-strand or double-strand form.
  • Genomic DNA from the lymphoblastoid cell line and HHW661 line are each digested to completion with Mbol, and the HHW661 Mbol fragments are biotinylated according to Example 5A.
  • the biotinylation is preferably carried out by a method, such as nick translation or terminal tailing with T4 DNA polymerase, which does not alter the sticky end sequences of the fragments.
  • the fragments are hybridized under F- PERT conditions, as above, yielding homoduplexes and heteroduplexes which contain both end-hybridized and non- end-hybridized fragments, as illustrated in Figure 6.
  • the reannealed material is fractionated on a streptavidin column as above, and non-biotinylated bound DNA strands are released by alkaline denaturation as above.
  • the released single-strand species contain both single-copy and repeat-sequence strands which are in com ⁇ mon between the two fragment mixtures.
  • the single-strand eluate fraction from above is ethanol precipitated and reannealed using the F-PERT procedure, resulting in two populations of double-strand fragments, as seen in Figure 5. These include fragments formed by reannealing of end-hybridizable strands, giving duplex fragments in which the original Mbol ends are restored, and fragments formed by reannealing of non-end- hybridizable strands, which do not have defined sticky ends.
  • the reannealed fragments from above are mixed with pUC18 plasmid which has been linearized by digesting with BamHI, and the Mbol-ended fragments are ligated into the cut plasmid according to standard procedures. Suc ⁇ cessful recombinants are selected as in Example 1, and the colonies are screened with repeat-sequence probes, also as above, to identify single-copy clones.
  • Genomic DNA from the HHW661 cells is cut to com ⁇ pletion with Mbol, and the digest fragments are density labeled with N nucleotides which are incorporated into the two fragment strands by nick translation, or terminal tailing with T4 polymerase, according to methods described above for biotinylating fragments.
  • DNA rom the lymphoblastoid cell line above is cut to completion with Mbol for hybridization with the labeled HHW661 fragments.
  • the density-labeled Mbol fragments (wavy-line duplex fragments in Figure 7) are mixed with the unlabeled ly phoblastoid-cell fragments (straight-line duplex frag ⁇ ments in the figure), denatured, and reannealed using-the F-PERT method, as described above. As shown in the figure, the annealing/reannealing process yields homoduplexes labeled at neither or both strands, and
  • heteroduplexes labeled in one strand only.
  • homoduplex and heteroduplex there are matched-strand fragments formed predominantly from single-copy, same-size strands, and unmatched-strand frag ⁇ ments formed predominantly from repeat sequences with dif- ._ ferent sizes.
  • the matched-strand fragments will have Mbol sticky ends, whereas the unmatched-strand frag ⁇ ments will not.
  • the reannealed mixture is fractionated by equilibrium centrifugation in a CsCl gradient, according 20 to classical techniques (Meselson) .
  • the fragments will have partitioned into three gradient bands, as indicated at the right in Figure 7.
  • These three bands progressing toward greater density, are: unlabeled homoduplexes; heteroduplexes (containing a single labeled _,. strand), and labeled homoduplexes. These bands are identified by UV absorption, and the heteroduplex band is removed by aspiration.
  • End-hybridized heteroduplex fragments are selected by cloning into a vector BamHI site, as in
  • Example 5 and the cloned inserts may be further screened to remove repeat sequences.
  • Genomic DNA from the HHW661 and human lymphoblastoid cell lines are digested to completion with EcoRI and Hindlll.
  • the digest fragments from the HHW661 line (DNA-I fragments in Figure 8) are cloned into the EcoRI/HindllI site of vector M13/mpl9 which carries an Ecol to Hindlll orientation in its polylinker, to place the fragments in a "5'-3'" orientation in the double- strand vector.
  • the digest fragments from the 0 lymphoblastoid line are cloned into the HindiII/EcoRI site of vector M13/mpl9 which carries a Hindlll to EcoRI orientation in its polylinker, to place the fragments in a "3'-5'" orienta ⁇ tion in the vector.
  • C The two vectors with their two inserts are grown under conditions of phage production, and the phage harvested from the colony supernatant by conventional methods.
  • the phage from the M13/mpl9 vector, which produce the "plus” strand of the fragment are mixed with Q the phage of the M13/mpl8 vector, which produce the "minus" strand of the fragment insert.
  • the two phage populations are rapidly annealed using the F-PERT method described above.
