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  • B01J2219/00718—Type of compounds synthesised
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  • B01J2219/00722—Nucleotides
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  • C12Q2600/00—Oligonucleotides characterized by their use
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  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
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  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

• 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.
• 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 carried 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.
• 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
• 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 target nucleic acid product.
• 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 or within two to four bases of 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
• 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.
• 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.
• the hybridizable probe Upon incorporation of the hybridizable probe into amplified product, fluorescence occurs, making detection of the amplified product distinguishable from non-specific background products.
• 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.
• 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.
• 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
• 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.
• 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.
• PEG polyethylene glycol
• thermostable polymerase that lacks 5' to 3' exonuclease activity, or a thermostable polymerase that lacks 3' to 5' exonuclease activity, or a thermostable polymerase that lacks both 5' to 3' and 3' to 5' exonuclease activity.
• thermostable polymerases which lack 5' to 3' exonuclease activity include Stoffel fragment, IsisTM DNA polymerase, PyraTM exo(-) DNA polymerase, and Q-BioTaqTM DNA polymerase.
• An example of a thermostable polymerase which lacks both 5' to 3' and 3' to 5' exonuclease activity is Q-BioTaqTM DNA polymerase.
• Suitable dyes include SYBR ® Green I and SYBR ® Green ⁇ , YOYO ® -l , TOTO ® -l , POPO ® -3, ethidium bromide, or any other dye that allows rapid, sensitive detection of amplified target nucleic acid sequence using fluorescence.
• 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.
• 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.
• 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.
• a method of identifying a SNP in a target sequence of nucleic acid 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
• 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).
• 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 selective and so selective amplification of the desired target sequence containing the SNP of interest also occurs.
• 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.
• 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.
• 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 (T m s), or may comprise 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 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 the fluorescent group fluoresces.
• 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 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.
• 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.
• 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.
• FRET Fluorescence Resonance Energy Transfter
• Yet another embodiment provides a kit for use in identification of amplified target nucleic acid sequences, the kit comprising a sample platen having one hydrophobic surface and having a high-density microfluidic array of hydrophilic through-holes.
• 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.
• 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.
• the thermostable polymerase may lack 5' to 3' exonuclease activity.
• Fig. 1-A shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP).
• 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-A shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP).
• 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.
• RS random sequence
• 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 PI.
• Fig. 2-A shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP).
• SNP single nucleotide polymorphism
• 2-B shows primers PI 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 PI, and each of primers PI and P2 contain a random sequence at their 5 '-end and 3 '-end, respectively.
• Fig. 2-C shows ligated P1-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
• Figs. 2-E1 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.
• 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.
• 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 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 fluorophore 2 in a second amplified product.
• 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 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. 5B 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-
• Fig. 5C shows extension of ligation products A-B-C-D and F-E-C-D by general extension primer D'.
• Fig. 5D 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. 5E 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.
• Target nucleic acid 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.
• 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 ohgonucleotide 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.
• 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.
• 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 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.
• oligonucleotide primer which is most complementary to 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 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.
• 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.
• four different nucleotide bases means deoxythymidine triphosphate (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.
• dTTP deoxythymidine triphosphate
• dATP deoxyadenosine triphosphate
• dCTP deoxycytidine triphosphate
• dGTP deoxyguanosine triphosphate
• dUTP deoxyinosine triphosphate
• rITP riboinosine triphosphate
• any other modified base 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.
• dATP, dCTP, dGTP and dTTP deoxynucleoside triphosphates
• ATP deoxynucleoside triphosphates
• 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.
• random sequence as used herein means a sequence unrelated to the 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.
• 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.
• first and second primers being ligatable upon binding to the nucleic acid target sequence 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.
• polymerase as used herein, means any oligomer synthesizing enzyme, including polymerases, helicases, and other protein fragments capable of polymerizing the synthesis of oligomers.
• controlled-temperature reaction mixture 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- 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 (PI, P2, P3) or more (A-B, C-D, F-E, D') primers, and is able to detect one or more SNPs in a single target simultaneously.
• the nucleotide complementary to the SNP of the target nucleotide is present at or near the 5'-end of the second primer P2.
• the nucleotide complementary to the SNP of the target nucleotide is present at or near the 3'- end of the first primer PI .
• there are more than one first primers and 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, T m s.
• Assays may be done with first and second primers that contain degenerate base- pairing positions which allow hybridization of variable regions in target sequences 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 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 (PI) or the 5' end (P2) to enable the primers to bind to SNPS in a target sequence (see Figs. 1-2).
• 5'-end of P2 can be modified to prevent undesirable ligation to fragments other than PI.
• the 5'-end of PI is phosphorylated to facilitate ligation with P2, and the
• 3' end of PI 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 PI 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.
