# Patent

## Title
Method to produce DNA of defined length and sequence and DNA probes produced thereby

## Abstract

A method for producing a single stranded DNA (ssDNA) molecule of a defined length and sequence is disclosed. This method enables the preparation of, inter alia, probes of greater length than can be chemically synthesized. The method starts with a double stranded molecule, such as genomic, double stranded DNA (dsDNA) from any organism. A fragment of the starting molecule (dsDNA) is amplified by specific primers engineered to introduce cleavage sites on either side of the desired sequence. Cleavage steps on the amplified, engineered fragment are combined with a phosphate removal step, thereby creating a construct that can be digested with an exonuclease without damage to the desired ssDNA. Probes, which hybridize with large gaps between the ends of the probes, are also disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of application Ser. No. 11/805,676 filed on May 24, 2007 and claims priority from U.S. Provisional Patent Application No. 60/808,490 filed on May 25, 2006, both of which are hereby incorporated by reference in their entirety.


FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under contracts HG000205 and GM062119 awarded by the National Institutes of Health. The Government has certain rights in this invention.


REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK
The present application includes sequences to be included in a Sequence Listing in computer readable form found on an accompanying computer file. The present application further includes a lengthy table submitted in electronic form. 







LENGTHY TABLES




The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08795968B2). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).






BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of nucleic acid synthesis and analysis, and particularly to the field of preparing single stranded DNA probes or primers of defined sequence and length.
2. Related Art
The generation of single-stranded DNA has a large number of applications in understanding biological functions of gene expression and function, treatment of diseases in plants and animals, and in applications to diagnostics and forensics. There are currently several applications that rely on the use of long oligonucleotides as probes. These include molecular inversion probes (Willis et al., 2000 U.S. Pat. No. 6,858,412), wherein probes termed “pre-circle” probes are hybridized at either end to a target, then circularized by filling the gap between the ends. It is said that the gap may be between 1 and 2000 nucleotides (Col. 14 1.35), but the examples are directed to single nucleotide gaps. This method is based on the fact that the two targeting domains of a pre-circle probe can be preferentially ligated together, if they are hybridized to a target strand such that they abut and if perfect complementarity exists at the two bases being ligated together. Perfect complementarity at the termini allows the formation of a ligation substrate such that the two termini can be ligated together to form a closed circular probe. If this complementarity does not exist, no ligation substrate is formed and the probes are not ligated together to an appreciable degree. Once the precircle probes have been ligated, the unligated precircle probes and/or target sequences are optionally removed or inactivated. The closed circular probe is then linearized by cleavage at the cleavage site, resulting in a cleaved probe comprising the universal priming sites at the new termini of the cleaved probe. The patent further states that, due to the length of the precircle probes, it is preferred that each target domain range in size from about 5 bases to about 100 bases, with from about 5 to about 40 being especially preferred.
Padlock probes are described in Landegren et al., U.S. Pat. No. 6,235,472, and Landegren et al., 2001). The term “padlock probe” refers to a probe designed to be circularized in the presence of a target sequence, so that it may be caused to close around the target-containing nucleic acid strand such that the cyclic probe will interlock with and thereby be efficiently linked to the target nucleic acid to be detected. In other words, because of the helical nature of double-stranded nucleic acids, such as DNA, circularized probes will be wound around the target strand, topologically connecting probes to target molecules through catenation, in a manner similar to “padlocks”. Such covalent catenation of probe molecules to target sequences results in the formation of a hybrid that resists extreme washing conditions, serving to reduce non-specific signals in genetic assays. Any probes hybridizing in a non-specific manner may therefore be efficiently removed by subjecting the target to non-hybridizing conditions and/or exonuclease activity. Further, the novel method may be performed with even very short synthetic probes since only part of the probe molecule needs to form a rigid double-stranded DNA molecule with the target molecule, whereas the rest of the probe molecule may be highly flexible, optionally branched single-stranded DNA or any other spacer material. In this system, a probe is hybridized to a target nucleic acid sequence, such as a DNA strand, via two end segments of the detecting reagent, designated Probe 1 and Probe 3, the latter being complementary to two respective non-contiguous sequences of the target molecule. An additional probe, designated Probe 2, is hybridized to the intermediate segment of the target molecule with the probe ends in juxtaposition to Probe 1 and Probe 3, respectively, and then ligated to the two ends.
Another application of single stranded DNA molecules is described in Fredriksson S., et al., “Protein detection using proximity-dependent DNA ligation assays,” Nat Biotechnol, 2002 May; 20 (5):473-7. This paper describes a technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein.
Another method for nucleic acid formation is strand displacement amplification (SDA), which is generally described in U.S. Pat. Nos. 5,455,166 and 5,130,238. A single stranded target nucleic acid, usually a DNA target sequence, is contacted with an SDA primer. An “SDA primer” generally has a length of 25-100 nucleotides and is substantially complementary to a region at the 3′ end of the target sequence, and the primer has a sequence at its 5′ end (outside of the region that is complementary to the target) that is a recognition sequence for a restriction endonuclease, sometimes referred to herein as a “nicking enzyme” or a “nicking endonuclease”, which is chosen to cleave a strand either at the recognition site, or either 3′ or 5′ to it, without cleaving the complementary sequence, either because the enzyme only cleaves one strand or because of the incorporation of the substituted nucleotides.
For many of the assays described above, single stranded DNA probes are synthesized chemically. Currently, these probes are very expensive to manufacture to the required specificity and purity that these applications demand.
Various attempts have been made to produce defined single-stranded DNA. Nikiforov and Knapp (U.S. Pat. No. 5,518,900) describe a method for producing single-stranded DNA from a PCR fragment where one of the primers used for amplification has a modification that makes that strand resistant to exonuclease digestion. This method suffers from the drawback that every probe requires the synthesis of oligonucleotides with chemically modified nucleotides, which is not economically feasible in large-scale genomic studies.
Higuchi et al., (1989) describe a method for producing single-stranded DNA from PCR fragments where one of the amplification primers is phosphorylated and the corresponding strand with the phosphorylated primers is a preferential substrate for nuclease digestion. The drawback of this method is that the non-phosphorylated strand from a blunt-end DNA molecule (as in a PCR product) acts as a substrate, though with reduced efficiency.
Binkowski, et al., “Correcting errors in synthetic DNA through consensus shuffling,” Nucleic Acids Res, Mar. 30, 2005; 33 (6): e55, describe a method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS).
BRIEF SUMMARY OF THE INVENTION
The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary. The present methods are useful for producing any single stranded DNA molecule where sequence exactness, including exact beginning and ending of all molecules, is needed. The DNA thus produced need not therefore be a probe. In general, the present synthetic methods involving cleavage and digestion of dsDNA permit creation of ssDNA of greater lengths than previously possible, e.g., lengths of 100 to 1000 or even higher nt.
The present invention, in one aspect, comprises a polynucleic acid probe, having a defined length between two ends and a defined sequence, for hybridization to a target polynucleic acid sequence, comprising specified subsequences which are designed to allow the probe to hybridize to relatively widely spaced target regions, have the gap between the target regions filed, and then be circularized, and, finally, be amplified in circular form. The probe will contain the following subsequences: (a) a first target sequence, at one end of the probe, complementary to a first target region of the target polynucleic acid, for specifically binding thereto (the target region being, e.g., human genomic DNA); (b) a second target sequence, at an opposite end of the probe, complementary to a second target region of the target polynucleic acid for specifically binding thereto, where said first and second target regions are separated on the target polynucleic acid by a gap of at least 25-250 nt of target sequence, more preferably between 250 and 1000 nt; (c) at least one amplification primer site, adjacent the target sequence, and connected to a backbone sequence, for specifically binding a PCR primer, said primer oriented in a direction for amplification of target sequences only when nucleic acids are joined to the target sequences as complementary to the target polynucleic acid and further oriented to not amplify the backbone sequence; and (d) a backbone sequence of at least 25 nt, preferably 125-400 nt chosen to be non-complementary to the target polynucleic acid.
The probe will typically be DNA, but can include modified nucleic acids or hybrids. The probe may comprise two amplification primer sites, one adjacent the first target sequence and one adjacent the second target sequence, oriented towards each other, whereby the circularized probe is selectively amplified after linear nucleic acids are digested with an endonuclease. The probe is circularized by gap filling and ligating between the target sequences of the probe, thereby forming a circular probe.
The probe may be formed with different sizes, but contain the afore-mentioned sequences, wherein the backbone region is from a non-human organism and the target sequences hybridize to human genetic sequences.
In one aspect of the invention, the target sequences are adjacent to a 5′ end and a 3′ end of an exon of a eukaryotic gene, so that an entire exon sequence is obtained and amplified for further study. In certain aspects the invention comprises a primer for performing PCR amplification comprising a homology region for hybridization to a target under annealing conditions and a non-homologous restriction endonuclease recognition sequence. The probe may further comprise a non-homologous region for forming a primer-binding site to another primer.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram representing the creation of a single stranded polynucleotide (e.g., DNA) probe having a defined length and sequence;
FIG. 2 is a diagram (2A) of a probe having common amplification primer regions and a spacer backbone, a diagram showing binding of a probe (2B) and a diagram of a circularized probe being amplified while still in a circle (2C);
FIG. 3 is a diagram showing a technique for spacer multiplex detection, (“SMART,” or Spacer Multiplex Amplification ReacTion) showing a target sequence and probe creation (3A), amplified sequence design (3B), and hybridization of the present probe (3C); and
FIG. 4 is a diagram showing a probe having a spacer backbone and adapter sequences.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Definitions
The term “probe” refers to a polynucleic acid that contains target regions that specifically bind to a target, complementary thereto. As is known, in order to be specific the target region must be at least 10 bases long, and should be between 10 and 50, or even longer, bases long. The present probes preferably comprise two target regions, and the regions should be selected to be non-complementary, i.e. not bind to each other. The present probes include nucleic acid sequences that are used to detect identical, allelic or related nucleic acid sequences. The probes are isolated oligonucleotides or polynucleotides and may or may not be attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
The term “target nucleic acid” refers to the polynucleic acids to be analyzed in the presently preferred method of using the probes to be synthesized. In most cases, the DNA will be human DNA, preferably genomic DNA. However, the present probes may be adapted for use with any sample, including bacteria, viruses, and, particularly in the case of viruses, may include RNA targets. Hybridization of DNA to RNA targets is described in Schwille et al., “Quantitative Hybridization Kinetics of DNA Probes to RNA in Solution Followed by Diffusional Fluorescence Correlation Analysis,” Biochemistry, 1996, 35, 10182-10193. A polymerase may be used to extend the probe bound to the target nucleic acid by inserting bases complementary to the target.
The term “restriction enzyme” is used in its conventional sense. Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. Of particular interest are Type II enzymes, which cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for DNA analysis and gene cloning. Rather then forming a single family of related proteins, type II enzymes are a collection of unrelated proteins of many different sorts. Type II enzymes frequently differ so utterly in amino acid sequence from one another, and indeed from every other known protein, that they likely arose independently in the course of evolution rather than diverging from common ancestors.
The most common type II enzymes are those like Hha I, Hind III and Not I that cleave DNA within their recognition sequences. Enzymes of this kind are the principle ones available commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as homodimers, but a few (e.g., BbvC I: CCTCAGC) (SEQ ID NO: 1) recognize asymmetric DNA sequences because they bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoR I: GAATTC) (SEQ ID NO: 2) in which the two half-sites of the recognition sequence are adjacent, while others recognize discontinuous sequences (e.g., Bgl I: GCCNNNNNGGC) (SEQ ID NO: 3) in which the half-sites are separated. Cleavage leaves a 3′-hydroxyl on one side of each cut and a 5′-phosphate on the other. They require only magnesium for activity and the corresponding modification enzymes require only S-adenosylmethionine. They tend to be small, with subunits in the 200-350 amino acid range.
The next most common type II enzymes, usually referred to as ‘type IIs” are those like Fok I and Alw I that cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400-650 amino acids in length, and they recognize sequences that are continuous and asymmetric. They comprise two distinct domains, one for DNA binding, and the other for DNA cleavage. They are thought to bind to DNA as monomers for the most part, but to cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme molecules. For this reason, some type IIs enzymes are much more active on DNA molecules that contain multiple recognition sites.
The third major kind of type II enzyme, more properly referred to as “type IV” are large, combination restriction-and-modification enzymes, 850-1250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recognition sequences; those that recognize continuous sequences (e.g., Eco57 I: CTGAAG) (SEQ ID NO: 4) cleave on just one side; those that recognize discontinuous sequences (e.g., Bcg I: CGANNNNNNTGC) (SEQ ID NO: 5) cleave on both sides releasing a small fragment containing the recognition sequence. The amino acid sequences of these enzymes are varied but their organization is consistent. They comprise an N-terminal DNA-cleavage domain joined to a DNA-modification domain and one or two DNA sequence-specificity domains forming the C-terminus, or present as a separate subunit. When these enzymes bind to their substrates, they switch into either restriction mode to cleave the DNA, or modification mode to methylate it.
Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests. No laboratory uses have been devised for them, and none are available commercially.
The terms “complementary” and “complementarity” refer to the natural binding of polynucleotides by base pairing. For example, the sequence “5′ A-G-T 3′” bonds to the complementary sequence “3′ T-C-A 5′.” Complementarity between two single-stranded molecules may be “partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acid strands, in the design and use of peptide nucleic acid (PNA) molecules, and in the design of the present primers for adding restriction sites.
“Hybridization” refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the “washing” step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68° C. in the presence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/ml denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Generally, such wash temperatures are selected to be about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y.; specifically see volume 2, chapter 9. As described below, a primer that does not entirely match the target is used with appropriate stringency. The “stringency” here is achieved by varying the temperature, magnesium concentration, or both, in the annealing steps where primer and target bind to each other in PCR, or probe and target bind to each other in the SMART reaction. The important point here is that the annealing take place under the buffer conditions of the enzymatic reaction.
The phrases “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
“PCR” refers to the polymerase chain reaction, as originally developed and covered by U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, as well as variations on polymerase chain reaction. In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication. The amplified sequence(s) may be readily identified by a variety of techniques.
“Primers” are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 10 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Overview
1. Preparation of Probe Using Particular Restriction Endonuclease Reactions
The present method to produce single-stranded DNA molecules of defined length and sequence uses a double stranded DNA molecule as the template. To generate the desired single-stranded DNA molecule from the double stranded DNA template, we employ a series of enzymatic reactions that involves restriction endonucleases, a phosphatase, and an exonuclease. The DNA template can be either a PCR product or any other DNA molecule such as plasmid, viral, genomic DNA, or synthetic DNA.
Referring now to FIG. 1, the starting double stranded DNA is illustrated at the top. Within that molecule, the final, desired final strand is shown as box 10 on the top strand. In a PCR reaction, first shown as “step a,” primers 12, 14 are hybridized to the strands and standard PCR amplification is carried out for the number of cycles desired, typically 10-30 cycles. The PCR products will have added sequences, shown with hatching, as non-complementary, containing restriction sites for yielding the defined ends. By using PCR primers having non-complementary portions, PCR products are obtained which have any predetermined 5′ and 3′ ends. This feature is exploited to engineer specific restriction enzyme sites where the cut site can precisely define any end of the final product. Furthermore, in the creation of SMART probes, as described below, the non-complementary portions of the PCR primers can be designed to include target sequences and primer sequences.
After the PCR product is obtained, in step b, shown in FIG. 1, the amplified double stranded material from step a is reacted with a first restriction endonuclease to create a cut 16 to create the desired sequence at the 5′ end of desired top strand 10 of the molecule and create a 5′ overhang whereby the desired 5′ end overhangs the 3′ complementary strand. The cut site should be at the nucleotide that is the desired 5′ end, typically next to the first mismatch with the target sequence in the primer. That is, the desired 5′ end is assumed to be present in the starting material, although it can be tailored by adding nucleotides in the primer.
After the step b digestion with the first restriction endonuclease (to create the desired 5′ end), in step the phosphate group on the overhang is removed by a phosphatase (e.g., shrimp alkaline phosphatase) as shown at 18. Removal of this phosphate makes the top strand (containing the desired product) resistant to exonuclease digestion. After the phosphatase reaction, the double stranded DNA molecule is digested with the second restriction enzyme that creates the desired sequence at other end (3′ end) of the molecule in step d.
The second restriction endonuclease is used as shown at 20 to create the desired sequence at the 3′ end of the molecule. This should be a blunt end, but may also result in a 3′ overhang of the desired strand, as shown at 23. We prefer to use restriction enzymes that generate a blunt-ended molecule in step d, but this is not the only option since there are other ways of generating a blunt end molecule that has a phosphate group. This series of digestions results in the generation a double-stranded DNA molecule with a blunt end that has a 5′ phosphate group, and an overhang on the opposite end that has no phosphate group on the 5′ end.
After restriction endonuclease and phosphatase treatment, the double-stranded molecule is converted to single strand by digestion with lambda exonuclease, as shown at 21. This generates the final 5′ molecule 10, as shown in step e. The exonuclease (e.g., lambda exonuclease, exonucleases I-III from E. coli, nuclease Bal-31, exoribonucleases, and the exonuclease activities of DNA polymerases) will selectively degrade the strand with the phosphorylated 5′ end and will keep the other strand intact. The method we have described requires the sequential digestions with restriction enzymes and a phosphatase step in between that allow the formation of a suitable substrate for an enzymatic exonuclease digestion. It does not rely on chemical modification of the primers to generate this molecule.
It is possible to carry out the above described steps with several different dsDNA starting materials and produce multiple, different ssDNAs. In this case the restriction enzymes used require a unique recognition site that is common to all the molecules. To achieve the goal to generating defined ends with shared restriction enzyme recognition sequences, the examples below describe two enzymes that cut in a sequence independent manner. That is, the cut site is outside of the recognition site. There are a few enzymes that have this flexibility of cutting in a sequence independent manner, and we chose two for purposes of illustration—BsaI and MlyI, discussed further below.
The order of the steps in certain respects is important. The amplification must be carried out first in order to generate a large number of final product molecules of the desired sequence, and to add the tailored ends for restriction cleavage. The first cleavage of step b must be done prior to phosphate removal in order to create the desired 5′ nucleoside. The 3′ cleavage of step d must follow this because a 5′ phosphate on the bottom strand (to be removed) is needed for exonuclease digestion.
2. Selection of Restriction Enzymes
Restriction enzymes are chosen based on the desired 5′ and 3′ ends of the sequence. The most flexibility is obtained with restriction enzymes that cut outside the recognition site, and the recognition site is outside the desired sequence. Thus, there will be one enzyme where the cut is 5′ of the recognition site, and one enzyme where the cut is 3′ of the recognition site.
BsaI
Digestion with BsaI generates a 5′ overhang five bases inward from the recognition site. The recognition site is GGTCTC, (SEQ ID NO: 6) however the cut site is outside the recognition sequence. It cuts in a sequence independent manner to generate a molecule with a 5′ overhang that has a phosphate group.
Before Digestion:








