CA2549849A1

CA2549849A1 - Improved selective ligation and amplification assay

Info

Publication number: CA2549849A1
Application number: CA002549849A
Authority: CA
Inventors: Tom Morrison
Original assignee: Individual
Current assignee: Life Technologies Corp
Priority date: 2003-12-10
Filing date: 2004-12-10
Publication date: 2005-06-30
Also published as: EP1692314A1; JP2007515956A; WO2005059178A1

Abstract

An improved assay for identifying and distinguishing one or more a single nucleotide polymorphisms in one or more target sequences of nucleic acid comprises, in a single-tube reaction system, three or more primers, two of which bind to a target nucleic acid sequence, flanking a SNP, so that the 3'-end of one or more first primers is adjacent to the 5'-end of a second primer, the two primers being selectively ligated and then amplified by a third primer to exponentially produce the complementary strand of the one or more target sequences. The other strand of the one or more target sequences are exponentially amplified by one or more hybridizable probes, each labeled with a different fluorophore, the fluorophore-labeled hybridizable probes being quenched until incorporation into and amplification of target nucleic acid products. Also provided is a method for identifying one or more SNPs in one or more target sequences of nucleic acid in each single through-hole of a nanoliter sampling array, and a kit for such a method containing a nanoliter sampling array chip, primer sequences, and reagents required to selectively ligate primers for amplification of desired target nucleic acid sequences.

Description

Improved Selective Ligation and Amplification Assay Technical Field The present invention relates to assays for amplifying and identifying target sequences of nucleic acids involving a combined ligation and amplification protocol, and the use of nanoliter sampling arrays to perform such assays.
Sack~round Art Genetic variations are increasingly being linked to a multitude of disease conditions and predispositions for disease, including cancer, multiple sclerosis, autoimmune diseases, cystic fibrosis, and schizophrenia. The ability to identify genetic variations rapidly and inexpensively will greatly facilitate diagnosis, risk assessment, and determination of the prognosis for such diseases and predispositions for these diseases.
One possibility for identifying genetic variations involves combining selective ligation and amplification techniques, disclosed in U.S. Patent No. 5,593,840 to Bhatnagar et al. and U.S. Patent No. 6,245,505 to Todd et al, both of which are hereby incorporated by reference herein. Both patents disclose the use of at least three primers, two of which are complementary to adjacent regions of the 3'-end of one strand of a target nucleic acid sequence which, after hybridization, can be ligated and then extended.
In Todd et al., the third primer is a random sequence, complementary to the random sequence at the 3'-end of the downstream primer (that ligates to the upstream primer) and identical to the random sequence on the 5'-end of the first primer. In Bhatnagar et al., the third primer is complementary to the upstream primer, and also to the opposite strand of the target sequence. In both cases, there must be complementarity at the 3'-end of the third primer to allow amplification to occur.
A heat-stable polymerase is used to amplify the target nucleic acid sequence, and both the ligation and amplification reactions can be carned out in the same reaction mixture. An optional gap between the adjacent primers may be present, which may be filled by a polymerase to allow successful ligation of the adjacent primers.
Such a system allows identification of genetic variability in target nucleic acid sequences, and identification of multiple alleles.