  • the duplex material representing homologous strands from the different DNA mixtures, is 5 separated from single-strand DNA by hydroxyapatite (HAP) chromatography, according to standard procedures (Britten). Briefly, HAP is suspended in 0.15 PB, 2 mM EDTA,_. and poured into a water-jacketed column maintained at the reannealing temperature. After washing the column 0 with several volumes of the reannealing buffer, the DNA material is loaded onto the column and the single-strand material is eluted with several volumes of the buffer. The duplex material is eluted at elevated temperature with buffer.
  • HAP hydroxyapatite
  • the heterologous duplex fragments include end-hybridized inserts in which the vec ⁇ tor polylinker sites EcoRI (R) and Hindlll (H) on the op ⁇ posite sides of the insert are aligned and homologous, and non-end-hybridized fragments in which at least one of the polylinker ends is unmatched.
  • the heterologous duplex material is now digested to completion with EcoRI and Hindlll to release end-hybridized heteroduplex inserts with opposite EcoRI and Hindlll ends, and these fragments are cloned in the EcoRI/HindiII site of a pUC18 vector, as above.
  • the relatively small EcoRI/ Hindlll fragments can be separated by gel electrophoresis, or by hybridization of the fragments with opposite-strand, biotinylated M13 vector, and removal by streptavidin af ⁇ finity chromatography, as in Example 5.
  • This section describes the isolation and cloning of sequences from a unique Sail fragment from the human genome.
  • the method involves first performing a partial digestion of the genome with Sail.
  • the partial digest fragments which have size ranges from a few to up to several thousand kilobases, are fractionated by pulsed- field gel electrophoresis, and the gel is probed with a radiolabeled, selected-sequence probe.
  • Two of the gel regions which are positive for hybridization to the probe are eluted, digested completely with Mbol, and the co ⁇ incidence cloning method of the invention is used to identify and isolate sequences from the unique Sail frag ⁇ ment in each fragment mixture which binds to the selected probe. Details of the method are as follows:
  • Peripheral blood lymphocytes are pelleted by low speed centrifugation, and washed two times with 10 ml of phosphate-buffered saline (PBS) .
  • the cells are suspended to a final concentration of about 1 x 10 cells/ ml and a portion of the suspension is mixed with an equal volume of 1% low-gelling temperature agarose.
  • the agarose mixture is cooled to 45-50° C and immediately pipetted into a mold that makes 100 ul blocks, each about 2mm x 5 mm x.10 mm.
  • the blocks are solidified by contacting the mold.with ice.
  • the cells are disrupted in the agarose blocks by incubating the blocks for 2 days at 50 C with gentle shaking in ESP buffer (0.5 M EDTA, pH 9.0, 1% sodium dodecyl sulfate (SDS), and 1 mg/ml proteinase K) . After incubation the samples are stored at 4 C in ESP.
  • ESP buffer 0.5 M EDTA, pH 9.0, 1% sodium dodecyl sulfate (SDS), and 1 mg/ml proteinase K
  • the blocks Prior to restriction endonuclease digestion, the blocks are treated with PMSF to inactivate proteases in the block. This is done by treating each block twice with PMSF to inactivate proteases in the block. This is done by treating each block twice with PMSF to inactivate proteases in the block. This is done by treating each block twice with PMSF to inactivate proteases in the block. This is done by treating each block twice with PMSF to inactivate proteases in the block.
  • Partial digestions are carried out in 1.5 ml microfuge tubes containing 100 ug/ml bovine serum albumin and Sail in 10 mM Tris-HCl buffer, pH 7.4, to a final volume of 250 ul.
  • the agarose blocks are added to the tubes before the addition of Sail.
  • the final concentra- tion of Sail is either 2, 5, or 10 units/ug DNA in the block.
  • Sail is added to the tubes to a final amount of 10, 5.0, or 100 units.
  • the tubes are incubated at 37° C for increasing time periods ranging from 30 minutes to 12 hours.
  • the buffer in a tube is carefully aspirated, and replaced with 1 ml of ES buffer (ESP without proteinase K) , and the block is incubated in this buffer for 1 hour at 4°C.
  • the buffer is then removed, replaced with 250 ul of ESP, and incubated an additional 2 hours at 50°.
  • the block may be placed directly- in an agar slab (below) for pulsed field gel electrophoresis (PFGE), or stored at 4° C until use.