5′ GGTCTCN ↓ NNNNN
(SEQ ID NO: 7)


 


3′ CCAGAGNNNNN ↑ NNNNN
(SEQ ID NO: 8)






After Digestion:











5′ GGTCTCN P5′NNNNN



 



3′ CCAGAGNNNNN 3′NNNNN
(SEQ ID NO: 603)






MlyI

The recognition sequence of MlyI is GAGTC, but the cut site is 5 bases inward, and it generates a blunt ended molecule with a 5′ phosphate group. The cut site is not dependent on the sequence between the recognition site and the cut site. The cut site is marked with arrows.
Before Digestion








5′ GAGTCNNNNN ↓ NNNN
(SEQ ID NO: 9)


 


3′ CTCACNNNNN ↑ NNNN
(SEQ ID NO: 10)






After Digestion











3′ GAGTCNNNNN↓ 5′P NNNN
(SEQ ID NO: 604)


 



3′ CTCACNNNNN↑ 3′ NNNN
(SEQ ID NO: 605)






The advantage of using restriction enzymes that digest a DNA molecule outside their recognition sequence is the fact that the restriction enzyme digests the DNA molecule in a position that is independent from the sequence constraints of the recognition site. In our methodology we have appropriately placed the position of the recognition sequences for the two restriction enzymes used in the method outside the sequence of the single stranded molecule we want to generate (see Table 1). For some applications, restriction enzymes that digest the DNA molecule within the recognition sequence of the enzyme also can be used. That is, if the desired end corresponds to the recognition/cut site of a restriction enzyme to be used. Also, the restriction enzyme used in the first digestion should produce a 5′ overhang (recessed 3′ hydroxyl end) even though this is not absolutely necessary.
Any restriction enzyme that produces a 5′ overhang at the desired 5′ end of the probe may be used (See FIG. 1, cut 16). Below is a representative list of suitable restriction enzymes. This information is adapted from information provided by New England BioLabs, Inc. at their web site. All recognition sequences are written 5′ to 3′ using the single letter code nomenclature with the point of cleavage indicated by a “/”. Numbers in parentheses indicate point of cleavage for non-palindromic enzymes. Isoschizomers with alternative cleavage sites are indicated with a “^”. Enzymes that are not currently commercially available are indicated with a “x”. The New England BioLabs® Catalog number is shown in the center column.