Summary of the Invention In a first embodiment of the invention, there is provided an improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled-temperature reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence l0 segment of the first and second primers, wherein the improvement comprises:
distinguishing in a single-tube reaction system between one or more SNPs in one or more target sequences of nucleic acid using two unique probes designed to hybridize to the target nucleic acid sequences with SNPs of interest, each hybridizable probe having a different fluorescent tag that is quenched until incorporation of the probe into amplified 15 target nucleic acid product.
In some embodiments of the improved assay, of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises one or more SNPs of interest that are not at an end of the target sequence, the assay being a selective ligation and amplification method of the type using a thermocycled reaction mixture including the 2o target sequence, a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the target sequence, a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the target sequence, the 5'-end of the second primer being adjacent to or within two to four bases of the 3'-end of the first primer wherein a nucleotide 25 complementary to the SNP of the target sequence is present at either the 3'-end of the first primer or at the 5'-end of the second primer, and a third primer that is substantially complementary to a random sequence segment at the 3'-end of the second primer and to a substantially similar sequence at the 5'-end of the first primer, at least four different nucleotide bases, a thermostable polymerise and a thermostable ligase, wherein the 30 improvement comprises distinguishing in a single-tube reaction system between one or more SNPs in one or more target sequences of nucleic acid using two unique probes designed to hybridize to the target nucleic acid sequences with SNPs of interest, each hybridizable probe having a different fluorescent tag that is quenched until incorporation of the probe into amplified target nucleic acid product. The first hybridizable probe with first fluorescent tag has a unique random sequence that hybridizes to a first amplified target nucleic acid generated by the third primer from a ligated first primer-second primer product having a first SNP of interest on the 3'-end of the first primer, the first hybridizable probe thereby becoming incorporated into amplified opposite strand target nucleic acid product to give a first fluorescent signal. The second hybridizable probe with second fluorescent tag has a unique random sequence that hybridizes to a second amplified product generated by the third primer from a different ligated first primer-second primer product having a second SNP of interest on the 3'-end of the first primer, the second hybridizable probe thereby becoming incorporated into amplified opposite strand target nucleic acid product to give a second fluorescent signal.
In a preferred embodiment, the random sequences of the first and second hybridizable probes are unique sequences, such that specific incorporation of each of the hybridizable probes into amplified target nucleic acid preferentially occurs after ligation of the first primer-second primer product having the particular SNP of interest that the hybridizable probe was designed to detect. Upon incorporation of the hybridizable probe into amplified product, fluorescence occurs, making detection of the amplified product distinguishable from non-specific background products. Additionally, the random sequence of the third primer is also a unique sequence, optimized for PCR to reduce non-specific amplified products that may be generated in the presence of human or other species chromosomes to a sufficiently low level that such non-specific products do not interfere with detection of amplified products having a SNP of interest.
Alternatively, the two hybridizable probes do not contain fluorescent tags, but are simply additional primers designed to distinguish different ligated products having different SNPs of interest. Detection of amplified product with a SNP of interest is then done using additional hybridizable probes, similar to the additional primers, but are developed in a manner not to interfere with amplification. These hybridizable probes have a fluorescent tag, or alternatively, each have a different fluorescent tag, and upon hybridizing to amplified product, fluoresce, thereby allowing detection of amplified product.
In another embodiment of the invention there is provided an improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises a SNP of interest that is not at an end of the target sequence, the assay being a selective ligation and amplification method of the type using a thermocycled reaction mixture including the target sequence, a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the target sequence, a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer wherein a nucleotide complementary to the SNP of the target sequence is present at either the 3'-end of the first primer or at the 5'-end of the second primer, and a third primer that is substantially complementary to a random sequence segment at the 3'-end of the second primer and to a substantially similar sequence at the 5'-end of the first primer, at least four different nucleotide bases, a thermostable polymerise and a thermostable ligase, wherein the improvement comprises homogeneously detecting amplified target sequence using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence. In a preferred embodiment, the random sequence of the third primer is a unique sequence, optimized for PCR such that no non-specific products are generated in the presence of human or other species chromosomes. In some embodiments, primers may be affixed on, within or under a biocompatible material such as a wax-like coating on the surface of the through-holes by drying the primers after application to the through-holes, wherein the biocompatible material may comprise, for example, a polyethylene glycol (PEG) material.
Alternatively, assays in accordance with the present invention may. use a thermostable polymerise that lacks 5' to 3' exonuclease activity, or a thermostable polymerise that lacks 3' to 5' exonuclease activity, or a thermostable polymerise that lacks both 5' to 3' and 3' to 5' exonuclease activity. Examples of thermostable polymerises which lack 5' to 3' exonuclease activity include Stoffel fragment, IsisTM
DNA polymerise, PyraTM exo(-) DNA polymerise, and Q-BioTaqTM DNA polymerise.
Examples of thermostable polymerises which lack 3' to 5' exonuclease activity include Taq polymerise, SurePrimeTM Polymerise, and Q-BioTaqTM DNA polymerise. An example of a thermostable polymerise which lacks both 5' to 3' and 3' to 5' exonuclease activity is Q-BioTaqTM DNA polymerise. Suitable dyes include SYBR°
Green I and SYBR° Green II, YOYO"-1, TOTO°-1, POPO°-3, ethidium bromide, or any other dye that allows rapid, sensitive detection of amplified target nucleic acid sequence using fluorescence.
In another embodiment, there is provided a nanoliter sampling array comprising a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. In this particular embodiment, each through-hole contains at least a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of a potential nucleic acid target sequence a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the potential nucleic acid target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer upon binding to the potential nucleic acid target sequence.
In addition, the sampling array may further comprise a second platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes wherein the first and second platen are fixedly coupled such that the through-holes of each are aligned.
In yet another embodiment, there is provided a method of identifying a SNP in a target sequence of nucleic acid, the method comprising providing a first sample platen having a high-density microfluidic array of through-holes, each through-hole having a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the target sequence, a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the target sequence, the,5'-end of the second primer being adjacent to the 3'-end of the first primer, and third primer that is substantially complementary to a random sequence segment at the 3'-end of the second primer and to the 5'-end of the first primer, introducing a sample containing a target sequence of nucleic acid having a SNP of interest to the array, introducing reagents to the through-holes in the array, the reagents including a thermostable polymerase, a thermostable ligase, and at least four different nucleotide bases, thermocycling the array, and detecting amplified target sequence. In a preferred embodiment, primers 1 and 2 are designed with a possible match to the target strand SNP
located at either the 3'-end of the 5' primer (the first primer) or located at the 5'-end of the 3' primer (the second primer). When the first and second primers hybridize to the target strand, adjacent to each other and flanking the SNP, ligation of the primers only occurs if there is a successful match to the SNP by one of the primers. In this way, the ligation is 3o selective and so selective amplification of the desired target sequence containing the SNP
of interest also occurs. As described above, in some embodiments, primers may be affixed on, within or under a biocompatible material such as a wax-like coating on the surface of the through-holes by drying the primers after application to the through-holes, wherein the biocornpatible material may comprise, for example, a polyethylene glycol (PEG) material.
In addition, the method of identifying a SNP in a target sequence of nucleic acid may additionally comprise using a thermostable polymerase that lacks 5' to 3' exonuclease activity, and detecting amplified target sequence using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.
Alternatively, detecting may comprise using first primers and second primers designed to generate amplified target sequences with differential melting curves to distinguish individual amplified target sequences by differences in melting temperatures (Tms), or may comprise using a probe specific for hybridizing across a ligation junction formed l0 between the first primer and second primer after binding to the target sequence wherein the probe specific for hybridizing across the ligation junction has a fluorescent group 'and a fluorescence-modifying group, or using a probe containing a fluorescent group and a fluorescence-modifying group specific for hybridizing to a region of the target sequence wherein upon extension of the probe, the fluorescence-modifying group is excised and i5 . the fluorescent group fluoresces. Additionally, detection may be done using a probe specific for hybridizing to any unique sequence in the amplified target nucleic acid, the probe having a fluorescent group and a fluorescence-modifying group such that the upon ' hybridization the probe fluoresces, allowing detection of the amplified target nucleic acid.
Other means of detection comprise the use of amplification primers which match 2o the random sequence of primer 2 wherein the primers are labeled with a fluorescent group that only fluoresces when incorporated in a PCR product, similar to LuxTM
primers known in the art. In such an embodiment, the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that prior to incorporation, a sequence in the primer/probe binds to a complementary sequence in the 25 primer/probe containing the fluorescent group, quenching the fluorescent group. In another embodiment, primers 1 and 2 are Fluorescence Resonance Energy Transfter (FRET) partners, such that when hybridized to the amplified target sequence, produced only after primers 1 and 2 are ligated and amplified, they fluoresce.
Yet another embodiment provides a kit for use in identification of amplified target 30 nucleic acid sequences, the kit comprising a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. In the array of the kit, each through-hole contains at least a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of potential nucleic acid target sequence, and a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the potential nucleic acid target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer upon binding to the potential nucleic acid target sequence. The kit also comprises a reagent platen having a high-density microfluidic array of through-holes, each through-hole containing a third primer that is substantially complementary to a random sequence segment at the 3'-end of the second primer and to a substantially similar sequence at the 5'-end of the first primer, at least four different nucleotide bases, a thermostable polymerase, and a thermostable ligase. In the kit of this embodiment, the reagent platen has a structural geometry that corresponds to the sample platen, thereby allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen. In other embodiments, the thermostable polymerase may lack 5' to 3' exonuclease activity.
Brief Descriution of the Drawings The foregoing features of the invention will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:
Fig. 1-A, shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP).
2o Fig. 1-B1 shows a denatured 3' to 5' target strand with primers 1 and 2 hybridized adjacent to the SNP, the base complementary to the SNP located at the 3'-end of primer 1 and shows the random sequence (RS) of primer 3 hybridized to 3'-end of the ligated Pl-P2 product.
Fig. 1-B2 shows a denatured 3' to 5' target strand with primers 1 and 2 hybridized adjacent to the SNP, the base complementary to the SNP located at the 5'-end of primer 2 and shows the random sequence (RS) of primer 3 hybridized to 3'-end of the ligated Pl-P2 product.
Fig. 1-C shows a denatured 5'-3' target nucleic acid strand being extended by un-ligated primer P1.
3o Fig. 2-A shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP).
Fig. 2-B shows primers P1 and P2 hybridized to a denatured target strand of nucleic acid (the 3' to 5' strand) wherein a base complementary to the SNP in the target strand is present on the 3'-end of Pl, and each of primers Pl and P2 contain a random sequence at their 5'-end and 3'-end, respectively.
Fig. 2-C shows ligated Pl-P2 product being amplified by primer P3 to produce P3-amplified product.
Fig. 2-D shows P3-amplified product being amplified by primer P3 to produce P3-ampflied product (3' to 5').
Figs. 2-El and 2-E2 show exponential amplification of P3-amplified product (5' to 3') and P3-amplified product (3' to 5'), respectively.
Fig. 3 shows a cartoon of the dye SYBR~ Green I binding to double-stranded amplified target nucleic acid and fluorescing.
Fig. 4-A shows upstream primer A-B, downstream primer C-D, and general extension primer D' with a target nucleic acid having a SNP of interest in a single-tube reaction system for distinguishing between one or more SNPs in one or more target sequences of nucleic acid, the single-tube reaction system also containing upstream primer F-E and a second nucleic acid target with a second SNP of interest.
Fig. 4-B shows ligation of upstream primer A-B with downstream primer C-D
when successful match-up occurs with a first SNP of interest in a first target sequence of nucleic acid, and also shows ligation of upstream primer F-E with down stream primer C-D when successful match-up occurs with a second SNP of interest in a second target 2o sequence of nucleic acid present in the same tube.
Fig. 4-C shows extension of ligation products A-B-C-D and F-E-C-D by general extension primer D'.
Fig. 4-D shows hybridization of hybridizable probe A with fluorescent tag 1 to extended product A'-B'-C'-D' and hybridization of hybridizable probe F with fluorescent tag 2 to extended product F'-E'-C'-D'.
Fig. 4-E shows incorporation and amplification of a first target nucleic acid with a first SNP of interest by hybridizable probe A, triggering fluorescence of fluorophore 1 in a first amplified product, and incorporation and amplification of a second target nucleic acid with a second SNP of interest by hybridizable probe F, triggering fluorescence of 3o fluorophore 2 in a second amplified product.
Fig. 5A shows upstream primer A-B, downstream primer C-D, and general extension primer D' with a target nucleic acid having a SNP of interest in a single-tube reaction system for distinguishing between one or more SNPs in one or more target sequences of nucleic acid , the single-tube reaction system also containing upstream primer F-E and a second nucleic acid target with a second SNP of interest in an alternative embodiment of the single-tube reaction system of Fig. 4.
Fig. 5S shows ligation of upstream primer A-B with downstream primer C-D
when successful match-up occurs with a first SNP of interest in a first target sequence of nucleic acid, and also shows ligation of upstream primer F-E with down stream primer C-D when successful match-up occurs with a second SNP of interest in a second target sequence of nucleic acid present in the same tube.
Fig. 5C shows extension of ligation products A-B-C-D and F-E-C-D by general extension primer D'.
Fig. SD shows hybridization of primer A with no fluorescent tag to extended product A'-B'-C'-D' and hybridization of primer F with no fluorescent tag to extended product F'-E'-C'-D'.
Fig. SE shows amplification of a first target nucleic acid with a first SNP of interest by primer A to produce a first amplified product, and amplification of a second target nucleic acid with a second SNP of interest by primer F, to produce a second amplified product.
Fig. 5F shows a competing reaction to the amplification reactions in Fig. SE, wherein incorporation and low-efficiency production of a first target nucleic acid with a first SNP of interest is carried out by hybridizable probe A, triggering fluorescence of 2o fluorophore 1 in a first product, thereby allowing detection of a first amplified target nucleic acid, and wherein incorporation and low-efficiency production of a second target nucleic acid with a second SNP of interest is carried out by hybridizable probe F, triggering fluorescence of fluorophore 2 in a second product, thereby allowing simultaneous detection of a second amplified target nucleic acid.
Fig. 6 shows a typical high-density sample array of through-holes according to the prior art.
Detailed Description of Specific Embodiments Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:
"Target nucleic acid," "target nucleic acid sequence" or "potential target nucleic acid sequence" means any prokaryotic or eukaryotic DNA or RNA including from plants, animals, insects, microorganisms, etc. It may be isolated or present in samples which contain nucleic acid sequences in addition to the target nucleic acid sequence to be amplified. The target nucleic acid sequence may be located within a nucleic acid sequence which is longer than that of the target sequence. The target nucleic acid sequence may be obtained synthetically, or enzymatically, or can be isolated from any organism by methods well known in the art. Particularly useful sources of nucleic acid are derived from tissues or blood samples of an organism, nucleic acids present in self-replicating vectors, and nucleic acids derived from viruses and pathogenic organisms such as bacteria and fungi. Also particularly useful are target nucleic acid sequences which are related to disease states, such as those caused by chromosomal rearrangement, insertion, deletion, translocation and other mutation, those caused by oncogenes, and those associated with cancer.
"Selected" means that a target nucleic acid sequence having the desired characteristics is located and probes are constructed around appropriate segments of the target sequence.
"Probe" or "primer" has the same meaning herein, namely, a nucleic acid oligonucleotide sequence which is single-stranded. The term oligonucleotide includes DNA, RNA and PNA.
A probe or primer is "substantially complementary" to the target nucleic acid sequence if it hybridizes to the sequence under renaturation conditions so as to allow target-dependent ligation or extension. Renaturation depends on specific base pairing between A-X (where X is T or U) and G-C bases to form a double-stranded duplex structure. Therefore, the primer sequences need not reflect the exact sequence of the target nucleic acid sequence. However, if an exact copy of the target sequence is desired, the primer should reflect the exact sequence. Typically, a "substantially complementary" primer will contain at least 70% or more bases which are complementary to the target nucleic acid sequence. More preferably 80% or the bases are complementary, and still more preferably more than 90% of the bases are complementary. Generally, the primer should hybridize to the target nucleic acid sequence at the end to be ligated or extended to allow target-dependent ligation or extension.
3o Primers may be RNA or DNA and may contain modified nitrogenous bases which are analogs of the normally incorporated bases, or which have been modified by attaching labels or linker arms suitable for attaching labels. Inosine may be used at positions where the target sequence is not known, or where it may be degenerate. The oligonucleotides should be sufficiently long to allow hybridization of the primer to the target sequence and to to allow amplification to proceed. They are preferably 15 to 50 nucleotides long, more preferably 20-40 nucleotides long, and still more preferably 25-35 nucleotides longs. The nucleotide sequence of the primers, both content and length, will vary depending on the target sequence to be amplified.
It is contemplated that a primer may comprise one or more oligonucleotides which comprise substantially complementary sequences to the target nucleic acid sequence.
Thus, under less stringent conditions, each of the oligonucleotide primers would hybridize to the same segment of the target sequence. However, under increasingly stringent conditions, only that oligonucleotide primer which is most complementary to 1o the target nucleic acid sequence will hybridize. The stringency of the hybridization conditions is generally known to those in the art to be dependent on temperature, solvent, ionic strength, and other parameters. One of the most easily controlled parameters is temperature and since conditions for selective ligation and amplification are similar to those for PCR reactions, one skilled in the art can determine the appropriate conditions 15 required to achieve the level of stringency desired.
Primers suitable for use in the present invention may be derived from any method known in the art, including chemical or enzymatic synthesis, or by cleavage of larger nucleic acids using non-specific nucleic acid-cleaving chemicals or enzymes, or by using site-specific restriction endonucleases.
2o In order for the ligase of the present invention to ligate the primers together, the primers used are preferably phosphorylated at their 5'-ends. This may be achieved by any known method in the art, including use of T4 polynucleotide kinase. The primers may be phosphorylated in the presence of unlabeled or radiolabeled ATP.
The term "four different nucleotide bases" means deoxythymidine triphosphate 25 (dTTP), deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP); and deoxyguanosine triphosphate (dGTP) when the context is DNS, and means uridine triphosphate (UTP), adenosine triphosphate (ATP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP) when the context is RNA. Alternatively, dUTP, dITP
(deoxyinosine triphosphate), rITP (riboinosine triphosphate) or any other modified base 3o may replace any one of the four nucleotide bases or may be included along with the four nucleotide bases in the reaction mixture so as to be incorporated into the amplified strand.
The amplification steps are conducted in the presence of at least the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP) or a modified nucleoside triphosphate to produce a DNA strand, or in the present of the four ribonucleoside triphosphates (ATP, CTP, DTP and UTPO or a modified ribonucleoside triphosphate to produce an RNA
strand from extension of the primer.
The term "adequate detection of desired amplified product" means detection of at least a two-fold increase in desired amplified target strand over competing linear products.
The term "target sequence detectable above linearly amplified product" means that target sequence is amplified at least two-fold over that of competing linearly amplified non-ligated primer product.
The term "random sequence" as used herein means a sequence unrelated to the to target sequence or chosen not to bind to the target sequence or other sequences that might be expected to be present in a test sample.
The term "biocompatible material" as used herein means that the material does not prevent biological processes, such as enzymatic reactions, from occurring when the biocompatible material is present, does not eliminate biological activity or required secondary, tertiary or quaternary. structure of biomolecules, such as nucleic acids and proteins, and in general, is not incompatible with biological processes and molecules.
The term "first and second primers being ligatable upon binding to the nucleic acid target sequence" as used herein, means that the first and second primers bind potential target nucleic acid with the 3'-end of the first primer adjacent to, or within about a one- to four- nucleotide gap of, the 5'-end of the second primer, such that subjecting the hybridized first and second primers to appropriate enzymatic or non-enzymatic ligation conditions, including optionally adding a polymerase activity to fill in the gap, allows the first and second primers to be enzymatically or non-enzymatically ligated into a single ligated nucleic acid product.
The term "polymerase" as used herein, means any oligomer synthesizing enzyme, including polymerases, helicases, and other protein fragments capable of polymerizing the synthesis of oligomers.
The term "controlled-temperature reaction mixture" as used herein means, any reaction mixture wherein temperature is controlled by means of a thermocycle apparatus, an isothermal apparatus, or any other means known to allow temperature control of a reaction, including temperature-controllable environments such as water, oil and sand baths, incubation chambers, etc.
The general assay for identifying single-nucleotide polymorphisms (SNPs) that are not at an end of a target sequence through detection of amplified target sequences, using a dye specific for binding to double-stranded DNA that fluoresces upon binding target sequence according to the present invention, is described below and illustrated in Figs. 1-5. The assay can be performed in a single-reaction chamber or container, in a series of reaction chambers or containers, in a nanoliter sampling array having a high-s density microfluidic array of hydrophilic through-holes, or in a kit comprising such an array plus necessary reagents. Detection may be homogeneous, and may employ a polymerase that lacks 5' to 3' exonuclease activity, or a polymerase that lacks 3' to 5' exonuclease activity, or a polymerase that lacks both exonuclease activities.
The assay can be done with three (P1, P2, P3) or more (A-B, C-D, F-E, D') 1o primers, and is able to detect one or more SNPs in a single target simultaneously. In some versions of the assay, the nucleotide complementary to the SNP of the target nucleotide is present at or near the 5'-end of the second primer P2. In other versions,.the nucleotide complementary to the SNP of the target nucleotide is present at or near the 3'-end of the first primer Pl. In other versions, there are more than one first primers and 15 second primers, these first and second primers designed to generate amplified target sequences having different melting temperatures, such that the assay is able to distinguish individual amplified target sequences because of their individual, and distinct, Tms.
Assays may be done with first and second primers that contain degenerate base-I
pairing positions which allow hybridization of variable regions in target sequences 20 . adjacent to the SNP, in this way expanding the general flexibility and utility of the assay.
Primers 1 and 2, corresponding to 5' and 3' ligation primers, respectively, may be fully or partially complementary to the target sequence. Primer 3 is a generic primer complementary to a random sequence (RS) located at the ends of primers 1 and/or 2 (see Figs. 1 and 2). The 3' end of primer 1 and the 5' end of primer 2 can hybridize either 25 immediately adjacent to each other on the target sequence or can hybridize on the target sequence with a separation, or gap, or one or more nucleotides between them (see Figs. 1-2 and 4-5). Primers 1 or 2 contain a variant base at or near the 3' end (P1) or the 5' end (P2) to enable the primers to bind to SNPS in a target sequence (see Figs. 1-2). There is also a 3'-hydroxyl group on P2, to facilitate enzymatic or non-enzymatic ligation between 3o P1 and P2 or polymerase extension prior to ligation (to fill in any gap).
In addition, the 5'-end of P2 can be modified to prevent undesirable ligation to fragments other than P1.
Similarly, the 5'-end of Pl is phosphorylated to facilitate ligation with P2, and the 3' end of P1 may be modified to prevent ligation to fragments other than P2.
Amplification of target nucleic acid is illustrated in Figs. 1 and 2.
Temperature is used to denature and anneal target nucleic acid and primers, as required, to allow selective extension of ligation of primers Pl and P2.
Detection of single-stranded ligation product is carried out using several strategies, some employing a dye specific for binding to double-stranded DNA
that is generated either using hybridization probes which hybridize to single-stranded amplified product, or generated after extension and amplification of both the sense and non-sense strands of the ligation product. Other detection strategies employ molecular beacons attached to hybridizable probes. And still other detection strategies employ the use of FRET pairs on hybridizable probes. In some assays, the fluorescent dye is merely added 1o to the reaction mixture, and change in fluorescence intensity is monitored to detect ligated product. In other assays, hybridizable probes are added after generation of ligation product which are specific to the ligation product, and which also contain a molecular beacon, or a fluorescent group and a fluorescence-modifying group. The hybridizable probe may bind to extended ligation product, remaining quenched by the fluorescence-modifying group until extended into amplified product, whereupon the fluorescent group fluoresces and amplified target sequence is detected (see Fig. 4), or the hybridizable probe may be specific for hybridizing across the ligation junction, wherein the probe is again quenched until after hybridizing (see Fig. 5). In the assay illustrated in Fig. 4, one or more hybridizable probes may be used, each having a distinct fluorophore and unique l 2o sequence that hybridizes to and amplifies each of one or more target nucleic acid sequences, thereby allowing multiple SNPs to be detected in a single-tube reaction system.
Any of the assays may also be carried out in a nanoliter sampling array. The nanoliter array may comprise one or more platens having at least one hydrophobic surface and a high-density microfluidic array of hydrophilic through-holes.
The inner surfaces of the through-holes may be coated with a biocompatible material such as a wax-like polyethylene glycol material, or other biocompatible material. Primers may be applied into the through-holes and then dried, either before or after application of the biocompatible material coating, thereby affixing the primers on, within or under the 3o biocompatible material. Target nucleic acids and reagents for processes used in the selective ligation and amplification assay can be loaded in liquid form into the sample through-holes using capillary action, with typical volumes of the sample through-holes being in the range of from 0.1 picoliter to 1 microliter. The interior surfaces of the through-holes may also have a hydrophilic surface or be coated with a porous hydrophilic material, or as described above, be coated with a biocompatible material such as PEG, to enhance the drawing power of the sample through-holes, attract liquid sample and aid in loading.
Kits for performing the assay may also be prepared, comprising one or more sample platen as described, the primers being affixed within the hydrophilic sample through-holes of the microfluidic array, and also comprising reagents required for the selective ligation and amplification assay. Target nucleic acid sequences) can then be added as desired to perform the assay. If not already provided with the kit, enzymes required to carry out the ligation and amplification reactions can also be added along with l0 the target nucleic acid sequence(s).
. E~PLES
Example 1. Homogeneous detection of amplified target sequence .
Homogeneous detection of amplified target sequences may be carried out using a dye specific for binding to double-stranded DNA or RNA. Primers Pl and P2, upstream and downstream primers, respectively, do not participate in amplification of target sequence, but rather, are responsible for identifying the target sequence containing a SNP.
When either primer P1 or P2 contains a match to the SNP of interest in the target sequence, ligation of P1 and P2 occurs, and then primer P3, the general extension primer, amplifies the Pl-P2 product. Consequently, concentrations of primers 1 and 2 are preferably optimized and adjusted to not interfere with exponential amplification of the target sequence such that only linear amplification of competing non-target sequences occurs. Examples of ds-DNA- and/or RNA-specific dyes that may be used include SYBR~ Green I and SYBR° Green II, YOYO°-1, TOTO~-1, POPO~-3 (see Appendix A, attached hereto), ethidium bromide (EtBr) and any other dye providing adequate sensitivity and ease of detection of desired amplified product.
In a particular embodiment, a sample target sequence of,nucleic acid, optionally containing a single nucleotide polymorphism, is mixed with at least three primers - a first upstream primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the target sequence, a second downstream primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer wherein a nucleotide complementary to the SNP of the target sequence is present at either the 3'-end of the first primer or at the 5'-end of the second primer, and a third general extension primer that is substantially complementary to a random sequence segment at the 3'-end of the second primer and to a substantially similar sequence at the 5'-end of the first primer. Additionally, at least four different nucleotide bases, a thermostable polymerise and a thermostable ligase are included in the reaction mixture, the thermostable polymerise preferably one that lacks 5' to 3' exonuclease activity, such as the Stoffel Fragment (see Appendix B, attached hereto). Examples of other thermostable polymerises which lack 5' to 3' exonuclease activity include IsisTM DNA
polymerise,.
PyraTM exo(-) DNA polymerise, and Q-BioTaq DNA polymerise (see Appendix C, attached hereto). Alternatively, the assay may use a thermostable polymerise that lacks 3' to to 5' exonuclease activity, or a thermostable polymerise that lacks both 5' to 3' and 3' to 5' exonuclease activity. Examples of thermostable polymerises which lack 3' to 5' exonuclease activity include Taq polymerise, SurePrimeTM Polymerise, and Q-BioTaqTM
DNA polymerise (id.). An example of a thermostable polymerise which lacks both 5' to 3' and 3' to 5' exonuclease activity is Q-BioTaq DNA polymerise (id.).
Addition of a dye specific for ds-DNA such as SYBR~ Green I, or specific for RNA such as SYBR~
Green II, allows detection of amplified product, by monitoring fluorescence emission of dye-bound nucleic acid product at 520 nm(see Appendix D, attached hereto).
As can be seen in Figure 1-A, a target nucleic acid may contain a SNP within the target sequence. Upon denaturation, Primer 1 (P1) and Primer 2 (P2) bind to the 3' to 5' 2o strand of the target sequence, adjacent to the SNP. There may be a gap of several (approximately 2-4) bases between the 3'-end of P1 and the 5'-end of P2, or there may be no gap. In Figure 1-B1, the base complementary to the SNP of the target sequence is at the 3'-end of P1. Alternatively, the base complementary to the SNP of the target sequence may be at the 5'-end of P2, as shown in Fig. 1-B2. The third primer (P3) contains a random sequence (RS) complementary to the random sequence of the 3'-end of P2, such that after ligation of P1 and P2, P3 binds and extends the ligated primer product, thereby amplifying the complementary strand (5'-3' strand) of the target sequence. As discussed above, a competing reaction may occur, such that primer P3 binds to primer P2 and extends this sequence to produce a linear product based on the P2 sequence.
Preferably, concentrations of primers P1 and P2 are adjusted to minimize the competing linear reaction. As shown in Figure 1-C, un-ligated primer Pl extends the 3' -5' strand of the target sequence.
In another, preferred embodiment shown in Figure 2 (A - E), the first primer (P1) also has a random sequence at the 5'-end. When a primer containing the complement to the SNP, either Pl on its 3'-end or P2 on its 5'-end (see Fig. 1-B), binds to the target strand (see Fig. 2-B), primers P1 and P2 are ligated, and the third primer (P3) then binds to the 3'-end of the ligated Pl-P2 product and produces the (3' to 5') P3-amplified strand (Fig. 2-C). At this point, primer P3 now also binds to the (3' to 5') P3-amplified product and produces the other (5' to 3') amplified product (see Fig. 2-D). Both target strands have now been produced, and can go on to yield exponentially amplified target sequence (Fig. 2-E1 and 2-E2). Additionally, detection with a fluorescent dye, such as SYBR~
Green I (SGI) may be done at temperatures above the Tm of the linear product, i.e., any product produced non-exponentially, thereby removing competing signal from any dye to bound to linear product. SYBR~ Green I and other dyes that bind to double-stranded nucleic acids do not bind to nucleic acids above their Tms because at those elevated temperatures, the nucleic acids are denatured. As seen in the cartoon of Figure 3, a dye such as SYBR° Green I binds to double-stranded amplified target nucleic acid with a concomitant laxge increase in fluorescence. Although SGI is shown in Figure 3 as intercalating into the amplified target ds-nucleic acid, nothing in the figure is intended to suggest either an actual structure, or actual mode of binding, for SGI with ds-nucleic acids.
Alternatively, the use of molecular beacon probes, having a fluorescent group on one end and a fluorescence-quenching group on the other, may be used. In this system, the molecular beacon remains quenched until being bound to amplified product (see, for example, Appendix E, attached hereto) because the molecular probe is typically in a hairpin conformation with the fluorescent group in close proximity to the fluorescence-quenching group, until the probe binds to the target amplified product (causing the hairpin structure to unfold, separating the fluorescent group from the quenching group).
Examples of fluorescence-quenching groups appropriate for embodiments of the present invention include the dark quencher dabcyl, and the EclipseTM Quencher from Epoch (id.). Examples of appropriate fluorescent groups that may be used in accordance with the present invention include Epoch's Yakima YellowTM and Redmond RedTM (id.), and any other appropriate fluorescent dye whose fluorescence may be quenched to an appropriately positioned quencher molecule.
In another embodiment, real-time amplification may be measured using a TaqMan" probe that is homologous to an internal sequence of the target nucleic acid, and having a fluorogenic 5'-end and a quencher 3'-end. During PCR amplification and extension, the quencher molecule is removed from the probe by 5'-exonuclease activity, releasing the fluorescent reporter molecule from close proximity to the quencher molecule on the 3'-end of the probe, thereby producing an increase in fluorescence emission as amplified product is produced (see Appendices F and G, attached hereto). In this system, a polymerase having 5' to 3' exonuclease activity is required.
Another embodiment utilizes a detection method for real-time amplification measurement that involves the use of a pair of amplification primers, one of which matches the random sequence of primer 2. One of these primers in the pair is labeled with a fluorescent group that only fluoresces when incorporated into a PCR product, similar to LuxTM primers known in the art (see Appendix H, attached hereto). In such an 1o embodiment, the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that prior to incorporation, a sequence in the primer/probe binds to a complementary sequence in the primer/probe containing the fluorescent group, quenching the fluorescent group. In another embodiment, primers 1 and 2 are FRET partners, such that when hybridized to the amplified target sequence, . produced only after primers 1 and 2 are ligated and amplified, they fluoresce (see Appendices E and also A) and thus permit detection of amplified target sequence. In a preferred embodiment, fluorescence detection would be carned out above the either the Tm for primer Pl, or above the Tm for primer P2, or alternatively be carried out above the Tms of both primers Pl and P2, to avoid background signal from possible hybridization of P1 and/or P2 to amplified target.
In another embodiment, primer may be designed to exponentially amplify target nucleic acid products that are distinguishable by an increase or decrease in melting temperature (Tm), wherein the exponentially amplified target sequence is either stabilized as indicated by an increase in Tm or de-stabilized, as indicated by a decrease in Tm, relative to the melting temperatures of linearly produced non-target product produced from non-ligated primers. Variability in the random sequence, or elsewhere in the primers, may be used to produce such exponentially amplified target nucleic acid sequence distinguishable by melting temperature from the linear product.
In another embodiment, a probe specific for hybridizing across the ligation junction formed after ligation of the first and second primers may be used.
Such a probe may have a hairpin conformation with a fluorescent reporter group on one end and a fluorescence-quenching group on the other end whereby no fluorescence occurs when the probe is not bound across the ligation junction. By optimizing reaction (conditions, such as temperature and/or ionic strength, the hairpin would be stabilized by binding across the ligation junction, whereupon fluorescence would occur and emission could be monitored to detect amplified product.
Example 2. Single-tube reaction system for distinguishing SNPs One preferred embodiment of the present invention is the single-tube reaction system shown in Figure 4. Similar to the embodiments shown in Figures 1 and 2 and discussed above in Example 1, a three-primer system is utilized to identify a SNP of interest in a target sequence of nucleic acid. Again, there is an upstream primer and a downstream primer that bind to the target nucleic acid, flanking the SNP of interest. The l0 3'-end of the upstream primer may be directly adjacent to the 5'-end of the downstream primer, or there may be a gap of between about 1 to 4 bases between the 3'-end of the upstream primer and the 5'-end of the downstream primer. Either the 3'-end of the upstream primer or the 5'-end of the downstream primer may contain the complement to the SNP of interest in the target nucleic acid.
Unlike the embodiments shown in Figures 1 and 2, however, the single-tube reaction system allows simultaneous single-tube identification and distinction between one or more SNPs of interest in one or more target nucleic acid sequences of interest.
This is accomplished by using unique sequences in each of the random sequence regions of the upstream primer and the downstream primer (the two which ligate) and the general extension primer. As see in Figure 4A, a single-tube reaction system may contain a first upstream primer A-B with random sequence A, which identifies a first SNP of interest in a first target nucleic acid segment, and a second upstream primer F-E with random sequence F, which identifies a second SNP or interest in a second target nucleic acid segment, and a general extension primer with random sequence D' complementary to random sequence D present in downstream primer C-D, wherein C is common to both target nucleic acid segments.
Upon successful identification and binding to a target nucleic acid having a SNP
of interest, upstream primers A-B and/or F-E will be ligated to downstream primer C-D, creating ligation products.A-B-C-D and/or F-E-C-D. If a gap is present between the 3'-end of the upstream primer and the 5'-end of the downstream primer, the gap will first be filled in by a polymerase activity, followed by ligation to form the ligation products.
Extension of both ligation products can then occur by general extension primer D', to produce extended products A'-B'-C'-D' and F'-E'-C'-D'.