  • PFGE pulsed field gel electrophoresis
  • Optimal partial digest conditions are determined by running each of the blocks from above on PFGE, and determining the optional incubation period and Sail concentration which give the desired size distribution of partial digest fragments. As seen in Figure ' 9, under optimal conditions, the genomic fragments will contain between zero to 3 or more internal Sail (S) sites.
  • S Sail
  • the Sail fragment of interest in the figure is the S,/S. fragment, which is also contained in the S 2 /S. and S./S. fragments shown in the figure..
  • the sys . fragment is a relatively large genomic fragment which contains (a) a single-copy gene ⁇ sequence which is homologous to the labeled probe, and (b) a gene region of interest.
  • the fragment of interest is too large to clone as a single piece, and the' probe-sequence region may be separated from the gene region of interest by typically more than about 50 and up
  • the Sail partial digest fragments from above are fractionated by PFGE, substantially according to published 25 methods (Smith; Schwartz). Briefly, a gel suspension containing 1.0% agarose, and TBE buffer (10 mM Tris/Borate buffer, pH 7.4 containing 0.1 mM EDTA) is poured into a 20 cm 2 mold to a depth of about 12 mm. After gel hardening, slots corresponding in size to the gel blocks are cut
  • the slab is placed in a horizontal gel box containing electrodes on all four sides, at an angle of 45 with respect to the sides of the box, i.e., such that the diagonals of the box
  • Electrophoresis is carried out with continuous circulation of TBE buffer, with cooling of the circulated buffer at 15 C, at pulse times of about 60 seconds at 200 volts.
  • the electrophoretic run is terminated when the marker bands have migrated to near the bottom of the gel, as indicated by ethidium staining. Typical electrophoresis times are between about 24 and 36 hours.
  • the gel is cut in half, providing one gel for use in Southern blotting, and a second gel for use in obtaining intact duplex Sail fragments. These two gels are referred to below as “probe” and “recovery” gels, respectively.
  • the probe gel is protected from light during subsequent manipulations prior to and during Southern blotting (Smith). Exposure to 254 n UV light is for one minute. Denaturation of gel DNA material is carried out for one hour in 0.5 NaOH, 0.5M NaCl, and neutralization is carried out for one hour in 1.5M Tris-Cl pH 7.5 with gentle agitation. The gel is blotted to nitrocellulose by ascending transfer overnight with a conventional sodium citrate buffer (Maniatis). The filter is baked for two hours in vacuo at 80 C, and stored in a tight container.
  • a Southern blot of the gel fragments is prepared, according to standard methods (Maniatis). From the blot, two probe-binding gel band regions, such as the regions identified as containing fragments S./S. and S,/S. in Figure 9, are identified. From the positions of these two gel regions, the cor ⁇ responding regions in the recovery gel are removed for recovery of the fragments in each region. The fragment material is eluted from the gels by electroelution accord ⁇ ing to standard procedures, and the eluted DNA fragments are ethanol precipitated.
  • the two Sail fragment mixtures obtained from the two gel regions above are each digested to completion with Mbol, and the resulting fragments in one of the mixtures is further treated with Klenow fragment in the presence of all four nucleotides, as in Example 1, to fill in the sticky Mbol ends.
  • the two fragments mixtures are then mixed, denatured at pH 13, and reannealed by the F-PERT method, as in Example 1, to generate end-hybridized heteroduplexes which have opposite blunt and Mbol sticky ends.
  • the hybridization fragments are then cloned into the BamHI/Smal site of pUC18, as in Example 1, and suc ⁇ cessful recombinants are identified and screened, both to remove repeat-sequence clones, and to identify clones which hybridize to the labeled probe used above to identify Sail fragments of interest on the PFGE gel.
  • the methods of Examples 2-8 could also be applied.
  • Genomic DNA is obtained from human PBLs as in Example 1, and this material is digested to completion with Mbol as in Example 1.
  • the digest material is divided into two equal portions, and one portion is further treated with Klenow fragment in the presence of all four nucleotides, as in Example 1, to fill in.the sticky Mbol ends.
  • the two fragment mixtures are then mixed, denatured at pH 13, and reannealed by the F-PERT method, as in Example 1, to generate end-hybridized heteroduplex sequences from the two mixtures which have opposite blunt and Mbol sticky ends.