• Still other detection strategies employ the use of FRET pairs on hybridizable probes.
• the fluorescent dye is merely added to the reaction mixture, and change in fluorescence intensity is monitored to detect ligated product.
• 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).
• one or more hybridizable probes may be used, each having a distinct fluorophore and unique 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 waxlike 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 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 sequence(s) 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 the target nucleic acid sequence(s).
• Homogeneous detection of amplified target sequences may be carried out using a dye specific for binding to double-stranded DNA or RNA.
• Primers PI 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.
• primer PI or P2 contains a match to the SNP of interest in the target sequence, ligation of PI and P2 occurs, and then primer P3, the general extension primer, amplifies the P1-P2 product.
• 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.
• ds-DNA- and/or RNA-specific dyes include SYBR ® Green I and SYBR ® Green fl, YOYO ® -l, TOTO ® -l, 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.
• a sample target sequence of nucleic acid 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.
• thermostable polymerase preferably one that lacks 5' to 3' exonuclease activity, such as the Stoffel Fragment (see Appendix B, attached hereto).
• thermostable polymerases which lack 5' to 3' exonuclease activity include IsisTM DNA polymerase, PyraTM exo(-) DNA polymerase, and Q-BioTaq DNA polymerase (see Appendix C, attached hereto).
• the assay may use a thermostable polymerase that lacks 3' to 5' exonuclease activity, or a thermostable polymerase that lacks both 5' to 3' and 3' to 5' exonuclease activity.
• thermostable polymerases which lack 3' to 5' exonuclease activity include Taq polymerase, SurePrimeTM Polymerase, and Q-BioTaqTM DNA polymerase (id.).
• An example of a thermostable polymerase which lacks both 5' to 3' and 3' to 5' exonuclease activity is Q-BioTaq DNA polymerase (id.).
• a target nucleic acid may contain a SNP within the target sequence.
• Primer 1 (PI) and Primer 2 (P2) bind to the 3' to 5' strand of the target sequence, adjacent to the SNP.
• the base complementary to the SNP of the target sequence is at the 3 '-end of PI.
• the base complementary to the SNP of the target sequence may be at the 5'-end of P2, as shown in Fig. 1-B2.
• RS random sequence
• 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.
• concentrations of primers PI and P2 are adjusted to minimize the competing linear reaction.
• un-ligated primer PI extends the 3' - 5' strand of the target sequence.
• the first primer (PI) also has a random sequence at the 5'-end.
• primers PI and P2 are ligated, and the third primer (P3) then binds to the 3'-end of the ligated P1-P2 product and produces the (3' to 5') P3-amplified strand (Fig. 2-C).
• 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).
• detection with a fluorescent dye such as SYBR ® Green I (SGI) may be done at temperatures above the T m of the linear product, i.e., any product produced non-exponentially, thereby removing competing signal from any dye 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 T ra s because at those elevated temperatures, the nucleic acids are denatured.
• a dye such as SYBR ® Green I binds to double-stranded amplified target nucleic acid with a concomitant large increase in fluorescence.
• 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.
• molecular beacon probes having a fluorescent group on one end and a fluorescence-quenching group on the other, may be used.
• 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).
• 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.
• 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.
• 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).
• 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).
• 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.
• 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.
• fluorescence detection would be carried out above the either the Tm for primer PI, or above the T m for primer P2, or alternatively be carried out above the T m s of both primers PI and P2, to avoid background signal from possible hybridization of PI and/or P2 to amplified target.
• primer may be designed to exponentially amplify target nucleic acid products that are distinguishable by an increase or decrease in melting temperature (T m ), wherein the exponentially amplified target sequence is either stabilized as indicated by an increase in T m or de-stabilized, as indicated by a decrease in T m , 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.
• 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.
• 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 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.
• 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.
• 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.
• upstream primers A-B and/or F-E 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'.
• 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.
• 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.
• FIG. 5A-5F 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.
• either the 3'-end of the upstream primers may contain the complement to the SNP of interest in the target nucleic acids
• the 5'-end of the downstream primer may contain the complement to the SNP of interest in 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.
• 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.
• 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).
• 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 primers being used to generate the C-X product to be ligated.
• 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.
• 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'.
• 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) 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 nanoliter sampling array 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, 2004, both of which are hereby incorporated by reference herein, is one preferred array.
• 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 1 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.
• 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.
• the target area of the receptacle interior walls 42 may have a hydrophilic surface that attracts a liquid sample.
• the sample through-holes 12 may contain a porous hydrophilic materiel that attracts a liquid sample.
• 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.
• 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.
• 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, PI 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 PI and P2 are further designed such that the base complementary to the SNP in the target sequence is either on the 3 '-end of PI, 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.