Next, hybridizable probe A with fluorophore 1 and hybridizable probe F with fluorophore 2, hybridize to extended products A'-B'-C'-D' and F'-E'-C'-D', respectively, which is followed by amplification such that each of the probes with its particular fluorescent tag is incorporated into amplified product (A-B-C-D or F-E-C-D), triggering fluorescence of either fluorophore 1 or fluorophore 2 or both. In this way, one or more SNPs may be identified and distinguished in a single-tube reaction system by monitoring the fluorescent signals of the two (or more) fluorophores upon incorporation into amplified product.
In another embodiment, an alternative single-tube reaction system for identifying and distinguishing one or more SNPs in one or more target nucleic acid segments is shown in Figures 5A-5F. Figures 5A through 5C are identical to Figures 4A
through 4C, in that upstream primers A-B and F-E, downstream primer C-D, and general extension primer D' are present in the single-tube reaction system. Again, either the 3'-end of the upstream primers may contain the complement to the SNP of interest in the target nucleic acids, or the 5'-end of the downstream primer may contain the complement to the SNP of interest in the target nucleic acids, and upon binding to the target nucleic acids, the two primers may be adjacent, or have a gap of about 1-4 bases between the 3'-end of the upstream primer and the 5'-end of the downstream primer, which must be filled by a polymerase, before ligation between the upstream and downstream primer can occur.
2o As shown in Fig. 5D, however, the alternative single-tube reaction system does not use hybridizable probes A and F with fluorophores 1 and 2 to amplify target nucleic acid, but rather, uses regular primers A and F to amplify extended products A'-B'-C'-D' and F'-E'-C'-D' into amplified target nucleic acids products A-B-C-D or F-E-C-D. Such a system may be advantageous when a particular target nucleic acid does not amplify ' efficiently with hybridizable probes that have bulky fluorophores attached to them. In this alternative single-tube reaction system, the amplified target nucleic acids are detected after amplification, by additional fluorescent-tagged hybridizable probes hyb-A and hyb-F, which differ from regular primers A and F in that they are shorter, and have secondary structure that dissolve at lower temperatures than the annealing temperatures of primers A
and F (or fluorescent probes A and F in Figure 4). This allows inefficient competition between hyb-A and hyb-F probes and regular primers A and F, in amplification of extended products A'-B'-C'-D' and F'-E'-C'-D' into target nucleic acid products A-B-C-D
or F-E-C-D, but allows enough competing reaction to occur to measure fluorescence of fluorophores 1 and 2, thereby allowing detection and quantitation of amplified target nucleic acid product.
Although use of a general extension primer such as D' that is complementary to a sequence D in segment C-D common to both target nucleic acid segments is convenient in the single-tube reaction systems described above and exemplified in Figs. 4 and 5, it is not required. It is envisioned that single-tube reaction systems could also be adapted for creating ligation products with with A-B and F-E using more than one extension primer simultaneously. The selectivity of the first primer A-B for the first SNP and the second primer E-F for the second SNP will ensure selective ligation, even with additional 1o primers being used to generate the C-X product to be ligated.
Upon successful identification and binding to a target nucleic acid having a SNP
.; of interest, upstream primers A-B and/or F-E will be ligated to downstream primer C-G
and C-H, respectively, creating ligation products A-B-C-G and/or F-E-C-H. If a gap is present between the 3'-end of the upstream primer and the 5'-end of the downstream 15 primer, the gap will first be filled in by a polymerase activity, followed by ligation to form the ligation products. Extension of both ligation products can then occur by extension primers G' and H', to produce extended products A'-B'-C'-G' and F'-E'-C'-H'.
As described above, one or more SNPs may be identified and distinguished in a single-tube reaction system by a) monitoring the fluorescent signals of two (or more) 2o fluorophores upon incorporation into amplified product, or b) detecting fluorescent signals after amplification, by use of additional fluorescent-tagged hybridizable probes hyb-A and hyb-F.
Example 3. A rcanoliter sampling array 25 Another embodiment of the present invention encompasses a nanoliter sampling array. Any array presently available in the prior art may be used, but an array of particular utility, similar to that described in U.S. Provisional Application Serial No.
60/518,240, filed November 7, 2003, and US regular application serial no. 10/984,027 filed on November 8, 200.4, both of which are hereby incorporated by reference herein, is one 30 preferred array. In this particular embodiment, the array comprises a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. A target nucleic acid sequence is selected, and the array is prepared wherein each through-hole in the array contains at least a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the nucleic acid target sequence and a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the nucleic acid target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer upon binding to the potential nucleic acid target sequence. Figure 4 shows such an array, known in the prior art. Array chip 10 typically may be from 0.1 mm to more than 10 mm thick; for example, from 0.3 to 1.52 mm thick, and commonly 0.5 mm.
Typical volumes of the sample through-holes 12 could be from 0.1 picoliter to microliter, with common volumes in the range of 0.2 to 100 nanoliters, for example, about 35 nanoliters. Capillary action or surface tension of the liquid samples may be used to load the sample through-holes 12. For typical chip dimensions, capillary forces are.
strong enough to hold liquids in place. Chips loaded with sample solutions can be waved in the air, and even centrifuged at moderate speeds, without displacing the samples.
To enhance the drawing power of the sample through-holes 12, the target area of the receptacle interior walls 42 may have a hydrophilic surface that attracts a liquid sample. Alternatively, the sample through-holes 12 may contain a porous hydrophilic materiel that attracts a liquid sample. In some embodiments, the sample through-holes in the array may be coated with a biocompatible material such as polyethylene glycol, and the primers may be affixed on, within or under the biocompatible material on the surface of the through-holes by drying the primers after application to the through-holes. To prevent cross-contamination (crosstalk),,the exterior planar surfaces 14 of chip 10 and a layer of material 40 around the openings of sample through-holes 12 may be of a hydrophobic material. Thus, each sample through-hole 12 has an interior hydrophilic .
region bounded at either end by a hydrophobic region.
The through-hole design of the sample through-holes 12 avoids problems of trapped air inherent in other microplate structures. This approach, together with hydrophobic and hydrophilic patterning enable self-metered loading of the sample through-holes 12. The self loading functionality helps in the manufacture of arrays with pre-loaded reagents, and also in that the arrays will fill themselves when contacted with an aqueous sample material.
Example 3. Method for identifying a SNP in a target sequence of nucleic acid.
Yet another embodiment is a method for identifying a single nucleotide polymorphism (SNP) in a target sequence of nucleic acid. A target sequence of nucleic acid is identified, and primers are prepared according to standard methods, such that two primers, Pl and P2, are designed to flank an internally-positioned SNP on one strand of the target nucleic acid sequence and are designed to be ligated with a thermally stable ligase. Primer Pl and P2 are further designed such that the base complementary to the SNP in the target sequence is either on the 3'-end of P1, or on the 5'-end of P2. In this particular method, a nanoliter sampling array is used. The method comprises providing a first platen having a high-density microfluidic array of through-holes is provided wherein each through-hole of the array contains a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of the target sequence, and a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the target sequence. Upon binding to the target 1o sequence, the 5'-erid of the second primer is adjacent to the 3'-end of the first primer.
The method further comprises introducing a sample containing the target nucleic acid sequence with internal SNP into the array, and introducing reagents into the through-holes in the array wherein the reagents include a third primer having a random sequence J capable of amplifying ligated primer Pl-P2 product, a thermostable polymerase, a i5 thermostable ligase, and at least four different nucleoside triphosphates.
Additional steps in the method comprise thermocycling the array with primers, target nucleic acid, and reagents, and detecting the resulting amplified target nucleic acid sequence.
Optionally, the thermostable polymerase may lack 5' to 3' exonuclease activity, or it may lack 3' to 5' exonuclease activity, or it may lack both 5' to 3' and 3' to 5' exonuclease activity.
20 It is also envisioned that the detecting step may comprise the use of a dye specific for binding to double-stranded DNA or to RNA that fluoresces upon binding amplified target sequence. Suitable dyes include SYBR~ Green I, SYBR~ Green II, YOYO~-1, TOTO~-l, POPO~-3, EtBr, and any other dye capable of providing low-sensitivity detection of amplified target sequence by fluorescence emission.
25 Alternatively, detection may occur through the addition of probes specific for hybridization across the ligation junction of the ligated P1-P2 primer product, where such probes contain a fluorescent group and a fluorescence-modifying group such as a fluorescence quencher.
In another alternative embodiment, detection may involve the use of a probe 30 containing a fluorescent group and a fluorescence-modifying group such as a fluorescence quencher that is specific for hybridizing to a region of the target sequence.
In this particular embodiment, the fluorescence-modifying group is excised upon extension of the probe, and the fluorescent group thus fluoresces, allowing detection of amplified product.

Additional embodiments of the present invention include a kit for use in identification of amplified target nucleic acid sequences, wherein the kit provides a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes. In one particular kit each through-hole contains at least a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of potential nucleic acid target sequence, a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a s ~ and end of the potential nucleic acid target sequence, the 5'-end of the second primer being adjacent to the 3'-end of the first primer upon binding to the potential nucleic acid target sequence and a reagent platen having a high-density microfluidic array of through-holes with each through-hole containing a third primer that is,substantially complementary to a random sequence segment at the 3'-end of the second primer and to a substantially similar sequence at the 5'-end of the first primer, at least four different nucleotide bases, a thermostable ligase and a fluorescent dye. In this particular embodiment, the reagent platen has a structural geometry that corresponds to the sample platen allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen. In some embodiments of the kit, the primers may be affixed on, within or under a biocompatible material such as a wax-like coating in the through-holes by drying the primers after being applied to the through-holes, wherein the biocompatible material may comprise, for example, a polyethylene glycol (PEG) material. To perform the selective ligation and amplification reaction for identification of an amplified target nucleic acid sequence, the user would merely add a sample containing the target nucleic acid, a thermostable polymerase, and optionally a buffer supplied with the kit to the through-holes.