TABLE 1







Restriction enzymes producing 5′ overhang














NEB
Catalog




Enzyme
Sequence
Enzyme
#
Sequence
Other Isoschizomers





Aar I
CACCTGC (4/8)







SEQ ID NO: 11






 


Acc36 I
ACCTGC (4/8)
BfuA I
R0701
ACCTGC (4/8)
BfuA I, BspM I, Bve I



SEQ ID NO: 12


SEQ ID NO: 12



 


Ace IIIx
CAGCTC (7/11)







SEQ ID NO: 13






 


AclW I
GGATC (4/5)
Alw I
R0513
GGATC (4/5)
Alw I, BspP I



SEQ ID NO: 14


SEQ ID NO: 14



 


Alw I
GGATC (4/5)
Alw I
R0513
GGATC (4/5)
AclW I, BspP I



SEQ ID NO: 14


SEQ ID NO: 14



 


Alw26 I
GTCTC (1/5)
BsmA I
R0529
GTCTC (1/5)
BsmA I, BsoMA I



SEQ ID NO: 15


SEQ ID NO: 15



 


Bbs I
GAAGAC (2/6)
Bbs I
R0539
GAAGAC (2/6)
Bpi I, BpuA I, BstV2 I



SEQ ID NO: 16


SEQ ID NO: 16



 


Bbv I
GCAGC (8/12)
Bbv I
R0173
GCAGC (8/12)
BseX I, BstV1 I



SEQ ID NO: 17


SEQ ID NO: 17



 


Bbv Iix
GAAGAC (2/6)
Bbs I
R0539
GAAGAC (2/6)
Bbs I, Bpi I, BpuA I, BstV2 I



SEQ ID NO: 18


SEQ ID NO: 18



 


BceA I
ACGGC (12/14)
BceA I
R0623
ACGGC (12/14)




SEQ ID NO: 19


SEQ ID NO: 19



 


BfuA I
ACCTGC (4/8)
BfuA I
R0701
ACCTGC (4/8)
Acc36 I, BspM I, Bve I



SEQ ID NO: 12


SEQ ID NO: 12



 


Bpi I
GAAGAC (2/6)
Bbs I
R0539
GAAGAC (2/6)
Bbs I, BpuA I, BstV2 I



SEQ ID NO: 20


SEQ ID NO: 20



 


BpuA I
GAAGAC (2/6)
Bbs I
R0539
GAAGAC (2/6)
Bbs I, Bpi I, BstV2 I



SEQ ID NO: 20


SEQ ID NO: 20



 


Bsa I
GGTCTC (1/5)
Bsa I
R0535
GGTCTC (1/5)
Bso31 I, BspTN I, Eco31 I



SEQ ID NO: 21


SEQ ID NO: 21



 


BsmA I
GTCTC (1/5)
BsmA I
R0529
GTCTC (1/5)
Alw26 I, BsoMA I



SEQ ID NO: 15


SEQ ID NO: 15



 


BsmB I
CGTCTC (1/5)
BsmB I
R0580
CGTCTC (1/5)
Esp3 I



SEQ ID NO: 22


SEQ ID NO: 22



 


BsmF I
GGGAC (10/14)
BsmF I
R0572
GGGAC (10/14)
BslF I



SEQ ID NO: 23


SEQ ID NO: 23



 


Bso31 I
GGTCTC (1/5)
Bsa I
R0535
GGTCTC (1/5)
Bsa I, BspTN I, Eco31 I



SEQ ID NO: 21


SEQ ID NO: 21



 


BsoMA I
GTCTC (1/5)
BsmA I
R0529
GTCTC (1/5)
Alw26 I, BsmA I



SEQ ID NO: 15


SEQ ID NO: 15



 


BspM I
ACCTGC (4/8)
BfuA I
R0701
ACCTGC (4/8)
Acc36 I, BfuA I, Bve I



SEQ ID NO: 12


SEQ ID NO: 12



 


BspP I
GGATC (4/5)
Alw I
R0513
GGATC (4/5)
AclW I, Alw I



SEQ ID NO: 14


SEQ ID NO: 14



 


BspTN I
GGTCTC (1/5)
Bsa I
R0535
GGTCTC (1/5)
Bsa I, Bso31 I, Eco31 I



SEQ ID NO: 21


SEQ ID NO: 21



 


Bst6 I
CTCTTC (1/4)
Ear I
R0528
CTCTTC (1/4)
Eam1104 I, Ear I, Ksp632 I



SEQ ID NO: 15


SEQ ID NO: 15



 


Eam1104 I
CTCTTC (1/4)
Ear I
R0528
CTCTTC (1/4)
Bst6 I, Ear I, Ksp632 I



SEQ ID NO: 15


SEQ ID NO: 15



 


Ear I
CTCTTC (1/4)
Ear I
R0528
CTCTTC (1/4)
Bst6 I, Eam1104 I, Ksp632 I



SEQ ID NO: 15


SEQ ID NO: 15



 


Eco31 I
GGTCTC (1/5)
Bsa I
R0535
GGTCTC (1/5)
Bsa I, Bso31 I, BspTN I



SEQ ID NO: 21


SEQ ID NO: 21



 


Esp3 I
CGTCTC (1/5)
BsmB I
R0580
CGTCTC (1/5)
BsmB I



SEQ ID NO: 22


SEQ ID NO: 22



 


Fau I
CCCGC (4/6)
Fau I
V0209
CCCGC (4/6)
Smu I



SEQ ID NO: 24


SEQ ID NO: 24



 


Fok I
GGATG (9/13)
BstF5 I{circumflex over ( )}
V0031
GGATG (2/0)
BseG I{circumflex over ( )}, BstF5 I{circumflex over ( )}



SEQ ID NO: 25


SEQ ID NO: 25



 


Hga I
GACGC (5/10)
Hga I
R0154
GACGC (5/10)




SEQ ID NO: 26


SEQ ID NO: 26



 


Ksp632 I
CTCTTC (1/4)
Ear I
R0528
CTCTTC (1/4)
Bst6 I, Eam1104 I, Ear I



SEQ ID NO: 15


SEQ ID NO: 15



 


Lwe I
GCATC (5/9)
SfaN I
R0172
GCATC (5/9)
SfaN I



SEQ ID NO: 27


SEQ ID NO: 27



 


Ple I
GAGTC (4/5)
Mly I{circumflex over ( )}
R0610
GAGTC (5/5)
Mly I{circumflex over ( )}, Pps I, Sch I{circumflex over ( )}



SEQ ID NO: 28


SEQ ID NO: 28



 


Pps I
GAGTC (4/5)
Mly I{circumflex over ( )}
R0610
GAGTC (5/5)
Mly I{circumflex over ( )}, Ple I, Sch I{circumflex over ( )}



SEQ ID NO: 28


SEQ ID NO: 28



 


Sap I
GCTCTTC (1/4)
Sap I
R0569
GCTCTTC (1/4)




SEQ ID NO: 29


SEQ ID NO: 29



 


SfaN I
GCATC (5/9)
SfaN I
R0172
GCATC (5/9)
Lwe I



SEQ ID NO: 27


SEQ ID NO: 27



 


Smu I
CCCGC (4/6)
Fau I
V0209
CCCGC (4/6)
Fau I



SEQ ID NO: 28


SEQ ID NO: 28



 


Sth132 Ix
CCCG (4/8)







SEQ ID NO: 29
















TABLE 2







Restriction enzymes producing 3′ overhang (see 23, step d, FIG. 1)















NEB
Catalog

Other



Enzyme
Sequence
Enzyme
#
Sequence
Isoschizomers
Enzyme





Acu I
CTGAAG (16/14)
Acu I
R0641
CTGAAG (16/14)
Eco57 I




SEQ ID NO: 30


SEQ ID NO: 30




 


AsuHP I
GGTGA (8/7)
Hph I
R0158
GGTGA (8/7)
Hph I




SEQ ID NO: 31


SEQ ID NO: 31




 


BciV I
GTATCC (6/5)
BciV I
R0596
GTATCC (6/5)
Bfu I




SEQ ID NO: 32


SEQ ID NO: 32




 


Bfi I
ACTGGG (5/4)
Bmr I
R0600
ACTGGG (5/4)
Bmr I




SEQ ID NO: 33


SEQ ID NO: 33




 


Bfu I
GTATCC (6/5)
BciV I
R0596
GTATCC (6/5)
BciV I




SEQ ID NO: 32


SEQ ID NO: 32




 


Bmr I
ACTGGG (5/4)
Bmr I
R0600
ACTGGG (5/4)
Bfi I




SEQ ID NO: 33


SEQ ID NO: 33




 


Bpm I
CTGGAG (16/14)
Bpm I
R0565
CTGGAG (16/14)
Gsu I




SEQ ID NO: 34


SEQ ID NO: 34




 












Bse3D I
GCAATG (2/0)
BsrD I
R0574
GCAATG (2/0)
BseM I, BsrD I



SEQ ID NO: 35


SEQ ID NO: 35



 


BseG I
GGATG (2/0)
BstF5 I
V0031
GGATG (2/0)
BstF5 I, Fok I{circumflex over ( )}



SEQ ID NO: 36


SEQ ID NO: 36



 


BseM I
GCAATG (2/0)
BsrD I
R0574
GCAATG (2/0)
Bse3D I, BsrD I



SEQ ID NO: 35


SEQ ID NO: 35



 













BseM II
CTCAG (10/8)
BspCN I{circumflex over ( )}
R0624
CTCAG (9/7)
BspCN I{circumflex over ( )}




SEQ ID NO: 37


SEQ ID NO: 37




 


BseR I
GAGGAG (10/8)
BseR I
R0581
GAGGAG (10/8)





SEQ ID NO: 38


SEQ ID NO: 38




 












BsrD I
GCAATG (2/0)
BsrD I
R0574
GCAATG (2/0)
Bse3D I, BseM I



SEQ ID NO: 39


SEQ ID NO: 39



 


BstF5 I
GGATG (2/0)
BstF5 I
V0031
GGATG (2/0)
BtsC I, BseG I, Fok I{circumflex over ( )} 



SEQ ID NO: 40


SEQ ID NO: 40



 













Eci I
GGCGGA (11/9)
Eci I
R0590
GGCGGA (11/9)





SEQ ID NO: 41


SEQ ID NO: 41




 


Eco57 I
CTGAAG (16/14)
Acu I
R0641
CTGAAG (16/14)
Acu I




SEQ ID NO: 42


SEQ ID NO: 42




 


Eco57M I
CTGRAG (16/14)








SEQ ID NO: 43







 


Gsu I
CTGGAG (16/14)
Bpm I
R0565
CTGGAG (16/14)
Bpm I




SEQ ID NO: 34


SEQ ID NO: 34




 


Hph I
GGTGA (8/7)
Hph I
R0158
GGTGA (8/7)
AsuHP I




SEQ ID NO: 31


SEQ ID NO: 31




 












Mly I
GAGTC (5/5)
Mly I
R0610
GAGTC (5/5)
Ple I{circumflex over ( )}, Pps I{circumflex over ( )}, Sch I