  • the hybridization products are then cloned into the BamHI/Smal site of pUC18, as in Example 1.
  • Successful recombinants can further be screened with repeat-sequence probes to remove remaining repeat-sequence clones in the library.

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Abstract

Procédé d'obtention de fragments d'ADN à séquence commune à partir de deux mélanges de fragments tels que les fragments obtenus à partir de deux génomes différents, ou à partir de deux régions différentes de séparation de fragments sur un gel. Les fragments d'au moins l'un des deux mélanges sont modifiés de sorte que des fragments hétéroduplex contenant un brin dérivé des fragments du premier mélange et un brin opposé dérivé des fragments homologues du second mélange peuvent être isolés à partir d'homoduplexes formés par hybridation des brins dans chaque mélange de fragments. Le procédé peut être utilisé dans des applications relatives à la topographie de gènes, à l'isolement de gènes, à la construction de chromosomes, au clonage de gènes conservés et à l'élimination de séquences répétitrices de l'ADN génomique. Sont également décrites des librairies de séquences-coïncidences formées par le procédé.
PCT/US1988/002631 1987-08-07 1988-08-02 Librairie et procede de clonage par coincidence WO1989001526A1 (fr)

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Cited By (27)

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GB2239456A (en) * 1989-12-27 1991-07-03 Us Commerce Cloning vector comprising heteroduplex dna
GR900100371A (el) * 1989-05-16 1991-10-10 Scripps Clinic Res Νέα μέ?οδος αντλήσεως εκ του ανοσολογικού ρεπερτορίου.
WO1992013100A1 (fr) * 1991-01-26 1992-08-06 Medical Research Council Analyse d'adn
EP0425661A4 (en) * 1989-05-16 1992-08-19 Scripps Clinic And Research Foundation A new method for tapping the immunological repertoire
EP0478627A4 (en) * 1989-05-16 1992-08-19 William D. Huse Co-expression of heteromeric receptors
EP0472638A4 (en) * 1989-05-16 1992-08-19 Scripps Clinic And Research Foundation Method for isolating receptors having a preselected specificity
US5179177A (en) * 1990-08-02 1993-01-12 Borden, Inc. Method for retarding ambient temperature hardening of a phenolic resin composition
US5180795A (en) * 1990-08-02 1993-01-19 Borden, Inc. Retarders for hardening phenolic resins
US5208274A (en) * 1990-08-02 1993-05-04 Borden, Inc. Retarders for hardening phenolic resins
EP0550645A4 (en) * 1990-09-28 1993-09-01 Ixsys, Inc. Surface expression libraries of heteromeric receptors
US5294649A (en) * 1990-08-02 1994-03-15 Borden, Inc. Accelerators for curing phenolic resole resins
GR900100370A (el) * 1989-05-16 1994-03-31 Scripps Clinic Res Μέ?οδος παραγωγής πολυμερών εχόντων μιαν προεπιλεγμένη δραστικότητα.
WO2000023622A1 (fr) * 1998-10-16 2000-04-27 Valigene Corporation Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique
WO2000024935A3 (fr) * 1998-10-26 2000-07-13 Univ Yale Procede fonde sur les differences de frequence d'alleles destine au clonage de phenotypes
US6291159B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
US6291160B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
WO2000055364A3 (fr) * 1999-03-12 2001-10-11 Amersham Pharm Biotech Uk Ltd Analyse genetique
WO2001077374A3 (fr) * 2000-04-08 2002-06-20 Biopsytek Analytik Gmbh Procede permettant d'identifier et d'isoler des fragments de genome a desequilibre de liaison
US6420111B1 (en) 1998-01-15 2002-07-16 Valigen (Us), Inc. Multiplex VGID
US6680192B1 (en) 1989-05-16 2004-01-20 Scripps Research Institute Method for producing polymers having a preselected activity
US6969586B1 (en) 1989-05-16 2005-11-29 Scripps Research Institute Method for tapping the immunological repertoire
US7858559B2 (en) 2000-11-17 2010-12-28 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
US11299780B2 (en) 2016-07-15 2022-04-12 The Regents Of The University Of California Methods of producing nucleic acid libraries
US11584929B2 (en) 2018-01-12 2023-02-21 Claret Bioscience, Llc Methods and compositions for analyzing nucleic acid