Section 8.7 - Analysis of DNA Sffiuucture, DNA Binding and DNA Damage ~ P P ~-~d ~ ~c ~1 ~~t~~
Updated: August 30, 20U3 Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage I ~t,s~er-xriePcxr~
Nucleic Acld Conformatir~nal Analyses A number of conventional dyes have been used to analyze nucleic acid conformation in vitro and In vivo:
~ Acridine orange (A-1301, A-3568; Section 8.1) is one of the most popular and versatile fluorescent stains for hlstochemistry and cytochemistry and can provide a wide variety of information about the in situ content, molecular structure, conformation and environment of many nucleic acid-containing cell constituents.
. Fluorescence photobleaching of DNA that has been photolytically labeled with ethidium monoazide (E-1374, Section 8.1) permits measurement of slow reorientational motions.e"
. The fluorescence intensity and binding affinity of the Hoechst dyes appear to be highly dependent on the sequence and conformation of the DNA base pairs.tFor example, staining by Hoechst 33258 (H-1398, H-3569; FluoroPure Grade, H-21491; Section 8.1) can discriminate parallel and anttparalfel stem regions in hairpin DNA
conformations.
~ The fluorescence lifetime of the PlcoGreen dye (P-7581, P-11495; Section 8.3 bound to single-stranded DNA is reported to be different when bound to double-stranded DNA.
Uwle also anticipate that several of our cyanine dyes (Section 8.1) - in particular the SYTO dyes (Table 8.3,) - may be useful in these applications because many of these stains appear to yield environment-sensitive rnetachromatlc shifts upon binding to nucleic acids.
Fluorescence of the TOTO-1, YOYO-1, BOBO-1 and POPO-1 dyes I'Table 8.2, Dimeric Cyanine Nucleic Acid Stains) is 'dependent on nucleic acid secondary structure; a shift to longer-wavelength emission and a concomitant drop in quantum yield are observed upon binding of these dyes to single-stranded nucleic acids at high dye:base ratios. Most of our unsymmetricai cyanine dyes show this spectral shift, and some show sequence selectivity in their fluorescence intensity as well.
Examining the Behavior of Single Nucleic Acid Mvlecu~es Once bound to nucleic acids, several of the cyanine dyes in section 8.1 are so bright that they can be used to directly visualize single nucleic acid molecules in the fluorescence microscope (~, 11~).
The YOYO-1 and POPO-3 dyes (Y- 601 P-3584) dyes have also been used to follow the making and breaking of single chemical bonds.cA number of laboratories have taken advantage of the high sensitivity of these dyes to detect single nucleic acid molecules and to study btopolymer behavior:
. Video microscopy has been used to observe relaxation of YOYO-1 dye-stained phage lambda DNA multirners, after stretching in a fluid flow,lon a surFace t1~ or with optical tweezers.
TOTO-1 dye (T-3600) has also been used in this application.?
. Individual YOYO-1 dye-ssDNA~ molecular complexes have been imaged ih solution by SUBSTITUTE SHEET (RULE 26) section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage fiJuorescence video microscopy.~Y
. Molecular combing, a technique that uses a receding fluid interface to elongate DNA , molecules for optical mapping of genetic loci, was developed using the YOYO-i dye.k . Adsorption and desorption of single molecules of YOYO-1 dye-stained phage lambda DNA
have been observed on fused-silica and Cl8 chromatographic surfaces.("
. The activity of a single Recl3CD enzyme, which unwinds and separates the strands of dsDNA, has been studied using YOYO-i dye-stained. dsDNA in conjunction with optical tweezers and eplfluorescence microscopy.
Our YOYO-1 dye (Y-3601 has been used to stain DNA manipulated in solution by~changlng electronic fields, a technique that could prove valuable in miniaturizing and automating analysis of DNA fragments,, . Staining with the YOYO-1 dye (Y-36(71) was used to observe the interacfiion of ANA with various llpasomes d~k and to size plasrnids in a flowing stream.' ~ The YOYO-1 dye was also used to detect radiation-induced double-strand breaks in. individual electrostretched bacterial DNA molecules.
. Single-molecule imaging of nucleic acids stained with either YOYO-1 or POPO-3 or a combination of the two dyes through collection of the entire fluorescence spectrum of their campiex has been reported.
. Highly sensitive sheath-flow techniques have also been developed for detecting and discriminating the size of single TOTO-1 dye-DNA molecular complexes.l~
Large fragments of DNA stained with our TOTO-1 dye (T-3600) have been sorted by flow cytometry. This extremely rapid analytical method yields a linear relationship between the fluorescence intensity and the fragment size over a IO-50 kilobase pair ranger:
. The POPO-1 (P-3580, Section 8.1) and POPO-3 (P-3584) stains have been used to sensitively detect single DNA fragments by flow cytometry using two-photon fluorescence excitation..
~ The POPO-3 dye P-3584 has been used to study a single chemical reaction wlth,an individual DNA molecule. POPO-3 dye-stained DNA molecules stretched taught on a glass surface relax when a focused laser beam causes fluorescence-related breakage of the DNA
backbone, forming a gap that is visible by fluorescence micrascopy.~
. The TOTO-1 (T-3600 , YOYO-1 (Y-3601), POPO-3 (P-3584) and SYBR Green I (S-7563, 5-7557, S-7585) dyes have been used to visualize lambda DNA that has been stretched between beads with optical tweezers.t"~, . Fragment sizing on single molecules of dsDNA stained with our PicoGreen reagent has also been reported.
. The SYTOx Orange dye S-11368 Is the preferred dye for single-molecule sizing of DNA
fragments by flow cytometry in an instrument equipped with a Nd:YAG laser.,tl~
~ DAPI (D-1306, D-3571; FluaroPure Grade, D-21490) has also been employed to detect a single DNA molecule in solution ,cue and by fluorescence microscopy a and to detect femtograms of TUNA in stngle cells and chloroplasts.t~k The high affinity and bright fluorescence of other cyanlne dimers has allowed researchers to follow stained and transfected plasmids or stained virus particles within a cell.f~k DNA Binding Assays r~iectraphoretic Mabiiity-Shift (i3andshlft) Assays .
Bandshift assays to analyze DNA-protein interactions are conventionally performed using radtaactively labeled DNA fragments. However, use of our high-sensitivity fluorescent dyes makes these assays much simpler to pertorm and eliminates radioactive waste issues.
For example, SYBit Green I nucleic acid get stain (S-7567, S-7563, S-7585; SYBR Green I Nucleic Acid Gel Stan) has been used to post-stain gels after electrophoresis and can detect bound and unbound DNA
fragments with high sensitivity (Figure 8.134?. The SYBR Goid nucleic acid gel stain IGS-11494,, SUBSTITUTE SHEET (RULE 26) Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage SYBR Gold Nucleic Acid Gel Stain) is potentially even more useful in bandshift experiments because of its higher sensitivity.
Molecular Probes has made bandshift assays easy and more convenient with our Electrophoretic Mobility-Shift Assay (EMSA) Kit (E-33075). Our EMSA Kit provides a fast and quantitative fluorescence-based method to detect both nucleic acid and protein in the same gel (lii~), doubling the information that can be obtained from bandshift assays. This kit uses two fluorescent dyes for detection - SYBR Green EM9A.nudeic acid gel stain for RNA or DNA and SYPRO
Ruby EMSA
protein gel stain for proteins. Because the nucleic acids and proteins are stained in the gel after electrophoresis, there is no need to prelabel the the DNA or RNA with a radioisotope, biotin or a fluorescent dye before the binding reaction, and therefore there Is no possibility. that the label will interfere with protein binding. Staining takes only about 20 minutes for the nucleic acid stain, and about 4 hours far the subsequent protein stain, yielding results much faster than radioisotope labeling (which may require multiple exposure times) or chernilumlnescence-based detection (which requires blotting and multiple incubation steps). This kit also makes it possible to perform ratiametric measurements of nucleic acid and protein in the same band, providing more detailed information on the binding interaction. The signal from the two stains is linear over a broad range, allowing accurate determination of the amount of nucleic acid and protein, even in a single band, with detection limits of N1 ng for nucleic acids and N20 ng for protein . Both stains can be detected using a standard 300 nm UV illuminator, a 254-nm epi-illuminator or a laser-based scanner ().
Digital images can easily be overlaid for a two-color representation of nucleic acrd and protein in the gel. The EMSA Kit contains sufficient reagents for 10 nondenaturing potyacrylamide minigei assays, including:
. SYBR Green EMSA nucleic acid gel stain ~ SYPRO Ruby EMSA protein gel stain ~ Trichloroacetic acid, for preparing the working solution of SYPRO Ruby EMSA
protein gel stain . Concentrated EMSA gel-loading solution . /ac repressor, a DNA-binding protein to be used as a control .
. !ac operator, control DNA
. Concentrated buffer for the !ac repressor:operatar controls ~ A detailed protocol (Elsctroahoretic Mobility Shift Assay (EMSA) Kit) Fluorescent dyes have also been used to stain the DNA fragments or proteins before electrophoresis. For instance, proteins or DNA labeled cavalently with a reactive fluorescent dye (hC~,apter 1, Section 8.2) can be easily,tracked during cap111ary electrophoresis to monitor DNA-protein interactions.l~~ High-afi'inity nucleic acid stains have also been used prior to electrophoresis, although they can potentially Interfere with protein binding and alter mobility on the gel. The ethidlum homodimer-Z (EthD-1., E~1,69; Section 8.1), YOYO=1 and 1'0T0-1 dyes have been shown by several laboratories to be useful tools far labeling DNA prior to electrophoresis in bandshlft assays. EthD-1 and TOTO-1 were used to examine interactions between the binding domain of the Kluyvemmyces lactic heat shock transcription factor and its specific binding site.~k YOYO-1 dye has been used to study the association of E, coif RNA polymerase with DNA templates d~ and the binding of a heat-shock transcription factor to its promoter.d~k All ten of our spectrally distinct (Figure 8.1), high-affinity dimeric cyanine dyes (Table 8.2) and the ethidiurn homodlmers are potentially useful for multlcomponent analysis in this application.
DNA Binding Assays in Solution Hlolecular beacons exploit fluorescence resonance energy transfer (FRET) to simplify detection of nucleic acid hybridization in solution (Section 8.5. Figure 8.104). This method has also proven useful for studying DNA-protein interactions In solution. Binding of a molecular beacon to lactic dehydrogenase separated the fluorophore from the quencher on the two ends of the labeled oligonucleotide, resulting in an increase in fluorescence. The assay is sufficiently accurate to SUBSTITUTE SHEET (RULE 26) 'Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage measure binding constants. A molecular beacon was also used to develop a solution-based binding assay for ~x-CP2, which is part of an RNA-binding complex.
Selective Cleavage of Nucleic Acids with a Chemical Nuclease The thiol-reactive iodoacetamide of i,10-phenanthroline (P-6879, Section 2.3) is a useful adjunct reagent for bandshift assays. Conjugation to thiot-containing Ilgands confers the metal-binding properties of this important complexing agent on the ligand. For example, the covalent copper-phenanthroline complex of oilganucleatides or nucleic acid-binding molecules in combination with hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or RNA.tThis reagent can also be conjugated to proteins iv detect nucleic acid binding and targeted cleavage.d~
Assessing DNA Damage Comet (Single~Cell Gef Electrophoresis) Assay to Detect Damaged DMA
The comet assay - or single-cell gel electrophoresis assay - is used for rapid detection and quantitation of DNA damage from single ceiis.The comet assay is based on the alkaline tysis of labile DNA at sites of damage. Cells are immobilized in a thin agarose matrix on slides and gently fysed. When subjected to electrophoresis, the unwound, relaxed DNA migrates out of the cells, After staining with a nucleic acid stain, cells that have accumulated DNA
damage exhibit brightly fluorescent comets, with falls of DNA fragmentation or unwinding (). In contrast, cells with normal, undamaged DNA appear as round dots, because their intact DNA does not migrate out of the cell. The ease and sensitivity of the comet assay has provided a fast and convenient way to measure damage to human sperm DNA,tmonitor the sensitivity of tumor cells to radiation damage and to assess the sensitivity of molluscan cells to toxins in the environment. The, comet assay can also be used in combination with FISH to identify specific sequences with damaged DNA.d' Comet assays have traditionally been performed using ethidium bromide ( -13 , -3565) to stain the DNA;dhowever, our YOYO-1 dye (Y-3601) increases the sensitivity of the assay elghtfold compared to ethidium bromide and the fluorescence background from unbound YOYO-1 dye is negligible. Use of the SYBR Goid and SYBR Green I stains {Section 8.~.) further Improves the sensitivity of this assay.
TUNEL Assay for ~'n Situ Detection of Fragmented DNA
To detect fragmented DNA in labeled cells, terminal deoxynucleotidyl transferase (TdT) along with a fluorophore-, biotin-, or hapten-labeled dUTP can be added to cells. TdT
adds the labeled nucleotide to all available 3'-ends - the mare fragmented the DNA, the more 3'-ends are available and the brighter the fluorescent signal. Direct TUNEL assays using ChromaTlde BODIPY FL-14-dUTP
--C 7b14) to visualize DNA fragment ends are four times more sensitive than TUNEL assays using fluorescetn-labeled dUTP (~). Terminal deoxynucleottdyl transferase (TdT)-catalyzed tncorporatlon of bromo dUTP into nucleic acids of apoptotic cells and detection of the incorporated BrdU with an ant)bady conjugate Is the basis of the AP4-BrdU TUNEL Assay Kit (A-23210, Section 15.5). Indirect TUNEL assays using probes such as biotinylated dUTP or our ChramaTide DNP-11-dUTP (C-~sxo, Section 8.2) allow for amplification of the signal with our fiuorophore- or erizyme-conjugated streptavidtn conjugates (Section 7.f. Table 7.20) or with anti-DNP
antibody (Section 7~4). Several additional assays for apoptosis can be found in Section 15.5. .
Mtcroplate-Based Assays for DNA Damage Abasic sites in DNA, generated spontaneously or caused by free radicals, ionizing radiation or mutagens like MMS (methyl methanesuifonate), are one of the most common lesions in DNA and SUBSTITUTE SHEET (RULE 26) Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage are thought to be important intermediates in mutagenesis. A quick and sensitive microplate assay for abasic sites can be performed using ARP (A-10550, Ficture 8.137), a biotinylated hydroxylamine that reacts with the exposed aldehyde group at abasic sites. Biotins tround to the abasic sites can be quantitated with our fluorescent- or enzyme-conjugated streptavldin complexes d~'k (Section 7~6, Table 7.20). ARP is permeant to cell membranes, permitting detection of basic sites in living celis.t~k The PicoGreen reagent has also been used to simplify denaturatton assays for DNA damage. Strand breaks in dsDNA that result from DNA damage can ba quantified by measuring the relative amounts of ssDNA and dsDNA in a damaged sample. The relative amounts of dsDNA
to ssDNA can be assessed by measuring the increase In absorbance at 260 nm or by separating the two farms of DNA by alkaline sucrose gradient centrifugatlon,filters,d~ or hydroxyapatlte chromatography.
~ However, the absorbance-based technique suffers from Ivw sensitivity and thus requires relatively large sample sizes k and separation of ssDNA from dsDNA is laborious. This assay becomes much simpler and mare sensitive using the PtcoGreen dsDNA quantitatlon reagent (P-7581, P-7589. P-11495, -1~ 1496, R-21495; Section 8.3), which preferentially detects dsDNA in the presence of ssDNA.sThe dye can be added directly to the sample and the fluorescence signal read on a fluorescence-based microplate reader. Thts method makes it possible to screen large numbers of very small samples in a high-throughput setting. The PicoGreen reagent was also used to develop a homogeneous PCR-based genotyping assay.d~ Because the products do not need to be run on a gel, the assay can be easHy adapted for high throughput particularly using the RediPIate 96 version of the PicoGreen dsDNA quantitation assay (R-21495, Section 8.3).
Assays for Enzymes that Modify Nucleic Acids Get-Based Assays for DNase t)etectlon Our SYBR Green I stain (S-7563, S-7567, S-7585; SYt3R Green I Nucleic Acid GeI
Stainl has been used to develop DNase assays that show up to a 64-fold increase in sensitivity over similar ethidium bromide-based assays and up to 10,00-fold higher sensitivity than the traditional UV
hyperchrornidty assay. In a fast and simple assay, a single-length fragment of ANA can be incubated with the sample, followed by a short gel electrophoresis. Staining the gel with the SYBR
Green I dye permits easy detection of less than 10-5 Kunitz units (N5 pg) of DNase activity.
Even greater sensitivity can be achieved using the single radial enzyme diffusion (SRED) method, In which the SYBR Green I stain is mixed with DNA in matted agarose and the mixture is poured into a 2 mm thick slab. The sample to be tested is poured into i.5 mm circular wells punched out of the solidified agarose slab. As the sample diffuses through the agarose, the DNase degrades the DNA, creating dark circles around the sample well that do not show staining with the SYBR Green I dye when illuminated with UV light. The radius of these dark circles is proportional to the level of DNase activity. This method allows detection of as little as 2 x IO'' units (N0.1 pg) of DNase I or 2 x 10-6 (N0.9 pg) of DNase II. A third DNase assay - called the dried agarose film overlay (DAFO) method - uses the SYBR Green I stain to detect the presence of DNase activity in a polyacrylamide gel, allowing the tdentii~ication of heterogenelties in DNase species,t~ This method allows the detection of 4 x 10-6 units (N2 pg) DNase I or DNase II.
Solution-Based Assays for Nuclease Detection .
Contaminating DNases are often responsible for poor resolution of DNA
fragments, degradation of samples and nicking of supercviled plasmids. Conventional DNase assays detect DNase activity by monitoring the increase tn UV absorbahce that occur$ when the base pairs unstack as the DNA is degraded. Trits absorbance method, however, is intrinsically insensitive as tt requires large sample volumes and relies on small changes in absorbance. In contrast, our dyes far nucleic acid detection show a tremendous fluorescence increase upon binding to nucleic acids, but their fluorescence is not affected by the presence of a large excess of a nucleotide or very short oligonucleotides. Thus, nuclease activity can be easily and accurately measured by the decrease in fluorescence in the SUBSTITUTE SHEET (RULE 26) Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage presence of one of these dyes. For instance, the YOYO-1 nucleic acid stain (Y-3601 has been used in a fluorescence-based mlcraplate assay for nuclease activity.tk This assay takes advantage of the large fluorescence enhancement of the YOYO-i dye upon binding to nucleic acids and corresponding Jack of fluorescence in the presence of released nucleotides and very small nucleic acid fragments. Other dyes - in particular our PIcoGreen dsDNA quantitation reagent (P-7581. P-1 495; Section 8.3) - are likely to be more suitable for this assay.
Similarly, use of the. RIboGreen , RNA quantitation reagent R-11490, R-11491; Section 8.3) should allow ultrasensitive detection of rlbonuclease (RNase) activity. ' Using a design similar to that of molecular beacons (Section 8.5), the stem sequence in an oligonucleotlde hairpin loop can be modified to be a substrate for specific DNA cleavage agents, including nucleases. Dubbed a "break light," this substrate shows increased fluorescence as the Geavage agent breaks the DNA strand, separating. the fluorophore Pram the quencher.
An Assay for Reverse Transcrlptase Actt~ity The EnzChek Reverse Transcriptase Assay Kit (E-22064) is a convenient, efficient and inexpensive assay for measuring reverse transcriptase activity (F39ure 8.138). The key to this method is our PlcoGreen dsDNA quantitation reagent, which preferentially detects dsDNA or RNA-DNA
heteroduplexes over single-stranded nucleic acids or free nucleotides. In the assay, the sample to be measured is added to a mixture of a long poly(A) template, an oligo(dT) primer and dTTP.
Reverse transcriptase activity in the sample results In the formation of long RNA-DNA
hekeroduplexes, which are detected by the PicoGreen reagent at the end of the assay. In less than an hour, samples can be read in a fluorometer or micropiate reader with filter sets appropriate for fluorescein (FITC). The assay Is sensitive, detecting as little as 0.02 units of HIV reverse transcriptase, and has about a 50-fold linear range (Figure 8.139), Because It is much more rapid and less expensive than standard isotopic assay or immunoassays, it is suitable For testing large numbers of biological samples. The assay's simplicity also makes It useful for automated high-throughput screening of reverse transcriptase inhibitors.
The EnzChek Reverse Transcriptase Assay IClC (E-22054) contains:
~ The PicoGreen dsDNA quantitation reagent ~ A lambda DNA standard . A poly(A) rlbonucleotide template ~ An oligo(dT)16 primer w TI= buffer, polymerization buffer and an EDTA solution . A detailed protocol (EnzChek Reverse Transcriiptase Assay Kit) Sufficient amounts of reagents are provided for approximately 1000 fluorescence micropiate assays.
Telomerase In a gel-based assay far detection of telomerase activity (the telomeric repeat amplification protocol or TRAP) in human cells,and tumors, SYBR Green I dye staining was found to be more sensitive than silver staining and gave results comparable to those achieved with a radioisotope--based TRAP assay.tl~ Moreover, unlike the silver stains, the SYBR Green I
stain did not label proteins carried over From the reaction mixture. The SYBR Gold stain was also shown to be more sensitive than silver staining in the TRAP assay, and much easier to use.ti(~-The SYBR Green I
stain (S-7567, 5-7563, S-7585) has also beep used to develop high sensitivity assaysto detect topoasomerase activity.
SUBSTITUTE SHEET (RULE 26) section 8.7 ~- Analysis of DNA Structure, DNA Binding and DNA Damage ~.~..~...,~.,...
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SUBSTITUTE SHEET (RULE 26) pe Research -- Abstracts: Lawyer. et al. 2 (4): 275 ~6'.ENOME Appendix S
PCR Methods and Applications, vol 2, 27S-287, Copyright ~ 1993 by Cold Spring , ~ ~~1 ~s article to a friend , Harbor Laboratory Press ! Similar atxit1es found in:
Ganome Online 1~ Search pubMed for articles by:
Lawyer. F. C.11 G~lfand, D. H.
' AlE2'FICLES ~ 1~ Alert me when:
new articl~~cite this article 1 Download to Citati~Man_a~er High-level expression, purification, and e~izymatic characterization of full-length- Thermos aquaticus I)NA poiylmerase and a truncated folrm deficient in 5' to 3' exonuclease activity FC Lawyer, S Stoffel, RIB Saiki, SY Chang, PA Landre, RD Abramson and DH
Gelfand Program in Core Research, Roche Molecular Systems, Alameda, California 9450I.
The Thermos aquaticus DNA polymerise I (Taq PoI l) gene was cloned into a plasmid expression vector 'that utilizes the strong bacteriaphage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832- amino-acid Taq Pol I
and a deletion derivative encoding a 544-amino- acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced .
544-amino-acid construct produced 3% of total protein as Stoffel fragment.
Enzyme purification included cell lysis, heat treatment followed by Polymitt P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography.
For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 16~ grams wet weight cell paste. For the 6I-1cD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and IO- 55 mM KCI. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half life of 9 min at 97.5 degrees C.
The Stoffel fragment has a half life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent f to f exonuclease activity whereas the Stoffel fragment, deleted for the f to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerises that have been characterized;
specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest.reported.

SUBSTITUTE SHEET (RULE 26) ~ Genome Research -- Abstracts: Lawyer et al. 2 (4): 275 i~

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SUBSTITUTE SHEET (RULE 26) For general laboratory use.
FOR !N VITRO USE ONLY.
r.~~'BR Green 1 Nucleic Acid Gel Stain Nighty sensitive fluorescent stain for detecting DNA in agarose and polyacrylamide gels Cat. No. 1 988123 2 x 500 wi Cat. No. 1 988131 500 Lt,l Store at -15 to -25' C
9. Product ovanriaw Product storage! SYBR Green I Is supplied In an anhydrous DMSO
stability solution and is shipped at ambient temperature.
The unopened vial is stable at -7 5 to -25° C through Caution Because the expiration this date printed reagent an the label.
binds to nucleic acids, h should be treated ~ Aitquote as the stock a solution potential in 5D ~t mutagen aliquots, and used with appropriate care.
The DMSO
stock solution should be handled brown tubes with should be particular used.
caution as DMSO
is known to anic molecules into tissues.
of or ilitate the ent f g Condition TemperaturgStability ry ac When undiluted -16 8 -12 handling stock to months the DMSDStock solution, double gloves, protective clothing and eyewear should be worn . -25 and C
safe laboratory practices should be followed.

diluted stain2-8 por in C several ContentsHa, Label contents pH 7.0-8.5 days Cat buffer . fr e n 1 988 SYBR 2 x 500 wi o1 723 Greenl rc lene Stn a p ~ py Nucleic10 000 x contatner,protected Acid concentrate GelStaln. InDMSD from IighU

1988 SYBR 500 wl 731 Green t NucleicActd10000 x concentrate Gel in DMSO Handlingprotect from Stain Ilght. , recvrnmendatlons . before opening, eachvlal should be allowed to warm l t flu ~
ee g s Tan principleThe 6a dye ttom of exhibits he v a teal.
preferential tion t affinity th for it the DMSO
nucfefe so acids tp dap and its store aqueous fluorescent stain solutions signal in polypropylene Is largely enhanced when bound rather than to glass. as DNA the stain .(more may adsorb than to glass one order of magnitude larger ~rfaces, than the fluorescent enhancement or bound ethldtum dye is not bromide). stable in water alone.

A~piicationSYBR
Green I
dye is a highly sensitive fluorescent stain for detecting nucleic acids In agarose and polyacrylamide 2. Product gels charaetetistirs (1,2).
' The exceptional sensitivity of SYBR
Green I
stain makes 5ensltiviThe detection it limit usin useful SYBR Green for f Is as those krw as applications where D
the Including the detection amount of DNA
is limiting , NA usin )112 of nm trans-low-cycle , number 100 pg per and band oI
tow-target ds number illumination DNA with the Luml Image F1 Instrument ampllflcadon from Roche products; Diagnostics the (Cat. No.
detection 2 015170).
and restriction analysis This is approximately of 25 -100 low-copy times more number sensitive of than DNA
and RNAvectors;
and the detection of products of nuclease protection and ethidlum bandshitt bromide assays, staining.
' SYBR The detection Green Ifmit for 1 oligonuoleotides stain-s stained superior sensitivity allows replacement with SYBR
of Green 1 radtolsotopes is t~elow In 5D pmot some with applications, e,g, 312 nm transiilumlnation RT (Lumi-imager PGR. F7) or d(gte: 254 nm eplillumination SYBR , Green I
stain van also be applied as detection format ToidcityThe Amas in mammalian the mlcrosome LJghtCyder reverse InsVUment mutation But the provided corxenVation fn DMSO
is not standardized for assay shows the slgniflcantty precise less mutegeniclty quantfflcation of SYBR
and Green I than detection ethidlum of bromide nucleic (3.4) acids with the LightCycier:
Please refer to specialized kits gpeotralSYBR Green and 1 is maximally reagents excited for at 497 nm this and has instrument Sample SYBR characteristicssecondary mateHat Green broad excitation 1 peaks at can 284 nrn detecC and 382 nm. The emission of DfVA
stained with SYBR

double Green I Is stranded centered and at520 nm, stn to stranded DNA
g RNA
(with DetecUonThe spectral tower characteristics sensitivity); of SYBR
for Green I
RNA makes staining we recommend to use SYBR
Green it Dllgonucleotides itepb~a with a wide variety of get Imaging ~(q~a.
The UV traps-illuminators detection limit for oligonucteotides stained with Green I
is hg with nm epi-illuminator - UV epi-illuminators or nm translilumination, argon Ion lasers.