SEQ ID NO: 28


SEQ ID NO: 28



 













Mme I
TCCRAC (20/18)
Mme I
R0637
TCCRAC (20/18)





SEQ ID NO: 44


SEQ ID NO: 44




 


RleA Ix
CCCACA (12/9)








SEQ ID NO: 45







 












Sch I
GAGTC (5/5)
Mly I
R0610
GAGTC (5/5)
Mly I, Ple I{circumflex over ( )}, Pps I{circumflex over ( )}



SEQ ID NO: 28


SEQ ID NO: 28



 













TspDT I
ATGAA (11/9)








SEQ ID NO: 46







 


TspGW I
ACGGA (11/9)








SEQ ID NO: 46







 


Tth111 IIx
CAARCA (11/9)








SEQ ID NO: 47









The enzymes in Table 2 are useful if a recessed 5′ end, which is also a suitable target for exonuclease digestion (as is a blunt end) is desired in the second digestion
3. Design of Probes for Multiplex Amplification
(a) Introduction
The polymerase chain reaction (PCR) is one of the most commonly used techniques in genomics. This method of amplifying DNA from a limited amount of template material has been used extensively in DNA sequencing, SNP genotyping, molecular diagnostics etc., and has become one of the most routine protocols in molecular biology. One of the challenges facing high-throughput genomics is expanding the scale of PCR while keeping costs low. Considerable efforts have been made during the past several years to increase the throughput of PCR by “multiplexing” the reactions. The goal of multiplex PCR is to amplify a large number of targets in the same reaction vessel. This has been a challenge because of the large number of spurious reaction products that arise when a mixture of oligonucleotides are used in the same PCR reaction. Cho et al., (1999) were successfully able to multiplex 50 primer pairs, but this is not the scale that is sufficient for high-throughput genomics.
The present methodology is useful for other “gap fill” techniques. For example, probe ligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M. Whiteley, et al., D. Y. Wu, et al., Genomics 4:560 (1989), U. Landegren, et al., Science 241:1077 (1988), and E. Winn-Deen, et al., Clin. Chem. 37:1522 (1991). In one approach, known as oligonucleotide ligation assay (“OLA”), two probes or probe elements that span a target region of interest are hybridized to the target region. Where the probe element bases pair with adjacent target bases, the confronting ends of the probe elements can be joined by ligation, e.g., by treatment with ligase. The ligated probe element is then assayed, evidencing the presence of the target sequence.
In a modification of this approach, the ligated probe elements act as a template for a pair of complementary probe elements. With continued cycles of denaturation, hybridization, and ligation in the presence of pairs of probe elements, the target sequence is amplified linearly, allowing very small amounts of target sequence to be detected and/or amplified. This approach is referred to as ligase detection reaction.
Another technique requiring “gap fill” is molecular inversion probe (MIP) technology (U.S. Pat. No. 6,858,412, described above). Using this technology, several thousand probes have been hybridized to genomic DNA in one reaction vessel. Both padlock probes and MIP technology use single-stranded “pre-circles” to hybridize to target DNA. These pre-circles contain sequences on the 3′ and 5′ ends that are complementary to the target DNA. The DNA in between the complementary target sequences in the probe does not hybridize with the target, and forms a loop between the two hybridized target sequences (HTS). In the presence of DNA polymerase and ligase, the molecule extends from the 3′ end of the annealed probe and synthesizes a complement of the target until it reaches the 5′ end of the annealed probe. This is the “gap-fill” reaction. The molecule is circularized at that point by DNA ligase. This molecule is then freed from genomic DNA by exonucleases. In the MIP protocol, the closed circle is linearized, and the target sequences amplified with primer sequences present in the loop. The circles are not opened in the padlock probes, and the sequences are amplified by the rolling circle method. Currently the MIP technology is extensively used in SNP genotyping where the hybridized target sequences are very close to each other with a gap length of just one base pair, and the “gap-fill” reaction is the polymerization of one nucleotide before the ligation reaction. The MIP technology can conceivably be used in multiplex PCR reactions where the HTS (hybridized target sequences) are far apart and the “gap-fill” reaction can involve the polymerization of several nucleotides.
Meng Li (2006) et al., have used the padlock probe method to perform a “gap-fill” reaction where the HTS were 20 bases apart, and rolling circle amplification was used to amplify the DNA.
In all of the above cases, as the distance between the HTS (hybridized target sequences) becomes farther apart, the “gap-fill” reactions are also longer. To our knowledge, there are no publications that perform these large gap-fill reactions extending hundreds of bases, and ligations using the padlock method. We hypothesize that the rate-limiting step to perform a padlock or MIP methodology where the HTS are few hundred base bases apart might reside in the physical constraints of the corresponding sequences present on the probe to find the appropriate target sequences in the template DNA. We hypothesize optimizing the length of the spacer molecule in the probe will improve the chances of the corresponding sequences on the probe of finding their counterparts on the template DNA more efficiently. Thus will facilitate a more efficient gap-fill reaction and the downstream steps of the MIP or padlock protocol.
The present gap filling protocols are similar to others in that the gap fill reaction mixture contains a polymerase and a mixture of all four deoxynucleoside-triphosphates (also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP) in a manner similar to the MIP reaction and other polymerizations. Suitable enzymes include the Stoffel fragment of Taq polymerase (Applied Biosystems) or T4 DNA polymerase or any other enzyme that has no strand displacement activity or 5′ exonuclease activity. It is important that the gap fill reaction be carried out with an enzyme that has a lack of 5 prime exonuclease activity and a lack of strand displacement activity.
Modified or substituted dNTPs may be also used, such as 2′ deoxyadenosine 5′-O-(1-thiotriphosphate), 5-methyldeoxycytidine 5′-triphosphate, 2′-deoxyuridine 5′-triphosphate, and 7-deaza-2′-deoxyguanosine 5′-triphosphate. In addition, the substitution of the dNTP may occur after incorporation into a newly synthesized strand; for example, a methylase may be used to add methyl groups to the synthesized strand. Peptide-nucleic acid residues may be incorporated, as described in US PGPUB 2005/0053944 “Methods and kit for hybridization analysis using peptide nucleic acid probes,” hereby incorporated by reference.
(b) Design of Spacer Probes—Spacer Multiplex Amplification ReacTion (SMART)
A procedure which solves the problem of performing gap-fill reaction and ligation where the HTS (hybridized target sequences) are few hundred bases apart on the template DNA has been developed. We call the new methodology Spacer Multiplex Amplification ReacTion (SMART) because the success of the method is based on the optimization of the length of spacer backbone sequence on the probe. SMART probes are single-stranded molecules that have target (corresponding) sequences to genomic DNA or other target to be analyzed on either end of the of the probe sequence. An amplification primer AP1 and AP2 that was common to all the probes flanked each target. To test the hypothesis that the length of the spacer would determine the efficiency of longer extensions, we used probes where the length between the two common amplification primers was either 221 bases, or 38 bases as in conventional MIP probes. The SMART probes synthesized by the method we describe above, and the conventional MIP-size probes were synthesized chemically. The final configuration of the SMART probes, made according to FIG. 1, is shown in FIG. 2.
That is, the length of the spacer backbone should be at least 50% of the length of the HTS (hybridized target sequence) gap to be filled.
We reasoned that we could create molecules with varying spacer lengths by using the method of probe synthesis that we have developed and is described above. We designed smaller sized probes that approximated the conventional MIP) sizes, and the larger probes for the corresponding targets to test our hypothesis.
As shown in FIG. 2, the present probes, as prepared by the present method, comprise target sequences 22, 24, which are exactly complementary to and bind to targets such as genomic DNA 29. The arrowheads 27 indicate the direction of polymerization on a single stranded target molecule 29 shown in 2B. As is conventionally known, all polymerases have 5′ to 3′ polymerization activity and require a template strand and either a DNA or RNA primer having a 3′ end to which newly added bases are joined. AP1 and AP2 (26, 28) are common amplification primer sites for later amplification. Each probe comprises target sequences 22,24, one on each end, primer sites 26,28 next to each target sequence, and a spacer backbone 30 in between. The spacer backbones 30 are of variable length depending on the application. They are random DNA sequences that do not hybridize to target sequences. They may include peptide nucleic acids or other polymers.
As shown in FIG. 2B, multiple probes may be used on a target sequence, having unique target sequences 22, 24, but having common primer amplification sites 26, 28. Thus, in the amplification step shown in FIG. 2C, common primers binding to sites 26 and 28 will be used to amplify all hybridized, ligated probes. FIG. 2B shows one probe shown in FIG. 2A in contact with one region of single stranded target DNA 29 with a gap filling reaction catalyzed by DNA polymerase adding residues 25 and continuing as shown at 27 until the newly added region is complete up to the 5′ end of probe target region 24. For purposes of illustration, another probe having a smaller spacer backbone and a smaller gap is targeted to a different sequence. The probes have different targeting regions, differing in size and sequence, but identical primer regions AP1 and AP2. This permits a high degree of multiplexing in the final amplification reaction. Gap filling reactions adding nucleotides 25 are carried out by DNA polymerase, preferably lacking 3′-5′ exonuclease activity and the strand displacement activity, such as T4 polymerase, Taq, PolI (stofffel fragment). It is not necessary that the polymerase be thermostable.
Then, as shown in FIG. 2C, the probes are circularized by joining the newly added nucleotides to the end of the opposite target sequence. DNA ligase such as Ampligase, which functions at a high temperature, or any other DNA ligase such as T4 or E. coli DNA ligase may be used once the gap is filled. Then, an exonuclease is used to digest unreacted probes—which are linear—and target DNA. The circular probes that remain are then amplified as circles, as shown in 2C, using amplification primers 34, 36 targeted to primer sites API and AP2. Multiple copies of the target sequences in the target DNA, including the filled in gaps, are then prepared for further analysis.
EXAMPLES
Example 1
Probe Synthesis
The desired single-stranded probe we want to create is 298 bp. and the sequence of the molecule is SEQ ID NO: 48







5′TTGTTTTCTCCGTCGCCGTATCCCTTTAGTGAGGGTTAATAGTACGCT


 


TACTTCCGCGAAACGTCAGCGGAAGCACCACTATCTGGCGATCAAAAGGA


 


TGGTCATCGGTCACGGTGACAGTACGGGTACCTGACGGCCAGTCCACACT


 


GCTTTCACGCTGGCGCGGAAAAGCCGCGCTCG1CCGCCTTTACAATGTCC


 


CCGACGATTTTTTCCGCCCTCAGCGTACCGTTTATCGTACAGTTTTCAGC


 


TATCGTCACATTATTTAGGTGACACTATAGCCACAAATCAAGATCCGAAT


 


T3′






The underlined portions of the sequence represent the amplification primer sites described in Example 2, it being understood that the 5′ primer is the reverse complement of the underlined sequence. The probe sequence of SEQ ID NO: 48 above is derived from a double stranded DNA product, and it may be present in any organism or vector, as part of a larger sequence, and is set forth below in double stranded form as SEQ ID NO: 49