US11629345B2 (en) 2018-06-06 2023-04-18 The Regents Of The University Of California Methods of producing nucleic acid libraries and compositions and kits for practicing same

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EP1026239A3 (fr) * 1989-05-16 2004-03-03 The Scripps Research Institute Nouveau procédé d'exploitation du répertoire immunologique
US6969586B1 (en) 1989-05-16 2005-11-29 Scripps Research Institute Method for tapping the immunological repertoire
US8338107B2 (en) 1989-05-16 2012-12-25 Scripps Research Institute Method for producing polymers having a preselected activity
EP0425661A4 (en) * 1989-05-16 1992-08-19 Scripps Clinic And Research Foundation A new method for tapping the immunological repertoire
EP0478627A4 (en) * 1989-05-16 1992-08-19 William D. Huse Co-expression of heteromeric receptors
EP0472638A4 (en) * 1989-05-16 1992-08-19 Scripps Clinic And Research Foundation Method for isolating receptors having a preselected specificity
US7858359B2 (en) 1989-05-16 2010-12-28 Stratagene Method for tapping the immunological repertoire
US7189841B2 (en) 1989-05-16 2007-03-13 Scripps Research Institute Method for tapping the immunological repertoire
US6291160B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
US6680192B1 (en) 1989-05-16 2004-01-20 Scripps Research Institute Method for producing polymers having a preselected activity
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
GR900100370A (el) * 1989-05-16 1994-03-31 Scripps Clinic Res Μέ?οδος παραγωγής πολυμερών εχόντων μιαν προεπιλεγμένη δραστικότητα.
US6291161B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
GR900100371A (el) * 1989-05-16 1991-10-10 Scripps Clinic Res Νέα μέ?οδος αντλήσεως εκ του ανοσολογικού ρεπερτορίου.
US6291159B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for producing polymers having a preselected activity
GB2239456B (en) * 1989-12-27 1994-05-25 Us Commerce System for cloning vectors containing heteroduplex sequences
GB2239456A (en) * 1989-12-27 1991-07-03 Us Commerce Cloning vector comprising heteroduplex dna
US5179177A (en) * 1990-08-02 1993-01-12 Borden, Inc. Method for retarding ambient temperature hardening of a phenolic resin composition
US5294649A (en) * 1990-08-02 1994-03-15 Borden, Inc. Accelerators for curing phenolic resole resins
US5180795A (en) * 1990-08-02 1993-01-19 Borden, Inc. Retarders for hardening phenolic resins
US5208274A (en) * 1990-08-02 1993-05-04 Borden, Inc. Retarders for hardening phenolic resins
EP0550645A4 (en) * 1990-09-28 1993-09-01 Ixsys, Inc. Surface expression libraries of heteromeric receptors
WO1992013100A1 (fr) * 1991-01-26 1992-08-06 Medical Research Council Analyse d'adn
US6420111B1 (en) 1998-01-15 2002-07-16 Valigen (Us), Inc. Multiplex VGID
WO2000023622A1 (fr) * 1998-10-16 2000-04-27 Valigene Corporation Procedes de manipulation de populations d'acides nucleiques au moyen d'oligonucleotides a marquage peptidique
AU769759B2 (en) * 1998-10-26 2004-02-05 Yale University Allele frequency differences method for phenotype cloning
WO2000024935A3 (fr) * 1998-10-26 2000-07-13 Univ Yale Procede fonde sur les differences de frequence d'alleles destine au clonage de phenotypes
WO2000055364A3 (fr) * 1999-03-12 2001-10-11 Amersham Pharm Biotech Uk Ltd Analyse genetique
WO2001077374A3 (fr) * 2000-04-08 2002-06-20 Biopsytek Analytik Gmbh Procede permettant d'identifier et d'isoler des fragments de genome a desequilibre de liaison
US7858559B2 (en) 2000-11-17 2010-12-28 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
US8741810B2 (en) 2000-11-17 2014-06-03 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
US11299780B2 (en) 2016-07-15 2022-04-12 The Regents Of The University Of California Methods of producing nucleic acid libraries
US11584929B2 (en) 2018-01-12 2023-02-21 Claret Bioscience, Llc Methods and compositions for analyzing nucleic acid
US11629345B2 (en) 2018-06-06 2023-04-18 The Regents Of The University Of California Methods of producing nucleic acid libraries and compositions and kits for practicing same

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