StainingWithin Nnta: Double time 30 stranded minutes, DNA-bound gels SYBR Green are t ready to image or photo-graph stain fluoresces w(thout green under destaining. UV transillumination, Gais Number 5D0 that oontatn of stainsp,1 DNA with stock single stranded solution raglans is may sufflc(ent show fluorescence to that is prepare more orange a than green.
total of 5 liters of working solution.
which can be used to stain more than agarose or polyacryl minigeis.

otes.s4s.zzoos ysoma Ma cPM ROCI18 SUBSTITUTE SHEET (RULE 26) ~~cluantaga 3.2 Preparation of working solution Benefit Featuro Caution Since SYBR Green 1 hinds Fast staining of Half the time to nucleic adds, it should DNA necessary as In agarose and for ethtdium be treated as a potential mutagen bromide. and used with -polyacrylamide gelsi~ss than a the DMSO stock solution should be quarter of appropriate care.
the time necessary handled with particular caution as for most DMSO is known to silver staining.. facilitate the entry of organic molecules into tissues.

When handling the DMSO stock solution double Lower mutageniclty Less mutagenic gloves, protective clothing and eyewear than ethidium should be bromide accordingfollowed to Ames b f ti h ld b d t l Test results .
sa e ora ory prac ces s ou e worn an a High sensiUvity Z5-100 times preparation at Dilute SYBR Green more sensitive I Stock soluUon 1:10 000 In TE, TBE, than ethidium SygR Green or TAE buffer. The diluted bromide solution has to be stored In working solutions polypropylene containers or bottles.

o eStalning with SYBR Green 1 is very pH sensitive.

For optimal sensftivity, verify that the pH of the staining 3. Procedures and solution at the temperature used required materials for staining Is - between 15 and 8 (preferably pH 8.0), 3.1 Before pisposat you Aswith begin all nucleic acid stains, solution of SYBR
Green 1 PrestainedWe do should be gets not poured recommend through prepar(ng activated prestalned charcoal gels before with disposal.
The charcoal must then be inc)nerated to SYBR destroy Green the dye.
I stain One gram more of activated than charcoal 1-2 easily days In advance.

Gels absorbs evious the dye stained from 10 with liters ethidlum of freshly bromide prepared can b e stained working with solution:
SYBR
Green I followln g subs quentiy the standard protocol for poststalning.
There may be some decreases in senskivity when compared to a gel stained 3.3 Staining only DNA following with electrophoresis SYBR
Green I.

HlectrophoieslsPerform elecVophoresis on an agarose gel ar denaturing Additional polyarxytamide UV trans-gel or epi-illuminators, using: or respective Imaging TBE equipment [89 and instruments, mM such as Tris Lumi-Imager base, F1, argon 89 ion mM
boric acid,1 mM
EDTA, pH 8j reagents or required lasers or respective imaging instrument TAE clear polyproylene [40 contalnerfor mM staining Tris-acetate,1 mM
EDTA, pH
8]
buffer Note: TBE buffer po or not add any SDS
to the electrophoresis buffer TAE buffer as this will dramatically reduce staining efficiency.

- Procedure Please refer to the following table for the protocol.

Additional Note: For buffers reaching TBE sharp bands [89 and low mM Tris background base, 89 mM
boric acid, required1 mM 'stain tt~e for EDTA, gel directly pH after electrophoresis.
B) or stainingTE [10 mM
Tris-HCt,1 mM
EDTA, pH
8]
or TAE StepAction [40 mM
Tris-acetate,1 mM
EDTA, pH
8]
buffer.

1 Place the gel in a fitting polypropylene container.

Handlingin the Notes following There table is no please need find to wash information urea about or instructfansworking formaldehyde for conditions out for of the successful) gels staining. prior staining.

proper 2 Add enough staining staining solution to cover the Gel t]se clear potyproytene et.
container.for .

containersta(ning gels with protect SYBR Green 1. the staining container from light by f0 ' Never use covering polyvinylchloride/ it with polysterene ar aluminum glass container , foh for the or placing staining of agarose in the or acrylamide dark.
gels.

Stain gets in a 3 Stain fitting container. the gel for approx.
30 min under constant The size of the C.
container should agitation be gently at 15-25 not larger than 4 Ilituninatlon the gel. of the stained gel:

Protect the staining container from You can nm lime light use.

WorkingMake sure that the pH of the ultraviolet312 1-20 s working solutionsolution was adjusted trans-to pH 7.0 to 8.5 pliute 1:10 000 illumination In running buffer, do not use water epI 25A 1-1.5 or minutes AgarosePreferably use eluminatlon,320 (required 0,8196 agarose for max.
gels, gets increase of in for greater sensitivity gal concenVattan increases background. sensWvity especially po not use agarose with gels thicker than a hand-help lamp]

1 cm. If you use thicker gels the diffusion of the stain into the Lumi312 .1-20 the gel is decreased s and the background lmager is increased. F1 ' 5 Photograph the gel with Polaroid black and white print film using a SYBR
Green gel stain photographic filter.

Note:
Stained gels have negligible background fluorescence, allowing long film exposure using an f-stop of 4.5 Is adequate.

Roche Molet~ular.Biochexnicals SUBSTITUTE SHEET (RULE 26) ~.4 Precasting gels with SYBR Green ii stain 3.5 Staining DNA belore Electrophoresis Gener,~l in the following.table please find the features of General precast gels. .
See references (5,6) for general methods on how to stain DNA
before electrophoresis.
It may be necessary to optimize the protocols In these references for the DetectionThe DNA detection specific Itmlt for gels application.
precast limitwith SYBR Green We I may be slightly have poorer stained (on the order 30 1 to 40 pg/band). mg molecular weight-marker DNA

with 1:10 ODO
dilution of SYBR
Green I
in a total volume MigrationThe rate of migration of of DNA fragments 16 ml.
SYBR
Green I
has also been tested as a of In gels containing prestaining DNA SYBR Green I stain label for DNA
templates In bandshift fragmentsmay be sfgniticandy assays, slower than the and rate has been prove to be useful in this of migration of application.
the same fragments In a get containing , no dye.

MobilityThe mobility of ProcedurePlease DNA smaller molecules refer tends to to l the d following table.

of arger be affecte more than that of fragmentsfragments. StepAction ' 1 Incubate DNA with a 1:10 000 dilution of the dye (In TE, TBE or TAE) for at least 15 min prior Procedure to electrophoresis.
The final dilution of the SYBR
Green t is best , determined ' Follow el etectro empirically, 2 hores(s as usual as there g p may be some nan .
linear effects on the migration of different fragment site.

StepAction 1 Dilute SYBR
Green I stock reagent 1:10 000 into the gel 3.6 Removing solutionJust SYBR Green prior 1 stain to pouring from double-stranded the gel. DNA
The liquid should be as cool as possible when the dye Procedure is added. At least Boiling 99.996 and near of SYBR
boiling Green temperatures I can be removed from double-stranded destroy DNA by the ability simple of SYBR ethanol Green precipitation.
I to stain nucleic gyppeon acid.
Do not heat SYBR
Green I in the microwave.

2 Follow 1 Bring a solution of gel electrophoresis DNA stained with as usual. SYBR

Green ! reagent up to 100 mM NaCI and add 3 Illumination 2.5 volumes of absolute of the or 9596 ethanol.
stained get:

2 In b i t i cu You can nm Time e use... n on ce a m - Centrifuge mixture for at least 1D m1n in ultraviolet3121-20 a microfuge at 9'C.
s trans-itlurnination Remove the ethanol and wash the pellet epi eluminatlon,2541-1.5 once w(th 70-9596 or minutes ethanol.

for greater320(required 3 Dry the pellet and for resuspend double-stranded sensitivity max. . DNA In buffer sensitivity.

especially with a hand-help lamp) the Lumi 3121-20 fmager s A Photograph the gel with Polaroid 667 black and white print film using a SYBR
Green get stain photographic filter Alote:
Stained gels have negligible background nuarescence, allowing tong film exposure using an f-slap of 4.5 is adequate.

Roche Molecular BJochemicals SUBSTITUTE SHEET (RULE 26) r4. References E-mail Adress Country 1 Schneeberger, C, et a1.1995. PCR Methods & App. 4. 234-238.
2 3hrger, V., et e! 1994. 8lomed Products 18. 68.
3 MutaUon Res.113,173 (1983) 4 PNA570, 2261 (1973) MeUz Enrymol 217, 414 (1993) 6 Nudelc AcklS Rea 20, 2803 (1992) aigenUnablochem~rocha.comArgentina blxhemau~roche,camAustralia Gerhard.MuehlbauerCAroche.comAustria bloche~roci>a.com Belgium blodtemcaCalroche.comCanada 6loshemcnQrochecomChina Dlochemcynicosla~mctte.com~yptus Bmcomp(s~mboxvotezCzech Republic ri SYBR blochersttid4odola.0Finland Green i is a trademark of Molecular probes, Inc., Eugene.
OR USA.

~ hanlImager blochem.tr~rocheoomFrmxe b a trademark of a Member of the Roche Group.

bkxlremirtfoda~rochecromGermany tubanegin~istntrostcomIran ageMekOtkmnat !steel ltbiochemct'roche.comItaly 4.1 Related bmirktrtomcatco,~ .!open products Bmsicorea~cholliaranetKorea blochemlnronl(4!mchecomNetherlands P PBCk Cat biochemnz4~rodae.camNew 2eaiand d SIZe NO

l7Ct . medinor~medlnocno Norway r0 .

TAE buffer 41 1 666 biochempc~roche,comportage!

, bbcham,sg~roche.camSingapore remlXed 10 x seatlt_alricablobofffnOroche.comSouthAMca TBE buffer, 4 I 1 666 btochemes~roche.comSpate remlxed 10 biochemse~roche.camSweden x Biochemlrr(o.CHai'rochecromSvAheNand Agarose, 500 1 368 rdr.blxheme7roche.eomunited Kingdom MP g 901 ~r~

LUmI-Imager 1 90i b odiodi ~ Au attar F1 800 ,oche ~m ccwtrles httpJ/btochem.rochecomJpack-insert/1888123a.pdt Argentina 541 954 5555; Australia (02) 9899 7999; Austria (01) 277 87; Belgium (02) 247 4930; Brazil *55 (11) 3666 3565; Canada (450) 666 7050; (800) 3fi1 2070; Chile 00 56 (2) 22 33 737 (central) 00 56 (2) 22 32 089 (Exec): China 86 216427 5586; Columbia 0057-1-34tZ197;
Czech Republic (0324) 45 54. 58 71-T; Denmark +45 363 999 58; Egypt 202 348 1715;
Finland (08) 429 2342; France 04 76 76 30 87; Germarry (0621) 759 8568; Greece 3 (0t) 67 40 236; Hong Kong (85~ 2485 7596; India 0031 (22) 4312312; Indonesia 62 (021) ext 755; Iran 00 98 212 08 2266 + 00 98 218 78 5656; !seal 972 3 6 49 31 11;
Italy 039 247 4109-4181; Japan 93 3432 3155; Kettpa 00254~7-74 46 77; Korea 82-2-3471-6500:
Kuwak 00965-483 26 00; Lummbourg 00352-4824821; Malaysia 60 (03) 755 5039; Mexico (5) 227 8967: Netherlands (036) 539 4911: New Zealand (09) 278 4157; Nigeria 00234-1-96 09 84:
Norway 22 07 65 OD; PhlBppinas (632) 810 7246; Poland +48 (22) 22 66 84 305;
Portugal (0t) 4171717: Republic of irefand 180D 4090 41; Russia (49) 621759 8636 Fax: (49) 6217698611;
Saudta Arabia +966 t 4010333; Sirrgaporo 0065 272 9200; South Ahica (011) 886 24Q0;
South Eastern Europe (01) 277 B7; South Korea 02 569 68D2; Spain (93) 201 4411; Sweden (OS) 404 8800: Switzerland +41 (41) 799 6161: Taiwan (02) 736 7125; Thailand 68 (2) 274 07 08 (121tne);Turkey OD90212216 3280: United Kingdom (D8D0) 521578: USA (80D) 4285933.
Roche Diagnostics GmbH.
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SUBSTITUTE SHEET (RULE 26) SUBSTITUTE SHEET (RULE 26) 'F~~t~~~ 1~'t~~rt~rt~tiQ~i ~, Rcaa=lmae PCR'1'aqman /~ ppendix c OVERVIEW
Real-time quantitative PCR is a powerful tool that can be used for gene expression analysis, genotyping, pathogen detectionlquantitation, mutation screening and DNA
quantltation. At the BRC, we use the ABi Prisrn 7900 Real Time Quantitative PCR
instrument (TaqMan~) to detect accumulatiorw of PCR product, allowing easy and accurate quantitation in the exponential phase of PCR reactions.
The ABI 7900 instrument continuously measures PCR product accumulation using a dual-labeled flourogenic oligonucleotide probe called a TaqMan~ probe. This probe is labeled with two different flourescent dyes, the 5' terminus reporter dye and the 3' terminus quenching dye. The sequence of the oligonucleotide probe is homologous to an internal target sequence present in the PCR amplicon. When the probe is intact, energy transfer occurs between the two flourophors, and the fluorescent emission is quenched.
During the extention phase of PCR, the probe is cleaved by 5' nuclease activity of Taq polymerase. Therefore, the reporter is no longer in proximity to the quencher, and the increase in emission intensity is measured.
Tha ABI 7900 Prism software aXamines, the fluorescence intensity of reporter and quencher dyes and calculates the increase in normalized reporter emission intensity over the course of the amplification. The results are , then plotted versus time, represented by cycle number, to produce a continuous measure of PCR
amplification.
To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined at a point during the early fog phase of product accumulation above background (defined as the threshold cycle number or GT). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each well.
Primers, Probes, and Reagents .
It is ess~ntial to have a well thought out experimental design for Real Time PC.R. Good primer and probe design is imperative. The BRC will design your probes and primers using Primer Express, the industry gold standard. Primers should be synthesized and purified at the BRC. This service is charged at our consultant rate of $50/hr.
We require purified primers. Probes should ' be synthesized by Biosearch Technology.
(wwinr.biosearchtech.com). Black hole quench probes give the most consistent data.
Average probe cost is $250.
If you plan to perform your own Taqman~ reactions, Applied Biosystems provides a number of kits specific to applications. See their web site www.AppliedBiosystems.com..
Pacfl~ty Acknowledgement so SUBSTITUTE SHEET (RULE 26) Re~-'~'irrie'~PCR"'~aqman The Taqman~ facility requests an aclmowledgement in the Methods section of any publications resulting from this data. An example is "Real time quantitative PCR was conducted by the Biomolecular Resource Center at the University of California, San Francisco." Additionally, if your project required special attention by a specific person at the BRC, an example would be "Technical expertise was provided by (specific name of BRC personnel) of the Biomolecular Resource Center at the University of California, San Francisco."
Biomolecular Resource, Center Genetic Analysis Faci 1 ity UCSF, Science 983, San Francisco, CA 94143-0541 Phone: (415) 514-0101 x1; FAX: 502-7649 Email: dnaC~3cgl.ucsf.edu sl SUBSTITUTE SHEET (RULE 26) l~~Qendt~t N
n~
fife ~~~hnr~~c~gi~~
tn~~t~uction manual LUXT"" FluorogeniC Primers For real-dime PCR and RT-PCR
Vexsion E
22 September 2003 SUBSTITUTE SHEET (RULE 26) PAGE INTENTIONNALLY LEFT BLANK

SUBSTITUTE SHEET (RULE 26) Table of Contents Introduction ...............................................................................
................:......................1 Designing and Ordering Custom LUX Primers ..............................................................3 Certified LUX Primer Sets for Housekeeping Genes.....................................................5 Storing and Reconstituting Primers............................................................:...........
..........5 Real-Time PCR............................................................................
....................................7 Multiplex Real-Time PCR............................................................................
...................11 Two-Step Real-Time RT-PCR.............................................,..............................
............12 One-Step Real-Time RT-PCR
..........................................................................:....
........14 Troubleshooting ... .
...............................................................................
.......................18 Accessory Products ...............................................................,...............
........................19 Purchaser Notification...................................................................
............'.....................20 Technical Service........................................................................
....................,..............21 ..
.
References ...............................................................................
...........
. ~ ......................

~

SUBSTITUTE SHEET (RULE 26) PAGE INTENTIONNALLY LEFT BLANK
SUBSTITUTE SHEET (RULE 26) lnt~rodrction wefView LUX"' (Light Upon e_Xtension) Primers are an easy to use, highly sensitive, and efficient method for performing real-time quantitative PCR (qPCR) and RT-PCR (qRT-PCR). LUX"' Primers combine high specificity and multiplexing capability with simple design and streamlined protocols. LUX"' Primers require no special probes or quenchers, and are compatible with melting curve analysis of real-tune qPCR products, allowing the differentiation of amplicons and primex dimer artifacts by their melting temperatures. You can-custom-design LUX"' Primers' from a target sequence using Web- or desktop-based software, or order predesigned and validated Certified LUX"' Primer Sets for Housekeeping Genes.
Each primer pair in the LUX"' system includes a fluorogenic primer with a fluorophore attached to its 3' end, as well as a corresponding unlabeled primer. The fluorogeruc primer has a short sequence tail of 4-6 nucleotides on the 5' end that is complementary to the 3' end of the primer. The resulting hairpin secondary structure provides optimal quenching of the fluorophore (see the figure below). When the primer is incorporated into the double-stranded PCR product, the fluorophore is dequenched and the signal increases by up to lt?-fold.
i:UX~" Primer . Itetative fluorescence:
Reaction .
0.1 Hairpin primer 1 rl~
0.4 Single-stranited primer 1.0 Extended primer (double~nded DNA) Labelitlg Each fluorogeruc LUX"' primer is labeled with one of two reporter dyes-P.A.M
(ti-carboxy-fluorescein) or ~OE (trcarboxy-4', 5'-dichIoro-2', T-dimethoxy fluorescein). Additional reporter dyes will be available in the future.
Continued on next page SUBSTITUTE SHEET (RULE 26) IfltPOdUCtiol1, Continued Applications LUX"" Primers can be used in real-time PCR and RT-PCR to quantify x00 or fewer copies of a target gene in as little as 1 pg of template DNA or RNA.
They have a broad dynamic range of 7-S orders.
Multiplex applications use separate FAM and JOE-labeled primer sets to detect two different genes in the same sample. Typically, a custom-designed FAM-labeled primer set would be used to detect the gene of interest, and a JOE-labeled Certified LUX"" Primer Set would be used to detect a housekeeping gene as an internal control.
ltlstfuit'1B11t ' LUX'" Primers axe compatible with a wide variety of real-time PCR
Compatlblllty instruments, including but not limited to the ABI PRTSM~
7700/7000/7900 and GeneAmp~ 5700, the Bio-Rad iCycler'", the Stratagene Mx4000"", the Stratagene Mx3000"", the Cepheid Smart Cycler'a, the Corbeu Research Rotor-Gene, and the Roche LightCycler~.
ABI PRISM is a registered trademark of Applere Corporation. GeneAmp is a registered trademark of Roche Molecular Systems, Inc. LightCycler is a registered trademark of Idaho Technologies, Ina iCycler, Mx4000, Mx3000, Rotor-Gene, and Smart Cycler are trademarks of their respective companies.