TTGTTTTCTCCGTCGCCGTATCCCTTTAGTGAGGGTTAATAGTACGCTT


 


ACTTCCGCGAAAACAAAAGAGGCAGCGGCATAGGGAAATCACTCCCAAT


 


TATCATGCGAATGAAGGCGCTT


 


ACGTCAGCGGAAGCACCACTATCTGGCGATCAAAAGGATGGTCATCGGT


 


CACGGTGACAGTGCAGTCGCCTTCGTGGTGATAGACCGCTAGTTTTCCT


 


ACCAGTAGCCAGTGCCACTGTC


 


TACGGGTACCTGACGGCCAGTCCACACTGCTTTCACGCTGGCGCGGAAA


 


AGCCGCGCTCGATGCCCATGGACTGCCGGTCAGGTGTGACGAAAGTGCG


 


ACCGCGCCTTTTCGGCGCGAGC


 


CCGCCTTTACAATGTCCCCGACGATTTTTTCCGCCCTCAGCGTACCGTT


 


TATCGTACAGTGGCGGAAATGTTACAGGGGCTGCTAAAAAAGGCGGGAG


 


TCGCATGGCAAATAGCATGTCA


 


TTTCAGCTATCGTCACATTATTTAGGTGACACTATAGCCACAAATCAAG


 


ATCCGAATTAAAGTCGATAGCAGTGTAATAAATCCACTGTGATATCGGT


 


GTTTAGTTCTAGGCTTAA






Step 1: Incorporation of Restriction Sites at the Ends of Double Stranded Template by PCR Amplification With Engineered Primers

The first step in the procedure is to incorporate the two restrictions sites at the appropriate position of the double stranded molecule. To achieve that, we amplified a double-stranded PCR product with PCR primers that had an MlyI sequence engineered into the one primer, and the BsaI sequence engineered into the other primer.









Reverse primer (MlyI_Target)



(SEQ ID NO: 50)



CATCGTGAGTCACTCGAATTCGGATCTTGATTTGTGG



 



Forward primer (BsaI_Target)



(SEQ ID NO: 51)



GTACGAGGTCTCACTTGTTTTCTCCGTCGCCGTA






Thus, the reverse primer of SEQ ID NO: 50 and the forward primer SEQ ID NO: 51 do not hybridize completely to the target sequence of SEQ ID NO: 48. They will still function to prime PCR amplification, and the amplification product will contain the engineered sequence shown in italics, which need not be complementary to the target sequence.








(SEQ ID NO: 52)



5′  ↓TTGTTTTCTCCGTCGCCGTATCCCTTTAGTGAGGGTTAATAGTACGC



3′CATGCTCCAGAGTAACA↑AAAGAGGCAGCGGCATAGGGAAATCACTCCCAATTATCATGCG


 


TTACTTCCGCGAAACGTCAGCGGAAGCACCACTATCTGGCGATCAAAAGGATGGTCATCG


AATGAAGGCGCTTTGCAGTCGCCTTCGTGGTGATAGACCGCTAGTTTTCCTACCAGTAGC


 


GTCACGGTGACAGTACGGGTACCTGACGGCCAGTCCACACTGCTTTCACGCTGGCGCGGA


CAGTGCCACTGTCATGCCCATGGACTGCCGGTCAGGTGTGACGAAAGTGCGACCGCGCCT


 


AAAGCCGCGCTCGCCGCCTTTACAATGTCCCCGACGATTTTTTCCGCCCTCAGCGTACCG


TTTCGGCGCGAGCGGCGGAAATGTTACAGGGGCTGCTAAAAAAGGCGGGAGTCGCATGGC


 


 


AAATAGCATGTCAAAAGTCGATAGCAGTGTAATAAATCCACTGTGATATCGGTGTTTAGT


 


A G A T C C G A A T T   G A G T G A C T C A C G A T G 3′


T C T A G G C T T A A        5′ MlyI






Step 2: Digestion at 5′ End

The first digestion with BsaI generates a 5′ overhang five bases inward from the recognition site on the top strand, and 1 base inward on the lower strand. This creates a molecule with a recessed 3′ end and a protruding 5′ end. The 5′ end now has a phosphate group. The molecule will be as in SEQ ID NO: 52, with the portions 5′ (top strand) and 3′ (bottom strand) of the single bold arrow removed.
Step 3: Phosphatase Treatment
The phosphate group on the 5′ overhang of this molecule (shown as double underlined TTGT in SEQ ID NO: 52) is then cleaved with a phosphatase making it resistant to lambda exonuclease cleavage.
Step 4: Second Digestion at 3′ End to Create a Blunt End
The molecule is then digested with the enzyme MlyI that cuts 5 bases inward from the recognition site, and generates a blunt-end molecule that has a phosphate group at the 5′ end. This can be seen in SEQ ID NO: 52 as removal of the portions 3′ of the double arrow (top strand) and 5′ of the double arrow (bottom strand). The 5′ adenosine on the bottom strand will have a terminal phosphate group after the restriction enzyme cleavage.
Step 5: Digestion of Second Strand
The molecule as shown in SEQ ID NO: 52 now has a 5′ phosphate on the adenosine on the blunt end, and a non-phosphorylated 5′ overhang (TTGT) on the other. As a result, when it is digested with Lambda exonuclease, the enzyme preferentially cleaves the phosphorylated strand, and generates the desired single-stranded molecule shown in SEQ ID NO: 48.
The desired ssDNA has now been formed. The target sequences, AP1 and AP2 are in bold and italic in SEQ ID NO: 52, above.
Example 2
Multiplex Probe Design
Enzymatic synthesis of the long single-stranded molecule was derived from a double-stranded PCR product following the procedure described above.
1) Generation of Double Stranded Templates Shown in FIG. 3A
As shown in FIG. 3, double stranded DNA is first amplified using primers 52, 54. A 220 by sequence from bacteriophage lambda, shown at 50, serves as starting point and a backbone for the probe. The phage was selected because its DNA would be non-hybridizable with human target DNA. The PCR amplification primers were synthesized with additional amplification primer 1 (AP1) sequence 33 on the forward primer and amplification primer 2 35 (AP2) sequence on the reverse amplification primers (FIG. 3A). These are common amplification primer targets for PCR using common primers for a multiple of probes in the downstream application. The molecule with the backbone and amplification primers was common for all the probes, except for the target sequences. The probes contain unique target sequences 56, 38.
The common lambda backbone was used as the template to make the template-specific probes (FIG. 3A). The forward primer had recognition sequences for MlyI (40) at the 5′ end. The reverse primer had sequences complementary to the target on the genomic DNA that was the desired distance apart from first target, as well as the amplification primer 2 on the target and a BsaI recognition sequence 42 at the 5′ end of the reverse primer. The resulting fragment has MlyI and BsaI adaptors flanking the two targets with the spacer DNA in between them (FIG. 3C) when the probe ends hybridize to the target.
2) Generation of Single Stranded Probe of FIG. 3B:
The PCR fragment generated as shown in FIG. 3A was digested with BsaI to generate the 5′ overhang. The molecule was then dephosphorylated with Shrimp alkaline phosphatase to remove the 5′ phosphate. It was then digested with MlyI to generate a specific 5′ end opposite the specific end created with the BsaI digestion. This molecule then was digested with lambda exonuclease. The final probe configuration is shown in FIG. 3B, wherein the target sequences 56 and 38 were targeted to human genomic DNA 44. The single-stranded probe was subsequently phosphorylated with T4 polynucleotide kinase prior to being hybridized to the genomic DNA. Ligation requires a 5′ phosphate group, added by this phoshporylation step, because it was removed earlier to protect from exonuclease digestion.
3. Amplification of Target
100 attomoles probe was annealed with 500 ng human genomic DNA. This was done by denaturation at 95° C. and annealing overnight at 58° C. As illustrated in FIG. 3C, the single stranded probe hybridized to the target, and was extended by polymerase as shown at 46. It was then ligated to form a circular probe.
The spacer backbone is illustrated in FIG. 3C as forming a loop between the hybridized targets. The DNA polymerase catalyzed the polymerization of DNA from the 3′ end to fill the gap between the two targets. The ampligase enzyme closes the circle by ligating the two ends of the probe when the enzyme reaches the 5′ end of the other target. The probes were extended and circularized using Stoffel polymerase and Ampligase in ampligase buffer (Epicenter). Following circularization, unreacted probe and genomic DNA were digested using Exonuclease I and III.
The exonuclease digestion freed the circles from genomic DNA. The circles were then amplified. It should be noted that, unlike the MIPS protocol, the molecules were not linearized, and amplification was performed using primer sequences present in the loop of probe that does not hybridize with the target. Specific amplification of probes that had hybridized was demonstrated by sequencing. The entire contents of the extension and ligation reactions were used for PCR amplification in a cocktail containing 10 mM tris-HCL (ph8.3), 50 mM Potassium Chloride, 0.2.5 mM Magnesium Chloride, and 2 units Amplitaq Gold. The primers used for amplification were done using the forward primer CGTCACATTATTTAGGTGACACTATAG (SEQ ID No: 606) and GCGTACTATTAACCCTCACTAAAGG (SEQ ID No: 607) as the reverse primer. The cycling parameters were 10 mins of heat inactivation at 95° C. followed by 40 cycles of 95° C. for 30 sec, 63° C. for 30 sec, and 72° C. for 30 sec.
Example 3
Probe Target Annealing
We compared the ability of the conventional sized MIPS that were chemically synthesized, and the SMART probes that we generated, in their ability to extend from the annealed target. The SMART probes had a spacer of 280 nucleotides in between the target sequences and the MIPS probe had a spacer of 80 nucleotides. This would typically be part of an overall SMART probe of a length of about 320 nucleotides. We discovered that the SMART probes we tested were able to amplify target molecules with gaps up to about 400 nt long. These gaps are filled in as part of the present process, and the content of the filled gap provides analytical information. The longest extension tested in Table 1 was 330, with the longest success at 175, but it is expected, based on DNA polymerization in other techniques, that the present methods and materials would work for longer extensions by varying the size of the spacer. The MIPs probes on the other hand were unable to extend molecules in the ranges that we routinely observed with the SMART probes. (Table 3).
We have shown that the SMART probes synthesized by the method described above are able to function robustly by extending over larger distances than the conventional-sized MIP probes. We believe that the optimal backbone length will depend on the target size being amplified, and that having the ability to vary this spacer length will be important to be able to efficiently multiplex PCR from thousands of exons in a single reaction. These probes have application not only in multiplex PCR, but also to generate the constructs necessary for Mismatch repair detection (MRD) assay.







TABLE 3







Comparison of MIP and SMART probes in amplification


of identical genomic targets.