SUBSTITUTE SHEET (RULE 26) ~Deaigning and Ordering Custom LUXTM Primers LUX'~ Desig~et' To design and order custom LUX"" Primers for your genes of interest, visit the Primer Design Invitrogen LLJX"" Web site at wwvw.invitrogen.com/L11?( and follow the link to Software the LUX"' Designer software. The software is available as either a Web-based application or a Microsofl'~ Windows~ compatible download. Follow the step-by-step instructions in the software to submit your target sequence and generate primer designs.
LUX'" Designer will automatically generate one or more primer designs based on each sequence you submit and the selected design parameters. The design software includes algorithms to minimize primer self complementarity and interactions between primers. It also assigns rankings to the generated designs--based on primer melting temperature, hairpin structure, self annealing properties, etc.-to aid in selection.
When the designs have been generated, you can review them, select a design, select the fluorophore labels, and place your order.
Guid811~es fol' When you submit a target sequence containing your gene of intexest, keep in Submiftitlg a mind the following design criteria:
°TePget Sequence , ~e optimal amplicon length for real-time PCR ranges fxom 80 to 200 bases. You can specify a minimum, optimal, and maximum amplicon length when you submit the sequence.
~ The target sequence should be at least 10 bases longer than the minimum amplicon size you select. The longer the sequence, the more likely that an optimal primer design can be developed.
The sequence must contain only standard ILTPAC (International Union of Pure and Applied Chemistry) letter abbreviations. ' ~ When you select the design parameters, the default melting temperature range is 60-b8°C. Do not change this default unless the design engine finds no primers in this range. For primers in this range, PCR annealing temperatures from 55° to 64°C axe appropriate.
When you first submit a sequence, the Disable Score-Based Rejection checkbox should not be checked; the resulting scores provide an important measure of primer suitability. Scores in the range of 0.0-4,0 are acceptable. If no primers with a score of 4.U or lower can be generated from a sequence, you can disable score-based rejection and redesign the primers. Note that if you select a primer with a highex score, the efficiency of the reaction may be less than optimal.
See the LUX"' Designer Help for additional guidance.
SeleCtillg a~Primec After you submit your sequence, LUX'" Designer will first generate one or Design more designs for the labeled primex. The labeled primer can be either the forward or the reverse prinner. After you select a design for the Labeled primer, you will be prompted to select a design for the corresponding unlabeled primer.
Continued on next page SUBSTITUTE SHEET (RULE 26) Designing and Qrdering Custom LUXTM Primers, continua Selecting Labels After you have selected a primer set (labeled and unlabeled) for a particular sequence, you can specify the particular label and synthesis scale. Custom LUX"' Primers axe provided ize 50 nM or 200 nM synthesis scale, When selecting labels in a multiplex reaction, we recommend using the FAM
label for your gene of interest and the JOE label for the housekeeping gene that you will use as the internal control. Certified LUXT" Primer Sets for Housekeeping Genes are recommended for the JOE-labeled control gene.
Placing the Order After you have selected the label and synthesis scale, you can submit your order to Invitrogen using the Web site or by e-mail or fax. Each.primer order will be shipped durectly from Invitrogen's Custom Primer Pacilities. Labeled primers are supplied in an amber tube; unlabeled primers are supplied in a clear tube. ' Each primer ordered from lnvitrogen's Custom Primer Facilities comes with a Certificate of Analysis (COA) verifying the amount and sequence.
PI'OdUCt Custom LUX"' Primers are tested pest synthesis by optical density (OD) ratio Qualification measurements and mass spectroscopy to ensure efficient dye labeling and correct molecular weight and composition.
See the Certificate of Analysis shipped with each primer for more information.

SUBSTITUTE SHEET (RULE 26) Storing and Reconstituting Primers Primer Storage atld Store primexs at 20°C in the dark LU7C"' Primers are stable for:
Stability , >t2 months when stoxed at-20°C in lyophilized form.
~ >6 months when stored at 20°C in solution.
Stability can be extended by storing at 70°C.
ReGOllstitutitlg Custom LUX"' Primers are provided lyophilized in 50-nmole or 200-nmole Primers synthesis scale. To reconstitute primers, centrifuge the tube for a few seconds to collect the oligonucIeotide in the bottom of the tube. Carefully open, add an appropriate volume of TE buffer or ultrapure water, close the tube, rehydrate for 5 minutes, and vortex fox 15 seconds.
We recommend that you rehydrate primers at concentrations greater than ltlvl. To prepare a 100 ExM primer stock solution, multiply the primer amount in nmoles by ten to determine the volume of diluent in p1.
.After reconstitution, store the pr'smer stock at-20°C in the dark, where it will be stable for 6 months or more.
SUBSTITUTE SHEET (RULE 26) Cerfiified LU~~' Primer Sets for Housekeeping Genes CeCEifled LUX~ Certified LUX"' Primer Sets for Housekeeping Genes are predesigned primer Primer Sets for . sets for genes that are commonly used as internal controls for normalizing , HOUSekeeplllg real-time RT-PCR experinnents. These primer sets have been optimized and Genes functionally validated to provide accurate, reproducible results using standard LUX'" protocols. They are supplied ready to use in TS buffer.
Each Certified LUX"' Primer Set includes a FAM- or JOE-labeled LUX'"' primer and a corresponding unlabeled primer. Each primer (labeled and unlabeled is supplied at 100 Etl and a concentration of 10 ltM. Available sets axe listed ' below. For additional informs#ionD visit r-irtvit:~~en.comlLLJX.
GenBank Forwardl Cat. Cat, RelativeCDS 1'CR
Product AccessionReverse no. no. reaslon LocationProduct no. Label FAM Ot3label Size label Ran a Human eves l8SrRNA X032Q5 Forward 115FiM-Ol115f1~I-02++f+ n!a l0I-150b h ACTlN Nlvt<001101Forward 101H-01101H-02+++ Bxons 101-I5D
2/3 b hATPSaseNI~Iv.OD1686Forward 108I3-OllOBH-02+++ n/a 101-150 b h132M 1VM_0020Q8Forward I13H-Ol113H-02+++ Exans 101-150 if b hGAPI~H t3M_002046Forward 100H-01100H-02++t- Exons 151-200 4/5 h hPGK1 NM_000291Porward 109H-Ol109Ii-02+++ nla 50-i00 hPPlA NM_021130Forward 106H-01lOdH-02+++ Faeons2/350-100b hRPL4 NM~000968Reverse lOt3H-Ol103H-02+++ E~tons8/910I-150b hEEFIG NM_001404Forward 147H-01107H-02++ nJa 50-IODb hHPRTI NM U00194Reverse 105H-01105H-02++ Hxons 50-100 5/6 b hSDHA NM_D04168Forward 102H-Ol102H-02++ fixons 50-100 ' 12/13 b hTPRC NM_003234Forward 111H-0I111H-02++ Futons 101-150 10/11 b hGI3S 13~OD0181Forward 112H-01I12H-02+ Rxons 101-150 7/8 b hHHMS NNiv000190Forward 110H-01I10H-02+ Facons 50-lOD
2/3 b hTBP NM_003194Porward 104H-01104Fi-02+ Bxons 101-150 3/4 b hLtBC NIv~021009Forward 114H-Ol114H-02+ n/a 5D-l0U
b Mouseirat eves 185 rRNAX03205 Forward 115HM-0i11513M-02t+++ n(a 10I-150 b m ACTIN NM UO?S93Forward 101M-01101M-02t++ fixons 1DI-150 2/3 b mB2M X01838 Forward 113M-01iI3M-02+++ n/a 50-100h mBfiPiG AP321I26Ponvard 107M-01107M-02+++ n/_a 101-150_ mGAFDFI Ntv1'008084Forward LOOM-01100M-02+++ Exons 151-200 4/5 b mPGKt N1V1_,008828Forward 109M 109M-(12t++ Exons 101-I50 01 1!2 b mPPIA. NM_008907Reverse 106M-Ol106M-02+++ Exons 50-100 1/2 b mRPh NM_02Z510Forward 103M-01103M-02+++ Exons 151-200b 2!3 irs-ipRT1NI~013556Forward 105M-Ol105MD2++ Fxons 50-100b mSDHA AF095438Forward 102M-Ol102M-02++ Exons 50-100b mATPSaseMt~016774Porwerd 108M-01I08M-02t n/a 50-1006 mGUS NM_OI0368Forward 112M-01112M-02+ Bxons 50-1DD

7/8 b mHBMS XM_129404Reverse 110M-01110M-02+ Fxons 50-IOD
4/5 b mTBP NM-013684Forward 104M-011021V1-02+ Exons 101-150 3/4 b mTPRC NIv~01I638Forward 111M-01111M-02+ Exons I01-I50 2/3 b Dros hila enea d185rRNAAYD37174Reverse 1151-01115D-02++++ n1a 101-150 . b dActln NM~079486Forward 101D-01'lOlD~02+++ Exons2/35p-lODb .

SUBSTITUTE SHEET (RULE 26) Reat-Time PCR
Int1'oduCtion . This section provides guidelines and protocols for performing real-time PCR
using LUX'" Primers.
Ten'ipiate The target template for real-time PCR is linear single-stranded or double SpeCifiCations stranded DNA, cDNA, or circular DNA (such as plasmids). The amount of DNA typically ranges from 10Z to 10' copies or 1 pg to 20 itg of template.
See page 12 far instructions on generating cDNA using reverse transcription as part of two-step real-time RT PCR.
Primer Far optimal PCR conditions, primer iitrations of 50-500 nM per primer are Concentration recommended. The sample reactions on pages 9-10 use 200 nM of each primer, equivalent to 1 ~xI of a 10 uM primer solution.
Magnesium The optimal Mg*'' concentration for a given target/primer/polymerase .Concentration combination can vary between 1 mM and 10 mM, but is usually in the range of 3 mM. See the sample reactions on pages 9-10.
dNTP The optimal concentration of dATP, dCTP, dGTP, and dTTP is 200 iaM each.
If COnCentt'atiott dUTP is used in place of dTTP, its optimal concentration is 400 ~M.
Enxyme We recommend using a "hot-start" DNA polymerase, preferably one that has SpeCi~CattonS been optimized for real-time PCR. Platinum~ Quantitative PCR
SuperMix UDG (Catalog no.11930-01~ is a 2X-concentrated, ready-to-use mixture containing all coxriponents except primers and template. It uses Platinum Tag DNA polymerase and has been specifically formulated to provide optimal performance in, real-time PCR systems.
Continued on next page SUBSTITUTE SHEET (RULE 26) R.eaf-Tfme PCR, continues instrument LUX'" Primers are compatible with a wide variety of real-time PCR
Specifications instruments with various detection capabilities. See page 2 for a partial list of compatible instruments. A protocol for instruments that use PCR tubes/plates is provided on page 9. A protocol for the LightCycler~ is provided on page 10.
At a n~ixumum, the instrument used to perform real-time PCR with LUX"'.
Detect fluorescence at each PCR cycle >3xcite and detect FAM-labeled LUXn' Primers near their excitation/emission wavelengths of 490/520 nm, and/or ~ Bxcite and detect jOE-labeled LU?C"' Pximers near their excitation/ennission yvavelengths of 520J550 nm Please refer to the specific instrument's user manual fox operating instructions.
itlstt'llmetlt Please follow the manufacturer's instructions for configuring your real tune Settings PCR instrument for use with LUX"' Primers. Note the following settings:
~ LUX'" Primers are compatible with standard melting curve analysis, if your instrument allows that option. Program your instrument accordingly., ~ The quencher setting on the insirument should reftect~the fact that LUX"' Primers do not contain a quencher.
~ We recommend the use of ROX Reference Dye (Cat. no. 12223-023) for normalization of well-to-well variation with instruments That are compatible with this option. Adjust your instrument settings accordingly.
Continued on next page SUBSTITUTE SHEET (RULE 26) Rest-Time PCR, Continued Protocol for The following protocol uses Platinum~ Quantitative PCR SuperMix-UDG with (nstrUtnent5 Using ROX reference reagent. It has been optimized for use with real-time PCR
PCR Tubes or instruments that use PCR tubes or plates. A protocol for the Roche Piates LightCycler~ is provided on the following page.
Note: The following protocol uses a 50-Etl reaction volume; smaller volumes may be used, depending on the requirements of your instrument. Before proceeding, see the real-time PCR guidelines on the previous pages. For multiplex reactions, see the guidelines on page 11.
1. To reduce well to-well variafi.on, prepare a Master Mix of all the reaction ingredients except template. The following table provides Master Mix volumes for one reaction and 50 reactions (scale up or down as needed):
Comp,Qnent o1/1 Vol/50 rxiis Platinum Quantitative PCR SuperMix-UDG' 25 ~l 1250 ~1 ROX Reference Dye 1 E.~l 50 ~.il Labeled LI7X"' Primer (10 uM) 1 gel 50 ~.il Unlabeled primer {10 uM) 1 w1 50 N.1 Sterile distilled water2 to 40 p1 to 2000 Nl '60 U/ml Platinum's Tag DNA polymerase, 40 mM Tris-HCl (pH 8.4),100 mM TCCI, 6 rnl4l MgClz, 400 f.~M dGTP, 400 ECM dATP, 400 tiM dCTP, 800 ~M dUTP, 40 U/ml UDG, and stabilizers.
zor use DNase/RNase Free DistHled Water (Cat. No.10977-015).
2. Program the real-time PCR instrument as follows:
3-Step Cycling (recommended) 2-Step Cycling (optional) 50°C, 2 min hold (TJDG treatment) 50°C, 2 min .hold (UDG
treatment) 95°C, 2 min hold 95°C, 2 min hold 45 cycles of: 45 cycles of:
95°C, x5 s , 95°C,15 s 55°C, 30 s 60-65°C, 30-45 s 72°C, 30 s Melting Curve Analysis (optional) Refer to instrument documentation 3. Add 40 ~.~1 of the Master Mix to an optical PCR tube or each well of a 96-well PCR plate.
4. Add 10 Eti of template diluted in TE or sterile dHzO to the tube or each well of the 96-well PCR plate. Cap or seal the tube/plate.
5. Gently mix and make sure that all components axe at the bottom of the tube/plate wells. Centrifuge briefly if needed.
6. Place reaction in the real-time PCR instrument and run the program.
Collect and analyze results.

SUBSTITUTE SHEET (RULE 26) Real-Time PC-R, contir~uea P1'otOCOI for the q'he following protocol uses Platinum~ Quantitative PCR
SuperMix-UDG and Roche LightCycler~ has been optimized for the Roche LightCycler~. Consult the LightCyclef~
documentation for detailed instructions on preparing the capillary tubes and operating the instrument. FAM-labeled LUX'" Primers are also compatible with Roche enzyme mixes.
Note: jOE-labeled LUX"' Primers are not compatible with the current version of the LightCycler~; use FAM-labeled primers only. The following protocol uses a 20-pt reaction volume. Before proceeding, see the real-time PCR
guidelines on the previous pages.
1. To reduce well-to-well variation, prepare a Master Mix of all the reaction ingredients except template. The following table provides volumes for one reaction and 34 reactions (scale as needed):
Component Vol/1 rxn Vo1/34 rxns Platinum~ Quantitative PCR SuperMix-UDG' 10 Eel , 340 Evl FAM labeled LUX'" Primer (10 uM) 1 pi 34 E.vl Unlabeled primer (10 ~ 1 N.l 34 Eel Bovine serum albumen (5 mg/ml)z 1 ~.il 34 Eel Platinum~ Tag DNA Polymerase3 0.12111 y,tl Sterile distilled water to 18 pi to 612 lsl '60 U/ml Platinum~ Tag DNA polymerase, 40 mM Tris-HCI (pH 8.4),100 mM KCI, 6 mM Mgt'1z, 400 ).tM dGTP, 400 ltM dATP, 400 ~M dCTP, 800 itM dUTP, 40 U/ml UDG, and stabilizers.
zValidated with non-acetylated Ultrapure BSA (10% solution) from Panvera (Cat.
nos. P2489 and P2046).
3Tota1 units of Platinum~ Tag DNA Polymerase in the reaction is 1.2 (including 0.6 U
in Platinum~ Quantitative PCR SuperMix-UDG
'or use DNase/RIVase Free Distilled Water (Cat. No.10977-015).
2. Set the fluorescence on the Roche LightCycler~ to the P1 channel. .
3. Program the instrument as follows: . ' Thermal Cycling Melting Cwrve Analysis (optional) Program choice: Amplification Program choice: Melting curve Analysis mode: Quantification Analysis mode: Melting curves Cycling: Cycling:
50°C, 2 min hold (UDG treatment) 95°C, 0 s 95°C, 2 min hold 55°C,15 sec 45 cycles of: 95°C, 0 (ixtcxease 0,1°C/s with 94°C, 5 s continuous acquisition) 55°C,10 s (single acquire) 40°C, 0 s 72°C,10 s 4. Add 18 ~1 of Master Mix to each capillary tube of the LightCycle~.
5. Add 2 E.vl of template to each tube, and cap the tube.
6. Centrifuge the tubes at 700 x g for 5 seconds.
Place the reaction tubes in the rotor of the LightCyclez~ and run the prograxn. Collect and analyze results.
SUBSTITUTE SHEET (RULE 26) Multiplex Real-Time PCR
MuitipleX In multiplex real-time PCR, different sets of primers with different labels are Real Time PCR used to amplify separate genes on the template DNA. Multiplexing with LUX"' Primers offers simplified kinetics when compared with probe based technologies, because only two oligos are used per target.
LUX'" Primers have been tested in multiplex reactions using a FAM-labeled primer set for the gene of interest and a jOE-labeled set for a housekeeping gene used as an internal control to normalize between different reactions. We recommend using Certified LUX'" Primer Sets for F3ousekeeping Genes for the internal control.
In a standard multiplex reaction, you can include the additional primers at the same volumes and concentration as the primers in a singleplex reaction, as shown in the example mixture below:
Component Volume Platinum~ Quantitative PCR SuperMix-UDG (2X) 25 E,~I
ROX Reference Dye (50X) 1 ~.~I
Template 10 ~I
Forward primer 1 (FAM label)1 i.i,I
(10 ~

Reverse primer 1 (10 ~.iM) 1 ial Forward primer 2 QOE label)1 ~.~1 (10 liM) Reverse primer 2 (1D ~.M) 1 N.I

Sterile distilled water to 50 ~l , Reduce the volume of wafer to compensate for the additional primer volume.
All other reaction volumes remain the same.
Follow the thermal cycling guidelines provided in Protocol for Instruments Using PCR Tubes or Plates on page 9. If you have difficulty performing the multiplex reaction using these guidelines, see the optimization hints below:
Optimizing If you notice a decline in real-lime PCR efficiency in your multiplex real-time Muitipiex PCR, you can optimize the reaction by performing the steps listed below.
Collditiolls Note: We recommend that you perform one optimization step and then repeat the reaction to test for efficiency before moving on to the next step:
1. Reduce the primer concentration of the gene with the highest abundance (typically the housekeeping gene) to 1 /4 the primer concenixation of the other gene. For example, in a standard 50 ~I reaction, you would add the primers for the less abundant gene at 1 NI each, and add the primers for the more abundant gene at 0.25 NI each.
2. Increase fine MgCl2 in the reaction from 3 mM to 5 mM.
3. Double the amount of polymerise enzyme (to 0.06 U per pi of reaction volume). If you are using Platinum~ Quantitative PCR SuperMix-UDG, add Platinurn~ Tag DNA polymerise stand-alone enzyme (Catalog no.
10966-018) to double the amount of enzyme.
4. Increase the dNTP concentrations in the reaction to 400 l.iM each.

SUBSTITUTE SHEET (RULE 26) Two-Step Reat-Time RT-PCR
IntroduCtlott For real time RT-PCR applications, vve recommend a two-step protocol so that the RT and PCR modules can be optimized separately for maximum efficiency and specificity.
This section provides an optimized protocol for performing reverse transcription as part of a two-step real-time RT-FCR protocol. You can use the resulting cDNA in the real-time PCR reaction on pages 7-10.
Templets ~ The target template for real-time RT-PCR is RNA-usually total cellular RNA
SpeCifICatiofls or mRNA. The amount of RNA typically varies from 1 pg to 100 ng of template per assay. The purity and integrity of the RNA have a direct impact on results. RNase and genomic DNA contamination are the most common problems, and purification methods should include RN 'se inhibitors and DNase digestion to minimize these.
We recommend using the Micro-to-Midi Total RNA Purification System (Catalog no. 12183-018) or TRIzoI~ reagent (Catalog no. 15596-026) to isolate total RNA. High-quality total RNA can be purified from as little as 100 cells up to 10' cells or 200 mg of tissue.
To isolate mRNA, we recommend using the FasfiTrack~ 2.0 mRNA Isolation Kit (Catalog no. K1593-02).
EnZyl'118 We recommend using Superscript'" II or Superscript'" 1TI RT for the reverse SpeCi#ICations transcription reaction. The sample protocol on page 13 uses the Superscript"' First-Strand Synthesis System for RT-PCR (Catalog no.11904-018), available from Invitrogen, which includes all components needed for the first strand synthesis reaction except the RNA.
Removing We recoxnrnend that you decrease the genomic DNA content in the RNA
Gel7omiC DNA f1'otll sample by performing a digest with DNase I, Amplification Grade (Catalog RNA Samples no.18068-015), as described below. The DNase I digest is designed for up to 1 Fg of RNA; for larger amounts of RNA, increase volumes accordingly.
Combiune the following in a tube on ice:
Component Conc. Volume RNA template - x E.il DNase reaction buffer 10X 1 1,i1 DNase I, Amplification1 U/ul 1 lil Grade DEPC-treated ddHzO to 10 E.~l 1. Incubate at room temperature fox 15 min.
2. Add 1 ~.~1 of 25-mM EDTA solution to the reaction mixture and incubate at 65°C for 10 min to inactivate the DNase I.
Continued on next page SUBSTITUTE SHEET (RULE 26) Two~Step Real-Tir~r~e RT-PCR, continues ReVePSe The following protocol can be used with either the SuperScript'" First-Strand Transcription Synthesis System for AT-PCR (with SuperScript~" II RT) or the SuperScript"' III
PI'OtOCO) First Strand Synthesis System for RT-PCR (with SuperScript'" III
RT). The protocol has been optimi2ed for LUX"' Primers. Follow this protocol to generate cDNA, which can then be used in real-time PCR (see pages 7 10).
1. Combine the following a tube on ice.
kit components in For multiplex reactions, a master mix without RNA may be prepared:

Oligo(dT),~-,s (0.5 pg/iil) or Oligo(dT)ZO (50 mM)" 0.5 ltl Random hexamers (50 ng/~1) 0.5 ~.i RNA (up to 1 fig) x lti lOx Buffer 2 ~1 25 mM MgClz 4 ~1 mM dNTP 1 ~1 0.1 M DTT 2 Etl RNase~UT"' (40 U/1.~1) 1 itt SuperScript"' II RT (50 U/Etl) or SuperScript'" III RT 1 i,il (200 U/NI) DBPC-treated ddHzO to 20 01 'Ollgo(dT juac is recommended for use with SuperScript"' lI RT; oligo(dTko is recommended for use with 5uperScript"' 1TI RT

2. Incubate tube at 25C
for 10 min.

3. Incubate tube at 42C
for 50 min.

4. Terminate the reaction at 70C at 20 min, and then chill on ice.

5. Add 1 E.~l (2 U) of E.
coTi RNaseH and incubate at 37C for 20 min.

Store the reaction at 20C
until use. Use 2,8 0.1 of cDNA for real-time PCR, as described on pages 7 10.

SUBSTITUTE SHEET (RULE 26) C)ne-Step Real-Time RT-PCR
Int1'oduEtion This section provides information and a protocol for performing one-step xeal-time RT-PCR using LUX'" Primers. One-step RT PCR is a complex reaction in which both reverse transcription and PCR are carried out in the same tube.
The one-step reaction described in this section uses the SuperScript'" III
Plafinum~ One-Step Quantitative RT-PCR System for superior specificity and sensitivity with LUX"' Primers.
Primer For optimal PCR, pximer titrations of 50--500 nM per primer are recommended.
Concentration! The 50-~1 sample reaction on page 16 uses 200 nM of each primer, equivalent to 1. g1 of a 10 ~IVI primer solution. See also the Important note below.
t ~ In one-step RT PCR, the reverse primer drives the xeverse ixanscription w, theN.p r reaction. We have found that doubling the concentration of the reverse primer "' from 200 nM to 400 nM can in some cases decrease the cycle threshold for detecting a given target concentration, and thus increase sensitivity. See . pages 3-4 fox guidance on primer design.
Template The target template for one-step teal-time RT PCR is RNA usually total SpeCificationS cellulax RNA or mRNA. The amount of template typically ranges from 1 pg to 100 ng per assay. The purity and integrity of the RNA have a dirrect impact on results. RNase and genomic DNA contamination are the most common problems, and purification methods should be designed to avoid these.
We recommend using the Micro-to-Midi Total RNA Puxification System (Catalog no 12183-018) or TRIzol~ reagent (Catalog no.15596-026) to isolate total RNA. High-quality total RNA can be purified from as little as 100 cells up r to 10' cells or 200 rng of tissue.
To isolate mRNA, we recommend using the FastTraclc~ 2.0 mRNA Isolation Kit (Catalog no. K1593-02).
EllZyhle The one-step RT PCR enzyme mix should be optimized for real-time PCR.
We Specifications recommend usiung the SuperScript"' III Platinum~ One-Step Quantitative RT
PCR System (Catalog nos.11732-020 and -088), which uses a SuperScript'" III
RT/Platinurn° Tag enzyme mix. It has been optimized for use in zeal-tune fluorescent PCR systems. See the sample reactions on pages 16-17.
Megtlesluto The optimal MgClz concentration for a given target/primer/polymerase Concentration combination can vary between 1 mM and 10 mM, but is usually in the range of 3 mM (see the sample xeaction on page 16).
dNTP The optimal concentration of dATP, dCTP, dGTP, and dTTP is 200 ~M each.
If COttce~ttt'ation dUTP is used in place of dTTP, its optimal concentration is X00 i.iM.
Continued on next page SUBSTITUTE SHEET (RULE 26) One-step Real-Time RT-PCR, continued lnStPUmellt LUX'" Primers are compatible with a wide variety of real-time PCR
Specifications instruments with various detection capabilities. See page 2 for a partial list of compatible instruments. A one-step real-time RT PCR protocol for instruments that use PCR tubes/plates is provided on page 16. A protocol for the LightCycle~ is provided on page 17. At a minimum, the instrument used to perform one-step real-time RT-PCR with LU?C"' Primers must be able to:
~ Detect fluorescence at each PCR cycle ~ Bxcite and detect FAM-labeled LUX"' Primers near their excitation/emission wavelengths of 490/520 nm, and/or ~ Bxcite and detect ]OE-labeled LUX"' Primers near their excitation/emission wavelengths of 520/550 nm Ittstl'umerit Please follow the manufacturers instructions for configuring your real-time Settings PCR instrument for use with LUX"' Primers. Note the following settings:
~ LUX"" Primers are compah'ble with standard melting curve analysis, if your instrument allows that option. Program your instrument accordingly.
~ The quencher setting on the instrument should reflect the fact that LUX'"
Primexs do not contain a quencher.
~ We recommend the use of ROX Reference Dye (Catalog no.12223-023) for normalization of well-to-well variation with instruments that are compatible with this option. Adjust your instrument settings accoxdingly_ Pxogram the instrument to perform cDNA synthesis irnrn, ediately followed by PCR amplification.
Removing We recommend that you decrease the genamic DNA content in the .RNA
GettOmiC DNA fPOlri sample by performing a digest with DNase I, Amplification Grade (Catalog RNA Samples no.18068-015), as described below. The DNase I digest is designed fox up to 1 ~,g of RNA; for larger amounts of RNA, increase volumes accordingly.
Combine the following in a tube on ice:
Component Conc. Volume RNA template - ~ x g1 DNase reaction buffer 10X , 1 g1 DNase I, Amplification Grade 1 1 ~1 U/ul DEPC-treated ddHzO to 10 ~.~1 1. Incubate at room temperature for 15 min.

2. Add 1 1.r1 of 25-mM EDTA solution to the reaction mixture and incubate at 65C for 10 min to inactivate the DNase I.

To verify the absence of genomic DNA iun the RNA sample, prepare a control reaction identical to the reactions on pages 16-17, using 2 U of Platinum~ Tag DNA polymerise (Catalog no.10966-018) in place of the SuperScript~° III
RT/Platinum~ Tag Mix.
Continued on next page SUBSTITUTE SHEET (RULE 26) One-Step Real-Time RT-PCR, Continued Protocol fob The following protocol using the SuperScript'" III Platinum~ One-Step ltlStl'tlmet7ts Using ~~~~dve RT PCR System has been optimized for LilX"' Prinners. Further PCR Tubes or op~a0on may be required.
Plates Note: Keep all components, reaction mixes and samples on ice. After assembly, transfer the reaction to a thermal cycler preheated to the cDNA
synthesis temperature and immediately begin RT PCR. We recommend performing the cDNA synthesis reaction at 50°C, but higher temperatures (up to 60°C) may be required for high GC content templates.
RNase inhibitor proteins, such as RNaseOLlT'" (Catalog no.10777-019), may be added to the reaction to safeguard against degradation of RNA.
1. The following table provides Master Mix volumes for a standard 50-~,~1 reaction size. Note that preparaiion of a master mix is crucial in quantitative applications to reduce pipetting errors.
Component Voll1 rxn Vol/100 rxns SuperScript''" III RT/Platinum° Tag Mix 1 ltl 100 Etl 2X Reaction Mixt 25 pl 2500 E.tl ROX Reference Dye (optional) 1 ul 100 Etl Labeled LU?C~' Primer (10 EtM} 1 ul 100 let Unlabeled primer (10 ttM)a 1 ul 100 E.tl RNaseOUT'" (optional) 1 itl 100 Id Sterile distilled water to 40 ul to 4000 pl ~SuppIied at 2X concentration includes 0.4 mM of each dNTP and 6 mM MgSO~
Seethe Important note on primer concentration on page 14.
2. Program the instrument with the following thermal cyclingprotocol (for cDNA synthesis, use a 15-min incubation at 50°C as a starting point):
cDNA synthesis:
54°C for 25 min hold PCR:
95°C for 2 min hold 40-50 cycles of:
95°C,15 s 60°C, 30 s Melting Curve Analysis (optional) Program according to instrument instructions 3. For each reaction, add 40 ~.l of the master mix to a 0.2-ml microcentrifuge tube or each well of a 96-well PCR plate on ice.
4. Add 10 ul of sample RNA (1 pg to 1 ttg total RNA) to each tube/plate well, and cap or seal.
5. Gently mix and make sure that au components axe at the bottom of the tube/plate wells. Centrifuge briefly if needed.
6. Place reactions in a preheated thermal cycler programmed as described above. Collect data and analyze results.
71 Continued on next page SUBSTITUTE SHEET (RULE 26) One-Step Reaf-Time RT-PCR, cont~ntaea PfotnCOl fof the The following protocol using the SuperScriptT" III Platinum~
One-Step RoChe LightCyClet~ ~~B~dve RT-PCR System has been optimized for LUX'" Primers and the Roche LighfCycler~. Further optimization may be required. FAM-labeled LUX'" Primers are also compatible with Roche enzyme mixes.
Note: ]OB-labeled primers are not compatible with the current version of the LightCyclex'~; use FAM labeled primers only.
After assembly, transfer the reaction to a thermal cycler preheated to the cDNA synthesis temperature and immediately begin RT-PCR. We recommend performing the cDNA synthesis reaction at 50°C, but higher temperatures (up to 60°C) may be required fox high GC content templates.
RNase inlu-bitor proteins, such as RNaseOUT"" (Catalog no.1OT77-019), may be added to the reaction to safeguard against iiegradation of RNA.
1. The following table provides Master Mix volumes for a standard 20,u1 reaction size. Note that preparation of a master mix is crucial in quantitative applications to reduce pipetting errors.
Component Vol 1 rxn Voll34 rxns SuperScript~' III RT/Platinum~ Taq Mix 0.8 E.t1 27.2 ttl 2X Reaction Mixl 10 ).1l 340 u) FAM labeled LUX"° Primer (10 p,M)2 1 N.1 34 it) Unlabeled primer (10 ~' 1 u) 34 NI
Bovine serum albumin (5 mg/mI)' 1 ~l 34 N.1 Sterile distilled water to 18 p) to 612 N.1 'Includes 0.4mM of each dIVTP and 6 mM MgSOa zIn the I,ightCycle~ reaction, the LUX'" Fiuorogenie Primer must be PAM
labeled.
35ee the Ianportant note on primer concentration on page I4.
"Validated with non-acetylated Ulbrapure BSA (10°I° solution) from Panvera (Cat.
pos. P2489 and P2046) 2. Set the fluorescence on the Roche LightCycler~ to the F1 channel.
3. Program the instrument as follows:
Thermal Cycling Melting Curvy Analysis (optional) Program chofce: Amplification Program choice: Melting curve Analysis mode: Quantification Analysis triode: Melting curves Cycling: Cycling:
45°C, 30 min hold (cDNA synthesis) 95°C, 0 s 95°C, 2 min hold 55°C,15 sec 50 cycles of: 95°G, 0 (increase 0.1°C/s with 95°C, 5 s continuous acquisition) 55°C,10 s (single acquire) 40°C, 0 s 72°C.10 s 4. Add 18 p) of Master Mix to each capillary tube of the LightCyclex'a on ice.
5. Add 2 NI of sample RNA (1 pg to 1 ttg total RNA) to each capillary tube and cap the tube.
6. Centrifuge the tubes at 700 x g fox 5 seconds.
7. Place the reaction tubes in the rotor of the LightCyclez'a and run the program. Collect and analyze results.

SUBSTITUTE SHEET (RULE 26) Troubleshooting Problem Cause Solution Signal in controls with no DNA contamination Hnsure that amplification reactions are assembled template in a DNA-free environment. Use of aerosol-resistant barrier tips is recommended. Take care to avoid cross-contamination between primers or template DNA in different reactions. Run PCR
product on an agarose gel in an area separate fxom the reaction assembly area to confirm product.
Amplification of PCR Analyze PCR product on an agarose gel in an area carryover products separate from the reaction assembly area.
Use Platinum~ Quantitative PCR SuperMix-UDG
as specified in the sample protocols on pages 9-10. Since dU'TP is substituted for dTTP in the reaction cocktail, any amplified DNA will contain uraal. UDG prevents rearnplification of PCR
carryover products by removing uracil residues from single or double stranded DNA. dU-containing DNA that has been digested with UDG
is unable to serve as template in future PCRs.
UDG is inactivated at high temperature during PCR thermal cyding, thereby allowing amplification of genuine target sequence(s).
Primer dimers Perform melting curve analysis of the PCR
product; identify dimers by lower melting point temperature. Confirm that primer designs have low scores (0.0-4.0) to minimize self annealing_ Redesign primers if necessary. When redesigning primers, note that you can first try redesigning only the unlabeled primer to save the cost of the LUX'" primer.
No or low signal Tnstruments setting not Confirm that the cycling parameters are correct, optimal the quencher is set to none, and the reference dye setting is correct.
Primer/template sequences do Confirm that the sequences match.
not match ' Primer designs are not optimal Confirm that the primer design scores are within the O.t~-4.0 xange and the optimal melting temperatures have been specified. Redesign primers if necessary. When redesigning primers, note that you can first try redesigning only the unlabeled primer to save the cost of the LUX"' primer.
Poor standard curve and $eaction is not optimized Reoptimize reacfion conditions. Prepare primer dynamic range titrations if necessary.
Reference dye not used Use ROX Reference Dye as specified.

SUBSTITUTE SHEET (RULE 26) Accessory Products P!'oduCts The following products are available for use with >a'CTX"' Primers in real-time PCR and RT-FCR protocols:
Product unt Cataloe no.

Platinum Quantitative PCR SuperMix-UDG100 rxns11730-017 500 rxns11730-025 SuperScript'" III First-Strand50 rxns 18080-051 Synthesis System for RT-PCR

Superscript'" III Platinum~ 100 rxns11732-020 Or,e-Step Quantitative RT 500 rxns11732-088 PCR System Platinum~ Taq DNA Polymerase 104 rxns10966-018 250 rxns10966-026 50Drxns 10966-034 5,000 10966-083 x~ms Micro-to-Midi Total RNA Purification50 rams 12183-018 System TRTzoh' Reagent 100 ml 15596-026 200xnI 15596-018 Micro-FastTrack'" 2.0 mRNA 20 rxns K1520-02 Isolation Kit ROX Reference Dye 500 Itl 12223-023 DNase I, AmpliC~cation Grade 100 U 18068-015 (1 Ul~.~t) RNaseOUT'" Recombinant Ribonuclease5,000 10777-019 Inhibitor U
(40 U/Ni) mM dNTP Mix 100 Ol 18427-013 DEPC-treated water 4 x 1.2510813-012 ml SUBSTITUTE SHEET (RULE 26) Purchaser Notificafiion limited Use The purchase of this product conveys to the Label buyer the non-transferable right to use the License NO. pm'~ased amount of the product and components 114: of the product in research LUXE P1UOPO~eIIiCconducted by the buyer (whether the buyer is an academic or for-profit entity). The Primer buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components fox commercial purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for the commercial purposes of the buyer, and that such collaborator agrees in . writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or informakion solely for research and not for commercial purposes. Commercial purposes means any activity by a party for consideration and may include, but is not limited to: (I) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or ifs components for therapeutic, diagnosiac or prophylactic purposes; or (4) resale of the product or its components, whether ox riot such product or its components are resold for use in research. ' Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogenbased upon the manufacture, use ox sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, lnvifrogen is wDling to accept return of the products with a full refund. For information on purchasing a license to this product for purposes other than researeh, contact Licensing Department,1600 Faraday Avenue, Carlsbad, California 92008.

Phone (760) 603-7200. Fax (760) 602-6500.