Length of gap-
outcome of MIPS
outcome of SMARTS


fill (bases)
(80 base spacer)
(280 base spacer)












1
successful
successful


161
successful
successful


186
successful
successful


141
successful
successful


175
successful
successful


244
failed
successful


249
failed
successful


290
failed
successful


330
failed
successful









The above Table 3 shows that a MIPS probe with an 80 base space (region between target sequences) would not amplify when the gap between the ends of the probe was 244 nucleotides (nt). On the other hand, the SMART probes were successful at all gap distances tested, even those longer than the spacer. The SMART probes may be made to any length. It is very hard to synthesize a ss DNA molecule bigger than 120-140 base pair total and at the same time accurate and reliable for high through put molecular biology analysis. The present method enables the convenient synthesis of large ssDNA molecules and provides the opportunity for the discovery of novel methodologies, such as long-gapped circular probes.
Example 4
Preparation of Single Stranded Probe
Step 1: Creation of the Lambda Backbone Common to all Probes
The template used was Lambda DNA, shown at 30 in FIG. 3. Lambda DNA is commonly used as a substrate in restriction enzyme activity assays and for preparation of DNA molecular weight standards. The phage is isolated from a heat-inducible lysogenic E. coli W3110 (cI857 Sam7) strain. Primers (52 and 54 in FIG. 3) were prepared by the addition of Amplification 1 (AP1) and Amplification 2 (AP2) sequences to backbone from bacteriophage lambda.









Primer 1 (SK51 has AP1)



SEQ ID NO: 53



   TAATGTGACGATAGCTG



 



Primer 2 (SK52 has AP2seq)



SEQ ID NO: 54



TGTCCCTTTAGTGAGGGTTAATAGTACGCTTACTTCCGCG






The sequence after amplification is







SEQ ID NO: 55


5′   TAATGTGACGATAGCTGAAAACTGTAC


 


GATAAACGGTACGCTGAGGGCGGAAAAAATCGTCGGGGACATTGTAAAGG


 


CGGCGAGCGCGGCTTTTCCGCGCCAGCGTGAAAGCAGTGTGGACTGGCCG


 


TCAGGTACCCGTACTGTCACCGTGACCGATGACCATCCTTTTGATCGCCA


 


GATAGTGGTGCTTCCGCTGACGTTTCGCGGAAGTAAGCGTACTA


 


   






The added sequences, from the primers, are bold and italicized.
Reaction Conditions


    2.5 μl PCRII 10× buffer (ABI)
    2.5 μl 25 mM MgCl2 
    2.5 μl 1.25 mM dNTP
    1.25 μl 5 uM SK51
    1.25 μl 5 uMSK52
    2.5 μl 10 ng/μl Bacteriophage lambda DNA
    12.5 μl dH20
    1.25 units amplitaq Gold (Applied Biosystems)









TABLE 4





PCR conditions
















Hold:
94° C. 10 mins


Cycle
94° C. 20 secs, 64° C. 30 sec 72° C. 30 sec.


Touch down in 0.5° C.



decrements 14 cycles.



Cycle
94° C. 20 sec, 56° C. 30 sec 72° C. 30 sec


25 cycles



Hold
72° C. 5 mins


Hold
 4° C. o/n









Step 2: Addition of MLYI and BSAI Adaptor Sequences and Probe Specific Sequences to Backbone








SK302_Probe_F:


SEQ ID NO: 56


GTACGAGGTCTCA*GAAATGACAAATATAGATGGCAAAAGCCATCCCTTT


 


AGTGAGGGTTAAT


 


SK302_Probe_R:


SEQ ID NO: 57


 CGTGAGTCACTCGTCACAGATAGGCATGGTGTCAAAGTCATCTATAG


 


TGTCACCTAAAT 3′






These adaptor sequences are analogous to those shown at 40 and 42 in FIG. 3, but used in a second amplification as shown in FIG. 4. The bold residues adjacent the * will represent the final 5′ end and will be seen in the amplified sequence below. The 5′ sequence TAC and reverse probe sequence CAT are in shaded text to show the correlation between the target and the probe.








Target 2 BsaI



SEQ ID NO 58



GTACGAGGTCTCA*GAAATGACAAATATAGATGGCAAAAGCCATCCCTTTAGTGAGGGTTA



CATGCTCCAGAGTCTTT*ACTGTTTATATCTACCGTTTTCGGTAGGGAAATCACTCCCAAT


 


ATAGTACGCTTACTTCCGCGAAACGTCAGCGGAAGCACCACTATCTGGCGATCAAAAGGA


TATCATGCGAATGAAGGCGCTTTGCAGTCGCCTTCGTGGTGATAGACCGCTAGTTTTCCT


 


TGGTCATCGGTCACGGTGACAGTACGGGTACCTGACGGCCAGTCCACACTGCTTTCACGC


ACCAGTAGCCAGTGCCACTGTCATGCCCATGGACTGCCGGTCAGGTGTGACGAAAGTGCG


 


TGGCGCGGAAAAGCCGCGCTCGCCGCCTTTACAATGTCCCCGACGATTTTTTCCGCCCTC


ACCGCGCCTTTTCGGCGCGAGCGGCGGAAATGTTACAGGGGCTGCTAAAAAAGGCGGGAG


 


 


AGCGTACCGTTTATCGTACAGTTTTCAGCTATCGTCACATTAATTTAGGTGACACTATAG


TCGCATGGCAAATAGCATGTCAAAAGTCGATAGCAGTGTAATTAAATCCACTGTGATATC


 


ATGACTTTGACACCATGCCTATCTGTGA**CGAGTGACTCACGATG   3′


TACTGAAACTGTGGTACGGATAGACACT**GCTCACTGAGTGC  5′






As can be seen in the amplified sequence above, the final 5′ end is bolded to the right of the *. The bolded region to the left of the * can be seen to be complementary to the bolded sequence of probe R, with the final, added pairs of residues indicated by shading. The ** indicates where the final 3′ end will be.

    2.5 μl PCRII 10× buffer (ABI)
    2.5 μl 25 mM MgCl2 
    2.5 μl 1.25 mM dNTP
    1.25 μl 5 μM SK302_Probe_F
    1.25 μl 5 μM SK302_Probe_R
    2.5 μl 1:10,000 dil of Step 1 PCR
    12.5 μl dH20
    1.25 units amplitaq Gold (ABI)









TABLE 5





PCR conditions
















Hold:
94° C. 10 mins


Cycle
94° C. 20 secs, 64° C. 30 sec 72° C. 30 sec.


Touch down in 0.5° C.



decrements 14 cycles.



Cycle
94° C. 20 sec, 56° C. 30 sec 72° C. 30 sec


25 cycles



Hold
72° C. 5 mins


Hold
 4° C. o/n









Step 3: Digestion With BsaI

The first digestion with BsaI cuts 1 base inward from the recognition site on the top strand, and 5 bases inward on the lower strand. This creates a molecule with a recessed 3′ end and a protruding 5′ end. The 5′ end (G in the sequence GAA adjacent *) now has a phosphate group. The sequence is shown at SEQ ID NO: 58, where the asterisks indicate cut sites.
Procedure:


    Clean up the PCR product on a Micro Biospin P-30 column (Bio-Rad).
    Spin the column at 1000 g for 2 min.
    Add 50 μl PCR product (approx 200 ng) and spin 1000 g 4 min.
    Use 45 μl of eluate for BsaI digestion
    Add 5 μl NEB3 buffer and 3 μl BsaI (New England Biolabs)
    Incubate at 50° C. for 2 hrs followed by 65° C. heat inactivation.

Step 4: Dephosphorylation



The phosphate group on the 5′ overhang of this molecule (G in the sequence GAA) is then cleaved with a phosphatase making it resistant to lambda exonuclease cleavage.

    Add 3 μl Shrimp Alkaline Phosphatase (1 unit/ul United States Biochemical)
    Incubate 37° C. for 60 min followed by heat inactivation at 80° C. 15 min

Step 5: Digestion with MlyI



The molecule is then digested with the enzyme MlyI that cuts 5 bases inward from the recognition site, and generates a blunt-end molecule that has a phosphate group at the 5′ end. The reaction was cleaned up as in Step 3 on the Micro Bio-Spin P-30 columns as described above:

    To 45 μl of the eluate add 5 μl NEB buffer I and 3 μl MlyI (NEB).
    Incubate at 37° C. for 60 min followed by heat inactivation for 15 min at 65° C.


After this step, the molecule has the sequence shown in SEQ ID NO: 58, with the portions 5′ of the * and 3′ of the ** (with reference to the top strand) removed.
Step 6: Digestion with Lambda Exonuclease
This enzyme preferentially cleaves the phosphorylated strand, and generates the desired single-stranded molecule. The resulting sequence is the top strand of STEP 5.
To achieve this, we added 0.2 units Lambda Exonuclease (NEB) to the MlyI digested DNA. Incubate at 37° C. for 15 min followed by heat inactivation for 15 min at 80° C.
Step 7: Phosphorylation of 5′ End:
Use 10 μl of the exonuclease digested DNA, 5 μl T4DNA ligase buffer (NEB), 1 μl T4 Polynucleotide kinase. Incubate 37° C. 60 min followed by heat inactivation for 15 min at 65° C.
Example 5
Spacer Multiplex Amplification ReacTion (SMART) Using the Above ssDNA Probe
Dilute kinased probe to 10 fmole/μl. Use this as the stock. Make a fresh 1:200 dilution to make a 50 amol/μl working solution.







TABLE 6





Hybridization of Probe to Target DNA

















Probe
2
μl


Human Genomic DNA
2
μl (500 ng)


Water
3
μl


10x Ampligase buffer (Epicenter Technologies)
0.7
μl


Mix well by pipetting.









Hold at 95° C. for 5 mins.
Gradually decrease temperature by 1° C. decrements to 58° holding at 1 min at each temperature. Hold overnight at 58° C. The target DNA is TLR10 (toll like receptor) of human genomic DNA2. Extension of hybridized probe and ligation

    Place tubes on a cold metal block on ice.
    After 2 mins add a 6.5 μl of a master mix containing
    0.8 μl 10× Ampligase buffer
    5 units Ampligase Epicenter)
    0.5 units Stoffel fragment of Taq polymerase (Applied Biosystems)
    5.2 μl dH20
    Incubate 58° C. for 2 mins.
    Place on ice block
    Add 1.5 μl of Cold dNTP mix (1.25 mM)
    Mix well by pipetting.
    Incubate 58° C. for 15 mins and hold at 37° C.

2. Exonuclease Digestion of Linear Probes/Template:



Add 10 units Exonuclease I (Ecpicenter Technologies) and 10 units Exonuclease III (Ecpicenter Technologies). Incubate at 37° C. for 15 mins and heat inactivate at 80° C. for 15 mins.
3. PCR Using Common Primers on Released Probes


    5 μl PCRII 10× buffer (ABI)
    5 μl 25 mM MgCl2 
    5 μl 1.25 mM dNTP
    5 μl 1 μM Amplification Primer 1
    5 μl 1 μM Amplification Primer 2
    5 μl 10 ng/μl extension and Ligation mix
    20 μl dH20
    2 units amplitaq Gold (ABI)
    PCR conditions
    95° C. 10 min.
    Cycle 40 times 94° C. 30 sec
    
        63° C. 30 sec
        72° C. 30 sec
    
    
    72° C. 5 mins
    hold 4° C.


The PCR step described above is carried out after the probe made by exonuclease digestion (i.e. after STEP 7) is hybridized to a target sequence, e.g., human genomic DNA. The hybridized DNA is detected and amplified by the PCR reaction described immediately above. The target sequence that is amplified after extension from the annealed probe is shown below,







SEQ ID NO: 59


ATGACTTTGACACCATGCCTATCTGTGA*GGAAGCTGGCAACATGTCACA


 


CCTGGAAATCCTAGGTTTGAGTGGGGCAAAAATACAAAAATCAGATTTCC


 


AGAAAATTGCTCATCTGCATCTAAATACTGTCTTCTTAGGATTCAGAACT


 


CTTCCTCATTATGAAGAAGGTAGCCTGCCCATCTTAAACACAACAAAACT


 


GCACATTGTTTTACCAATGGACACAAATTTCTGGGTTCTTTTGCGTGATG


 


GAATCAAGACTTCAAAAATATTA*GAAATGACAAATATAGATGGCAAAAG


 


CCA






In the above representation, the bold sequences can be seen to correspond to those underlined in SEQ ID NO: 58 (amplified sequence) and the remainder of the sequence is that which is filled in. The human DNA sequence is based on Homo sapiens toll-like receptor 10 (TLR10), transcript, which can be found in GenBank as, e.g. NM—001017388. In the sequences below, the target sequences are also shown in bold.
Example 6
Multiplex PCR Amplification
In this example, 3 different probes were mixed in the same tube to amplify products that are of three different lengths in the same tube. The present example includes three probes that bound to human genomic DNA. To prepare the probes we used 3 sets of primers. Each primer is identified with reference to the probe for which it is used, e.g. SK302-Probe_F is the forward primer for what will be the SK302 probe.
Probe 1—Extends 328 Bases
SK302_Probe_F:







SEQ ID NO: 56


GTACGAGGTCTCA*GAAATGACAAATATAGATGGCAAAAGCCATCCCTTT


 


AGTGAGGGTTAAT






It can be seen that the bolded bases of Probe F correspond to the bolded residues at the 3′ end of SEQ ID NO: 59.
SK302_Probe_R: This sequence is given above as SEQ ID NO: 57. Probe SK302 is also disclosed in Step 2 of Example 4.
The Target genomic sequence is as given in Example 5, SEQ ID NO: 59.
Probe 2—Extends 244 Bases
SK298_Probe_F and SK298_Probe_R sequences are given in Example 4
Their target genomic sequence 2 is







SEQ ID NO: 60


TTCTAGACATGCCCTTCATGTGATTCTTATG*AGAAAAAACCACCCAAAG


 


AATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGGTATCTACCTTAAGT


 


TTCATTTGATTTTCTGCTTTATCTTTACCTATCCAGATTTGCTTCTTAGT


 


TACTCACGGTATACTATTTCCACAGATGATTCATCAGCATCTGTCCTCTA


 


GAACACACGGAAGTGAAGATTCCTGAGGATCTAACTTGCAGTTGGACACT


 


ATGTTACATACTCTAATATAGTAGTGAAAGTCATTTCTTTGTATTCCAAG


 


TGGAGGAGTACAATATATTAGCGATGGGAAAAAAAAACTCATAAGTGTGC


 


AAAGTCAGGA**TTATTTCCCCATAATCACTATACAATAGTCT






This target sequence is from the human IL21 gene
SK298_Probe_F bolded residues can be seen to be the reverse complement of the 5′ end of Target genomic sequence 2 and can be visually matched at the asterisk. Similarly, for SK298_Probe_R, the bolded sequence can be found at the double asterisk in genomic sequence 2.

Probe 3 Extends 251 Bases








SK303_Probe_F:


SEQ ID NO: 61


CATCGTGAGTCACTCG*TAGGCATGGTGTCAAAGTCATTAAAAGAAAGAC


 


TATAGTGTCACCTAAAT


 


SK303_Probe_R:


SEQ ID NO: 62


GTACGAGGTCTCA**GAAATGACAAATATAGATGGCAAAAGCCAATTTCC


 


CTTTAGTGAGGGTTAAT






Target genomic sequence 3, also from TLR10







SEQ ID NO: 63


TCTTTCTTTTAATGACTTTGACACCATGCCTA*TCTGTGAGGAAGCTGGC


 


AACATGTCACACCTGGAAATCCTAGGTTTGAGTGGGGCAAAAATACAAAA


 


ATCAGATTTCCAGAAAATTGCTCATCTGCATCTAAATACTGTCTTCTTAG


 


GATTCAGAACTCTTCCTCATTATGAAGAAGGTAGCCTGCCCATCTTAAAC


 


ACAACAAAACTGCACATTGTTTTACCAATGGACACAAATTTCTGGGTTCT


 


TTTGCGTGATGGAATCAAGACTTCAAAAATATTA**GAAATGACAAATAT


 


AGATGGCAAAAGCCAATT






These three sets of primers (SK302 Probe F, SK302 Probe R; SK298 Probe F, SK298 ProbeR; and SK303 ProbeF and SK303 ProbeR) were used in each of three PCR amplifications with the template used in STEP 2 of the example where we describe preparation of the double stranded PCR product prior to digestion with MlyI (Example 2). All subsequent steps were performed exactly as in the example. After the single-stranded probes were phosphorylated using T4 polynucleotide kinase, the three single stranded probes were then mixed together such that their final concentration was 100 attomoles/μl. 2 μl of the mixed probe set was used in the SMART reaction exactly as described in the example. After amplification using the common PCR primers, we were able to identify the 3 discrete products that were the result of the three extensions and circularization of the three probes.
This example demonstrates that we can perform a multiplex PCR using the SMART probes.
Example 7
Multiplex PCR Amplification Using Over 500 Different Probes, which are Described in Accompanying CD
We designed probes to amplify exons from human genomic DNA in a multiplex fashion. To achieve this goal, we identified targets in the introns adjacent to the exons. For each exon, we designed two targets, one on each side of the exon, in the flanking intronic sequence. The distance between the targets varied based on the size of the exon. To connect the two targets we made a spacer backbone (see FIG. 4) from lambda DNA that was common to all the probes. To construct each exon probe, we designed a pair of primers (FIGS. 4, 61,62) that amplified a double-stranded PCR product using the backbone template. One primer for each exon had a BsaI site, a target sequence for the exon, followed by a region of homology to the backbone DNA (shown by vertical stripes in FIG. 4). The other primer for the exon probe had an MlyI site, the second target exon, followed by the homology to the backbone. The double-stranded probes for each exon were then converted into single-stranded probes. It should be noted that exons are about 150 to 400 nt long, so that the probes must obtain such information (gap filling) for the sequencing purpose described here. That is, one will obtain on the order of 106 amplified probes containing the exon sequence (30-40 PCR cycles), which is sufficient to determine the exon sequence in the gap. Another aspect of the present multiplex methodology is the high number of probes which can be amplified simultaneously, without artifacts. Aspects enabling this are the use of common amplification primers, and the use of a relatively low level of amplification primer (on the order of 100 attomoles).
Step 1. Preparation of the Backbone
The template used was Lambda DNA, shown at 64, FIG. 4, and at 30 in FIG. 2. Primers (52 and 54 in FIGS. 3, 61, 62, FIG. 4) were prepared by the addition of Amplification 1 (AP1) and Amplification 2 (AP2) sequences to backbone from bacteriophage lambda. The Primer SK618 has Amplification primer 1 shown below in bold italics, and the primer SK619 has Amplification primer 2 shown in bold italics. The PCR reaction was performed in 50 mM Potassium Chloride, 10 mM Tris-HCl (pH8.5), 2.5 mM Magnesium Chloride, 2 units Amplitaq gold. The cycling conditions were 10 min heat inactivation at 950 C followed by 25 cycles of 94° C. for 30 sec, 63° C. for 30 sec, and 72° C. for 30 sec per cycle.







Primer 1 (SK618 has AP1)


SEQ ID NO: 64


    TAATGTGACGATAGCTG


 


Primer 2 (SK619 has AP2seq)


SEQ ID NO: 65


 TCGATCCCTTTAGTGAGGGTTAATAGTACGCTTACTTCCGCG






The sequence after amplification (see FIG. 4B) is








SEQ ID NO: 66


5′    TAATGTGACGATAGCTGAAA


 


ACTGTACGATAAACGGTACGCTGAGGGCGGAAAAAATCGTCGGGGACATT


 


GTAAAGGCGGCGAGCGCGGCTTTTCCGCGCCAGCGTGAAAGCAGTGTGGA


 


CTGGCCGTCAGGTACCCGTACTGTCACCGTGACCGATGACCATCCTTTTG


 


ATCGCCAGATAGTGGTGCTTCCGCTGACGTTTCGCGGAAGTAAGCGTACT


 


    






For the convenience of the reader, three corresponding nucleotides are shaded to show correspondence between primer 2 and the amplified sequence.

Preparation of Single Stranded Probes

For each probe, two primers were used with this backbone as the template for a PCR reaction. One primer had a target sequence to genomic DNA with a BsaI adaptor, and the second primer had the target with a MlyI adaptor. These primers were used in a PCR reaction with the backbone DNA as template analogous to that shown in FIG. 4.

As an example:








Primer 292361_Bsa










GTACGAGGTCTCActgtaagccctgcaatttccccCCATCGATTCCCTTTAG
SEQ ID NO: 67



 


Primer 292361_Mly



CATCGTGAGTCACTCGtcatggggtaagacgatcatagaGGGGCGCGCCCTATAGTGT
SEQ ID NO: 68






The sequence of the double-stranded probe after amplification is shown below.