Limited Use Label 'rlus product is optimized for use in the Polymerase Chain Reaction (PCR) covered by LICSf7S~ P10. 4: patents owned by Roche Molecular Systems, Inc. and P.
Hoffmann-La Roche, Ltd.
PrOdUCtS fOr PCR (~~Roche"). No license under these patents to use the PCR
process is conveyed expressly which do not or by implication to the purchaser by the pur~ase of this product. A license to use the include an r1 h~ PCR process for certain research and development activities accompanies the purchase Y J of certain reagents From licensed suppliers such as Invitrogen, when used in t0 perform PCR conjunction with an Authorized Thermal Cycler, or is available from Applied Biosystems. Further information on purchasing licenses to practice the PCR
process may be obtained by contacting the Director of Incensing at Applied Biosystems, Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, A3ameda, California 94501.
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SUBSTITUTE SHEET (RULE 26) References Ailenberg, M., and Silverman, M. (2000) Controlled hot start and improved speaficity in carrying out PCR utilizing touch-up and Loop incorporated primers (TULIPS). BioT'echnigues 29,1018-1024.
Bustin, S. A. (2000) Absolute quantification of mRNA using real-lime reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25,169-193.
Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C., and W olf, D. E.
(2988) Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc.
Natl. Acad. Sci. USA 85, 8790-8794.
Crockett, A.~., and Wittsver, C.T. (2001) Fluorescein-labeled oligonucleofides for real-time pcr: using the inherent quenching of deoxyguanosine nucleotides. Anal. Biochem. 290, 89-97.
Higuchi, R., Focklei, C., Walsh, P.S., and Griffith, R (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology .20, 413-417.
Higurhi, R., Fockler, C., Dollinger, G., and Watson, R. (1993) Kinetic PCR
analysis: real-time monitoring of DNA amplification reactions. Biotechnology I2,1026--1030.
Holland et al. (1991) Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natt. Acad.
Sci. LISA 88, 7276-7280.
Kaboev, O. K., Luchkina, L. A., Tret'iakov, A. N., and Hahrmand, A.R. (2000) PCR hot start using primers with the structure of molecular beacons (hairpin-like structure).
Nucleic Acids Res. 28, e94.
Knemeyer, j.P., Marme, N., and Sauer, M. (2000) Probes for detection of specific DNA sequences at the single-molecule Level. Anal. Chem. 72, 3717 3724.
Murchie, A. I. H., Clegg, R. M., von Kitzing, E., Duckkett, D. R.,1?iektnann, S., and Lilley D. M. J. (1989) Fluorescence energy transfer shows that the four-way DNA junction is a right handed cross of antiparallel molecules. Nature 341, 763-766.
Myakishev. M.V., Kluipin, Y., Hu, S., and Hamer, D. H. (2001) High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers. Genorile Res.11,163--169.
Nazaxenko, L, Lowe, 8., Darfler, M., Ikonomi, P-, Schuster, D., and Rashtchian, A. (2002} Multiplex quantitative PCR using self quenched primers labeled with a single fluorophore. Nucl. Acids Res.
30, e37 Nazarenko, L, Pixes, R., Love, B., Obaidy, M., and Rashtchian, A. (2002) Effect of primary and secondary structure of oligodeoxyribonudeotides on the fluorescent properties of conjugated dyes.
Nucl. Acids Res. 30, 2089-2095 Nazarenko, LA., Bhatnagar, S.K., and Hohman, R.J. (1997) A closed tube format for amplification and detection of DNA based on energy transfer, Nucleic Acids Res. 25, 2516-2521.
Nuovo, G. J., Hohman, R. j., Nardone, G. A., and Nazarenko I. (1999) In situ amplification using universal energy transfer-labeled primers. J. Histochem. Cytochem. 47, 273-279.
Todd, A. V., Fuery, C. J., Impey, H. L., Applegate, T. L. and Haughton, M.A.
(2000) DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format Clin.
Chem. 46, 625rd30.
Tyagi, S., and Kramer, F.R. (1996) Molecular beacons: probes that fluoresce upon hybridization. Nature Biofechnol.14, 303-308.
Wittwer, C.T., Hemnann, M.G., Moss, A.A., Rasmussen, R.P. (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. HioTechnigues 22,130-138.
~2002 2003 Invitrogen Corporation. All rights reserved.

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Claims

1. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled-temperature reaction mixture including the target sequence, ligatable first and second primers having' at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a.
random sequence segment of the first and second primers, wherein the improvement comprises:
homogeneously detecting amplified target sequence using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.

2. An improved assay according to claim 1 wherein a nucleotide complementary to the SNP of the target sequence is present at the 5'-end of the second primer.

3. An improved assay according to claim 1, wherein the, dye.comprises SYBR® Green.

4. An improved assay according to claim 1, wherein the assay further comprises:
using a first primer and a second primer at concentrations such that a ligated product produces exponentially amplified target sequence detectable above linearly amplified non-ligated primer product.

5. An improved assay according to claim 1, wherein the assay further comprises:
using a plurality of first primers and second primers designed to generate amplified target sequences with differential melting curves;
distinguishing individual amplified target sequences by differences in melting temperatures (T m s).

6. An improved assay according to claim 1, wherein the first and second primers contain degenerate base-pairing positions to allow hybridization to variable regions in target sequences adjacent to the SNP.

7. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a temperature-controllable reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises:
-- -- detecting amplified target sequence using a probe specific for hybridizing across a ligation junction formed between the first primer and second primer after binding to the target sequence wherein the probe specific for hybridizing across the ligation junction contains a molecular beacon.

8. An improved assay according to claim 7, wherein the probe specific for hybridizing across the ligation junction has a fluorescent group and a fluorescence-modifying group.

9. An improved assay according to claim 8, wherein the fluorescent group is quenched when the probe is not bound across the ligation junction and the fluorescent group fluoresces when the probe is bound across the ligation junction.

10. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a temperature-controllable reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises:
detecting amplified target sequence using a probe specific for hybridizing to a region of the target sequence wherein the probe contains a fluorescent group and a fluorescence-modifying group.

11. An improved assay according to claim 10, wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces.

12. An improved assay according to claim 7 or 10, wherein the fluorescent group is quenched before incorporation into double-stranded product and is dequenched after incorporation into double-stranded product.

13. An improved assay according to claim 12, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the .probe containing the fluorescent,group, quenching the fluorescent group.

14. A nanoliter sampling array comprising:
a) a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes;
wherein each through-hole contains i) a first primer having at least a portion of its 3'-end substantially complementary to a first segment at a first end of a potential nucleic acid target sequence; and ii) a second primer having at least a portion of its 5'-end substantially complementary to a second segment at a second end of the potential nucleic acid target sequence, the first and second primers being ligatable upon binding to the potential nucleic acid target sequence.

15. A nanoliter sampling array according to claim 14, further comprising:
a second platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes;
wherein the first and second platen are fixedly coupled such that the through-holes of each are aligned.

16. A nanoliter sampling array according to claim 14, wherein at least one pair of aligned through-holes contains first reagents for a first assay process and second reagents for a second assay process.

17. An array according to claim 16, wherein one of the assay processes is PCR
amplification.

18. An array according to claim 16, wherein one of the assay processes is detection of amplified target nucleic acid sequence having a SNP.

19. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.

20. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises distinguishing individual amplified target sequences by differences in melting temperatures (T m s).

21. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises using a probe specific for hybridizing across a ligation junction formed between the first primer and second primer after binding to the target sequence, wherein the probe has a fluorescent group and a fluorescence-modifying group.

22. An array according to claim 18, wherein detection of amplified target nucleic acid sequence comprises using a probe containing a fluorescent group and a fluorescence-modifying group specific for hybridizing to a region of the target sequence wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces.

23. An array according to claim 22, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

24. An array according to claim 23, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

25. A nanoliter sampling array according to any of claims 14-24, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.

26. A method of identifying a SNP in a target sequence of nucleic acid, the method comprising:
providing a first sample platen having a high-density microfluidic array of through-holes, each through-hole having a first primer having at least a portion substantially complementary to a first segment of the target sequence, a second primer having at least a portion substantially complementary to a second segment of the target sequence, the 5'-end of the second primer ligatable to the 3'-end of the first primer after binding nucleic acid target sequence, and a third primer that is substantially complementary to a random sequence segment of the first and second primers;
introducing a sample containing a target sequence of nucleic acid having a SNP
of interest to the through-holes in the array;
introducing reagents to the through-holes in the array, the reagents including a reagent for effecting amplification, a reagent fox effecting ligation, and at least four different nucleotide bases;
effecting ligation of the first and second primers to produce a ligated product;
effecting amplification of the ligated product and target sequence;
detecting amplified target sequence.

27. A method of identifying a SNP in a target sequence of nucleic acid according to claim 26, wherein effecting ligation and effecting amplification comprises addition of a ligase and a pdlymerase followed by subjecting the array to controlled-temperature conditions.

28. A method according to claim 26 wherein detecting comprises using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding target sequence.

29. A method according to claim 26 wherein detecting comprises distinguishing individual amplified target sequences by differences in melting temperatures (T m s).

30. A method according to claim 26 wherein detecting comprises using a probe specific for hybridizing across a ligation junction formed between the first primer and second primer after binding to the target sequence, wherein the probe has a fluorescent group and a fluorescence-modifying group.

31. A method according to claim 26 wherein detecting comprises using a probe containing a fluorescent group and a fluorescence-modifying group specific for hybridizing to a region of the target sequence wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces.

32. An improved assay according to claim 30, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

33. A method according to claim 32, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

34. A kit for use in identification of amplified target nucleic acid sequences, the kit comprising:
a) a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes;
wherein each sample platen through-hole contains at least i) a first primer having at least a portion substantially complementary to a first segment of potential nucleic acid target sequence;
ii) a second primer having at least a portion substantially complementary to a second segment of the potential nucleic acid target sequence, the first and second primers ligatable upon binding to the potential nucleic acid target sequence;
b) a reagent platen having a high-density microfluidic array of through-holes, each reagent platen through-hole containing at least i) a third primer that is substantially complementary to a random sequence segment of the first and second primers;
ii) at least four different nucleotide bases;
iii) a reagent for effecting ligation; and iv) a fluorescent dye the reagent platen having a structural geometry that corresponds to the sample platen allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen.

35. A kit for use in identification of amplified target nucleic acid sequences according to claim 34, wherein a PCR-compatible buffer is also included.

36. A kit according to claim 34, wherein the fluorescent dye comprises SYBR® Green I, SYBR® Green II, YOYO®-1, TOTO®-1, POPO®-3, or ethidium bromide.

37. A kit according to any of claims 34-36, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.

38. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled-temperature reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, wherein the improvement comprises:

detecting one or more amplified target sequences in a single-tube reaction system using one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences, wherein the one or more probes each contain a distinct fluorescent group and a fluorescence-modifying group and wherein hybridization of the one or more probes results in fluorescence of the distinct fluorescent group.

39. An improved assay according to claim 38, wherein upon extension of the probe, the fluorescence-modifying group is excised and the fluorescent group fluoresces.

40. An improved assay according to claim 38, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

41. An improved assay according to claim 40, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

42. An improved assay according to claim 38, wherein the one or more target nucleic acid sequences is 2, each having a distinct SNP of interest.

43. An improved assay according to claim 42, wherein the one or more hybridizable probes is 2, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 2 target nucleic acid sequences.

44. An improved assay according to claim 38, wherein the one or more target nucleic acid sequences is 3, each having a distinct SNP of interest.

45. An improved assay according to claim 44, wherein the one or more hybridizable probes is 3, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 3 target nucleic acid sequences.

46. An improved assay according to claim 38, wherein the one or more target nucleic acid sequences is 4, each having a distinct SNP of interest.

47. An improved assay according to claim 46, wherein the one or more hybridizable probes is 4, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 4 target nucleic acid sequences.

48. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, the assay being a selective ligation and amplification method of the type using a controlled-temperature reaction mixture including the target sequence, ligatable first and second primers having at least a portion substantially complementary to first and second segments of the target sequence, respectively, and a third primer that is substantially complementary to a random sequence segment of the first and second primers, Wherein the improvement comprises:

detecting one or more amplified target sequences in a single-tube reaction system using one or more fourth primers, each having a fluorescent group and a fluorescent-modifying group, and each being complementary to a unique region of a ligated template for the one or more target nucleic acid sequences, wherein upon fourth primer incorporation into and amplification of the one or more target nucleic acid sequences, fluorescence of the distinct fluorescent group occurs such that detection of one or more amplified target nucleic acid sequences in a single-tube reaction system results.

49. An improved assay according to any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 5' to 3' exonuclease activity.

50. An improved assay according to any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 3' to 5' exonuclease activity.

51. An improved assay according to any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.

52. An improved assay according to any of claims 7, 10, 38 or 48, wherein the distinct fluorescent groups comprise Redmond Red TM, Yakima Yellow TM, and the fluorescence-modifying group comprises an Eclipse TM non-fluorescent quencher, dabcyl, or other fluorescent-quenching molecule.

53. An improved assay according to claim 48, wherein the one or more target nucleic acid sequences is 2, each having a distinct SNP of interest.

54. An improved assay according to claim 53, wherein the one or more fourth primers is 2, each having a distinct fluorophore and unique sequence that incorporates into and amplifies each of the 2 target nucleic acid sequences.

55. An improved assay according to claim 48, wherein the one or more target nucleic acid sequences is 3, each having a distinct SNP of interest.

56. An improved assay according to claim 55, wherein the one or more fourth primers is 3, each having a distinct fluorophore and unique sequence that incorporates into and amplifies each of the 3 target nucleic acid sequences.

57. An improved assay according to claim 48, wherein the one or more target nucleic acid sequences is 4, each having a distinct SNP of interest.

58. An improved assay according to claim 57, wherein the one or more fourth primers is 4, each having a distinct fluorophore and unique sequence that incorporates into and amplifies each of the 4 target nucleic acid sequences.

59. A nanoliter sampling array comprising:
a) a first platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes;
wherein each first platen through-hole contains at least i) one or more first primers, each having at least a portion substantially complementary to a first segment of one or more target nucleic acid sequences;
and ii) a second primer having at least a portion substantially complementary to a second segment of the one or more target nucleic acid sequences, the first and second primers being ligatable upon binding to the one or more target nucleic acid sequences.

60. A nanoliter sampling array according to claim 59, further comprising:
a second platen having at least one hydrophobic surface and having a high-density microfluidic array of hydrophilic second platen through-holes;
wherein the first and second platen are fixedly coupled such that the through-holes of each are aligned.

61. A nanoliter sampling array according to claim 59, wherein at least one pair of aligned through-holes contains at least first reagents for a first assay process and second reagents for a second assay process.

62. An array according to claim 61, wherein one of the assay processes is PCR
amplification.

63. An array according to claim 61, wherein one of the assay processes is detection of one or more amplified target nucleic acid sequences, each having a SNP.

64. An array according to claim 63, wherein detection of one or more amplified target nucleic acid sequences comprises using one or more probes specific for hybridizing to a region of each of the one or more target sequences, each probe containing a distinct fluorescent group and a fluorescence-modifying group, wherein upon extension of the one or more probes into one or more amplified target nucleic acid sequences, each of the distinct fluorescence-modifying groups is excised and the distinct fluorescent group fluoresces.

65. An array according to claim 63, wherein detection of one or more amplified target nucleic acid sequences comprises using one or more probes specific for hybridizing to a region of each of the one or more target sequences, each probe containing a distinct fluorescent group and a fluorescence-modifying group, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

66. An array according to claim 65, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

67. A nanoliter sampling array according to any of claims 59-66, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.

68. A method of identifying one or more SNPs in one or more target nucleic acid sequences, the method comprising:
providing a first sample platen having a high-density microfluidic array of through-holes, each sample platen through-hole containing at least one or more first primers, each first primer having at least a portion substantially complementary to a first segment of the one or more target nucleic acid sequences, a second primer having at least a portion substantially complementary to a second segment of the target sequences, the 5'-end of the second primer ligatable to the 3'-end of the first primer after binding to the one or more target nucleic acid sequences, and a third primer that is substantially complementary to a random sequence segment of the second primer;
introducing a sample containing one or more target sequences of nucleic acid, each having a SNP of interest, to the sample platen through-holes in the array;
introducing reagents to the sample platen through-holes in the array, the reagents including a reagent far effecting amplification, a reagent for effecting ligation, and at least four different nucleotide bases;

effecting ligation of the first and second primers to produce a ligated product;
effecting amplification of the ligated product and one or more target sequences;
and detecting one or more amplified target sequences.

69. A method of identifying a SNP in a target sequence of nucleic acid according to claim 68, wherein effecting ligation and effecting amplification comprises addition of a ligase and a polymerase followed by subjecting the array to controlled-temperature conditions.

70. A method of identifying one or more SNPs according to claim 68, further comprising, before introducing reagents to the sample platen through-holes in the array:
introducing a sample containing one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences and amplifying the one or more target sequences, wherein the one or more probes each contain a distinct fluorescent group and a fluorescence-modifying group.

71. A method of identifying one or more SNPs according to claim 70, wherein upon extension of the one or more probes into one or more amplified target nucleic acid sequences, each of the distinct fluorescence-modifying groups is excised and the distinct fluorescent group fluoresces.

72. An method of identifying one or more SNPs according to claim 70, wherein the fluorescent group is quenched before incorporation into double-strand product and is dequenched after incorporation into double-stranded product.

73. An method for identifying one or more SNPs according to claim 72, wherein the fluorescent group is quenched by secondary structure before incorporation into double-stranded product, such that before incorporation, a sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group, quenching the fluorescent group.

74. A method according to claim 71, wherein identifying one or more SNPs in one or more target nucleic acid sequences comprises monitoring differential fluorescence of the one or more distinct fluorescent groups incorporated into the one or more amplified target nucleic acid sequences.

75. A method of identifying one or more SNPs in one or more target sequences of nucleic acid according to claim 68, wherein the polymerase lacks 5' to 3' exonuclease activity.

76. A method of identifying one or more SNPs in a target sequence of nucleic acid according to claim 68, further comprising using a polymerase that lacks 3' to 5' exonuclease activity.

77. A method of identifying one or more SNPs in one or more target nucleic acid sequences according to claim 68, further comprising using a polymerase that lacks 5' to 3' exonuclease activity and 3' to 5' exonuclease activity.

78. A method according to claim 70, wherein the one or more target nucleic acid sequences is 2, each having a distinct SNP of interest.

79. A method according to claim 78, wherein the one or more hybridizable probes is 2, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 2 target nucleic acid sequences.

80. A method according to claim 70, wherein the one or more target nucleic acid sequences is 3, each having a distinct SNP of interest.

81. A method according to claim 80, wherein the one or more hybridizable probes is 3, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 3 target nucleic acid sequences.

82. An improved assay according to claim 70, wherein the one or more target nucleic acid sequences is 4, each having a distinct SNP of interest.

83. An improved assay according to claim 82, wherein the one or more hybridizable probes is 4, each having a distinct fluorophore and unique sequence that hybridizes to and amplifies each of the 4 target nucleic acid sequences.

84. A kit for use in identification of one or more amplified target nucleic acid sequences, the kit comprising:
a) a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes;
wherein each sample platen through-hole contains at least i) one or more first primers, each first primer having at least a portion substantially complementary to a first segment of one or more target nucleic acid sequences;
ii) a second primer having at least a portion substantially complementary to a second segment of the one or more target nucleic acid sequences, the 3'-end of the one or more first primers ligatable to the 5'-end of the second primer after binding to the one or more target nucleic acid sequences;
b) a reagent platen having a high-density microfluidic array of through-holes, each reagent platen through-hole containing at least i) a third primer that is substantially complementary to a random sequence segment of the second primer;
ii) one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences and amplifying the one or more target sequences, wherein the one or more probes each contain a distinct fluorescent group and a fluorescence-modifying group;
iii) four different nucleotide bases;
iv) a ligase; and the reagent platen having a structural geometry that corresponds to the sample platen allowing delivery of reagent components and target nucleic acid sample to the primers in the sample platen.

85. A kit for use in identification of amplified target nucleic acid sequences according to claim 84, wherein a PCR-compatible buffer is also included.

86. A kit according to claim 84 or 85, wherein the primers are affixed on, within or under a coating of the sample through-holes by drying, the coating comprising a biocompatible material.