BsaI



SEQ ID NO: 69



5′GTACGAGGTCTCA*ctgtaagccctgcaatttccccCCATCGATTCCCTTTAGGGTTAATA



  CATGCTCCAGAGTGACAT*TCGGGACGTTAAAGGGGGGTAGCTAAGGGAAATCCCAATTAT


 


  GTACGCTTACTTCCGCGAAACGTCAGCGGAAGCACCACTATCTGGCGATCAAAAGGATGG


  CATGCGAATGAAGGCGCTTTGCAGTCGCCTTCGTGGTGATAGACCGCTAGTTTTCGTACC


 


  TCATCGGTCACGGTGACAGTACGGGTACCTGACGGCCAGTCCACACTGCTTTCACGCTGG


  AGTAGCCAGTGCCACTGTCATGCCCATGGACTGCCGGTCAGGTGTGACGAAAGTGCGACC


 


  CGCGGAAAAGCCGCGCTCGCCGCCTTTACAATGTCCCCGACGATTTTTTCCGCCCTCAGC


  GCGCCTTTTCGGCGCGAGCGGCGGAAATGTTACAGGGGCTGCTAAAAAAGGCGGGAGTCG


 


  GTACCGTTTATCGTACAGTTTTCAGCTATCGTCACATTAATTTAGGTGACACTATAGGGC


  CATGGCAAATAGCATGTCAAAAGTCGATAGCAGTGTAATTAAATCCACTGTGATATCCCG


 


  GCGCCCCtctatgatcgtcttaccccatga**CGAGTGACTCACGATG 3′


  CGCGGGGAGATACTAGCAGAATGGGGTACT**GCTCACTGAGTGCTAC MlyI






It will be apparent that the “tga” at the 3′ end adjacent the ** corresponds to the reverse complement of the “tca” at the beginning of Primer 292361_Mly shown in bold. The target sequences that hybridize to genomic DNA are shown in lower case and underlined. The restriction cut sites for BsaI and MlyI are shown with an asterisk. As can be seen in the amplified sequence above, the final 5′ end is bolded to the right of the *.
After the double-stranded probe was amplified, the PCR products were digested with 10 units of BsaI in NEB4 (New England Biolabs) at 50° C., followed by digestion with 3 units Shrimp Alkaline Phosphatase (United States Biochemicals) at 37° C. in the same buffer for 60 min. The reaction volume was increased to 100 μl, and the MlyI digestion was carried out in NEB buffer 4 at 37° C. using 20 units MlyI (New England Biolabs). 20 μl of this reaction was digested with 0.5 units of Lambda Exonuclease (NEB) at 37° C. for 10 mins. The reaction products were phosphorylated using 5 units of Polynucleotide Kinase in T4DNA ligase buffer (NEB).
Hybridization of Probes, Extension, Multiplex PCR
100 attomoles of each probe was hybridized to 500 ng of human genomic DNA in 7 μl of 1× ampligase buffer containing 20 mM tris-HCl (pH8.3), 25 mM KCl, and 10 mM magnesium chloride. The hybridization was carried out in a single tube in a thermal cycler by raising the temperature of the mix to 98° C. for 2 min, and gradually bringing the temperature to 58° C. by decreasing the temperature by 1° C. per minute. The reaction was held at 58° C. overnight. The extension reaction was carried out by adding 1.6 μl of 5×GC buffer (NEB) that was supplied with PhusionTM High-Fidelity DNA Polymerase, 100 nM dNTP, 0.4 units PhusionTM High-Fidelity DNA Polymerase (NEB), and 0.5 units ampligase (Epicenter). The reaction was incubated for 20 min at 58° C. and 10 min at 72° C. The excess probe and genomic DMA was digested with 1 unit Exonuclease I and 0.2 units Exonuclease III for 310 min at 37° C. followed by heat inactivation for 20 min at 80° C. The reaction volume was raised to 50 μl and the PCR reaction was carried out in 1×GC buffer (NEB), 200 mM dNTP, and 0.2 units PhusionTM High-Fidelity DNA Polymerase after addition of the two amplification primers AP1 and AP2. The reactions were cycled 40 times at 98° C. for 30 sec, 62° C. 30 sec, and 72° C. 30 sec per cycle.
Determining Success of Multiplex PCR
1.5 micrograms of the PCR products were digested with 0.04 units DnaseI (NEB) for 5 min. After a column cleanup on a Biorad P-30 chromatography column, the products were end-labeled with followed by end-labeling at 37° C. for 10 min with 1 nmole of Bio-N6-ddATP in 1×NEBuffer 4 (NEB) supplemented with 2.5 mM cobalt chloride using 1 unit Terminal Transferase (NEB).
The labeled products were hybridized to a DNA microarray chip containing tiling probes to all the exon targets under interrogation. Tiling probes are complementary to various subsequences within a DNA sequence set (here, the human genome). They are further described in European Patent EP1479782. The success of the multiplex PCR was determined by whether a sequence for each exon could be determined by analyzing the re-sequencing array.







TABLE 7







Success of multiplex PCR










Size of
Percent GC 35-50%
Percent GC >50%













amplicon(bp)
Success
Failures
Success
FAILURES
Total















150-200
78
1
27
7
113


200-300
187
2
67
46
302


300-400
57
0
25
14
96


400-500
9
0
5
8
22


Total
331
3
124
75
533









The above results show that overall, 331 probes out of 533 were amplified in the multiplex reaction, as evidenced by sufficient quantity of probe to show a signal when the amplicon, containing the target sequence, was hybridized to a DNA microarray containing human gene sequences. If the probes are designed to contain less than 50% GC content in the amplicon and target and primer sequences, the failure rate is 3/331 or less than 1%.
A Lengthy Table is submitted herewith on CD describing all 500 probes, SEQ ID NOs 70-601. Each entry on the CD ROM is organized as follows: an arbitrary probe ID NO (e.g. 29190), SEQ ID NO; the size of the amplified sequence in by (e.g. 235), including the target sequence, and the actual sequence filled in and amplified with the target sequences set off by slashes. These sequences represent human gene exon sequences obtained with the probes and are presented for purposes of exemplification of a large scale multiplex reaction with relatively large gaps filled in and amplified using the present SMART probes.
CONCLUSION
Other Embodiments
The present specific description is meant to exemplify and illustrate the invention and should in no way be seen as limiting the scope of the invention, which is defined by the literal and equivalent scope of the appended claims. Any patents or publications mentioned in this specification are indicative of levels of those skilled in the art to which the patent pertains and are intended to convey details of the invention which may not be explicitly set out but would be understood by workers in the field Such patents or publications are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference for the purpose of describing and enabling the method or material referred to.
Alternative embodiments may be carried out, given the present teachings. For example, US PGPUB 2005/0053990 to Roberts et al., published Mar. 10, 2005 entitled “Cleavage of RNA by restriction endonucleases,” hereby incorporated by reference, teaches that a number of restriction endonucleases have been shown to be capable of cleaving RNA in RNA/DNA duplexes although this property is not inherent in the universe of restriction endonucleases. It is taught there that restriction endonuclease cleavage of RNA/DNA duplexes have precise ends corresponding to the cleavage site of the restriction endonuclease. RNA is expected to be size limited only at the lower end of the range, typically 2-8 nucleotides longer than the length of the recognition sequence of the restriction enzyme. Given this teaching, one may prepare cDNA-RNA duplexes from RNA using reverse transcriptase and prepare ssDNA probes from cDNA according to the present teachings.
REFERENCES


    Cho R J, Mindrinos M N, Richards D R, Sapolsky R J, Anderson M, Drenkard E, Dewdney J, Reuber T L, Stammers M, Federspiel N, Theologis A, Yang W H, Hubbell E, Lashkari D, Lemieux B, Dean C, Lipshutz R J, Ausubel F M, Davis R W and Oefner P J. Genome-Wide Mapping with Biallelic Markers in Arabidopsis thaliana. Nature Genetics, 1999, 23:203-207.
    Crothers et al., Amplification of DNA to produce single-stranded product of defined sequence and length U.S. Pat. No. 6,815,167 (Nov. 9, 2004).
    Hardenbol P, Baner J, Jain M, Nilsson M, Namsaraev E A, Karlin-Neumann G A, Fakhrai-Rad H, Ronaghi M, Willis T D, Landegren U, Davis R W. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat Biotechnol. (2003) June; 21 (6):673-8.
    Higuchi, R and Ochman, H. Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Research, Vol 17, No. 14, 5865 (1989).
    Landegren et al., Rolling circle replication of padlock probes U.S. Pat. No. 6,558,928 (May 6, 2003).
    Landegren et al., Nucleic acid detecting reagent U.S. Pat. No. 6,235,472 (May 22, 2001).
    Li M, Diehl F. Dressman D, Vogelstein B, Kinzler K. BEAMing up for detection and quantification of rare sequence variants. Nature Methods 3, 95-97 (2006).
    Lizardi. Rolling Circle reporter Systems. U.S. Pat. No. 5,854,033. (1998).
    Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments Fredrik Dahl, Mats Gullberg, Johan Stenberg, Ulf Landegren and Mats Nilsson Nucleic Acids Research (2005) 33 (8):e71.
    Nikiforov et al., U.S. Pat. No. 5,518,900 Method for generating single-stranded DNA molecules (May 1996).
    Nilsson, M., et al., “Padlock Probes: Circularizing Oligonucleotides for Localized DNA Detection,” Science, 285:2085-2088 (1994).
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## Claims

1. A method for preparing a molecule for sequencing, comprising the step of hybridizing to said molecule for sequencing a probe molecule of defined sequence and length, having a non-human sequence, a predefined 5′ end, a predefined 3′ end and a portion of a double stranded precursor molecule, said probe molecule further being prepared from said double stranded precursor molecule by a method comprising the steps of:
(a) amplifying the double stranded precursor molecule with primers each containing a sequence homologous to a portion of the double stranded molecule and a sequence not homologous to the double stranded precursor molecule, said primers also containing a sequence comprising one of said predefined 5′ end or said predefined 3′ end, said primers further containing restriction enzyme cleavage sites to obtain an amplified double stranded molecule having cleavage sites at the 3′ and 5′ ends, said primers further comprising a first target sequence and a second, different, target sequence, wherein the first target sequence and the second target sequence hybridize to human genetic sequences;
(b) cleaving the amplified double stranded molecule at the 5′ end with a first restriction enzyme;
(c) cleaving the amplified double stranded probe molecule at the 3′ end with a second, different restriction enzyme to prepare the probe molecule of defined sequence and length; and
(d) hybridizing said double stranded probe molecule of step (c) through said first target sequence and said second target sequence to said molecule for sequencing, thereby preparing said molecule for sequencing.

2. The method of claim 1 wherein a cleaving step is done with a type IIs restriction enzyme.

3. The method of claim 2 further comprising the step of digesting with BsaI to generate a 5′ overhang outside of BsaI's recognition site.

4. The method of claim 1 wherein the cleaving with a restriction enzyme at the desired predetermined 3′ end is done with a restriction enzyme to create a 3′ blunt end or no 5′ overhang.

5. The method of claim 1 wherein the predefined 5′ end and predefined 3′ end are separated by at least 125 nucleotides of spacer backbone from the precursor molecule.

6. The method of claim 1 wherein the nonhuman organism is a microorganism.

7. The method of claim 1 wherein said molecule for sequencing spans an exon.

8. The method of claim 1 further comprising the step of sequencing said molecule after said hybridizing of step (d).

9. The method of claim 1 wherein said hybridizing is done at an elevated temperature.