• the 5'-e ⁇ d 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 capable of amplifying ligated primer P1-P2 product, a thermostable polymerase, a 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.
• 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.
• 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 ® -l, TOTO ® -l, POPO ® -3, EtBr, and any other dye capable of providing low-sensitivity detection of amplified target sequence by fluorescence emission.
• 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.
• detection may involve the use of a probe 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.
• the fluorescence-modifying group is excised upon extension of the probe, and the fluorescent group thus fluoresces, allowing detection of amplified product.
• kits 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.
• 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 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 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.
• 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.
• 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.
• PEG polyethylene glycol
• 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 Structure, DNA Binding and DNA Damage Nucleic Acid Conformatio ⁇ al Analysis
• 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 histochemistry 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-1374 ethidium monoazide
• Fluorescence of the TOTO-1, YOYO-1, BOBO-1 and POPO-1 dyes (Table 8.2, Pimeric 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 ratlos. ⁇ & Most of our unsymmetrical cyanine dyes show this spectral shift, and some show sequence selectivity in their fluorescence Intensity as well.
• 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 (131, "El).
• the YOYO-1 and POPO-3 dyes (Y-3601, P-3584) dyes have also been used to follow the making and breaking of single chemical bonds.
• ⁇ IS A number of laboratories have taken advantage of the high sensitivity of these dyes to detect single nucleic acid molecules and to study biopolymer behavior: • Video microscopy has been used to observe relaxation of YOYO-1 dye-stained phage lambda DNA multlmers, after stretching in a fluid flovt,0t on a surface ⁇ f& or with optical tweezers.
• the POPO-3 dye (P-3584 * ) has been used to study a single chemical reaction w!th,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 m ⁇ croscopy.* ⁇ t • The TOTO-1 (T-3600), YOYO-1 fY-360 * ), POPO-3 fP-3584) and SYBR Green I (S-7563, S ⁇ 7567, S-7585) dyes have been used to visualize lambda DNA that has been stretched between beads with optical tweezers.* ⁇ ',-*® • Fragment sizing on single molecules of dsDNA stained with our PicoGreen reagent has also been reported.
• the SYTOX Orange dye (S- 1368) is the preferred dye for single-molecule sizing of DNA fragments by flow cytometry in an Instrument equipped with a Nd:YAG laser.id ⁇ fr • DAPI (P-1306, P-3571; FluoroPure Grade, D-21490) has also been employed to detect a single DNA molecule in solution ⁇ &t and by fluorescence microscopy ⁇ $_? ⁇ and to detect femtogra s of DNA in single cells and chloroplasts.*lEEr
• SYBR Green I nucleic acid gel stain S-7567, S-7563, S-7585: SYBR Green I Nucleic Acid Gel Stain
• SYBR Gold nucleic acid gel stain S- 11494, "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 bandshlft assays easy and more convenient with our Electrophoretic Mobility-Shift Assay (EMSA) Kit (E-33075).
• EMSA Kit provides a fast and quantitative fluorescence-based method to detect both nucleic acid and protein in the same gel (fiS), doubling the information that can be obtained from bandshift assays.
• This kit uses two fluorescent dyes for detection — SYBR Green EMSA.nu leic acid gel stain for RNA or DNA and SYPRO Ruby EMSA protein gel stain for proteins.
• 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 ⁇ 1 ng for nucleic acids and ⁇ 20 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 (.£1). Digital images can easily be overlaid for a two-color representation of nucleic acid and protein in the gel.
• the EMSA Kit contains sufficient reagents for 10 nondenaturing polyacrylamide minigel assays, including: • SYBR Green EMSA nucleic acid gel stain • SYPRO Ruby EMSA protein gel stain • Trlchloroacetic acid, for preparing the working solution of SYPRO Ruby EMSA protein gel stain • Concentrated EMSA gel-loading solution • lac repressor, a DNA-binding protein to be used as a control • lac operator, control DNA • Concentrated buffer for the lac represso ⁇ operator controls • A detailed protocol (Electrophoretic Mobility Shift Assay (EMSA) Kit)
• Fluorescent dyes have also been used to stain the DNA fragments or proteins before electrophoresis.
• proteins or DNA labeled covalently with a reactive fluorescent dye (Chapter 1, Section 8.2) can be easily tracked during capillary electrophoresis to monitor DNA- protein Interactions.
• a reactive fluorescent dye Chosterin-1
• High-affinity 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 ethidium homodimer-1 (EthD-1, E-1169? Section 8.1), YOYO-1 and TOTO-1 dyes have been shown by several laboratories to be useful tools for labeling DNA prior to electrophoresis in bandshlft assays.
• EthD-1 and TOTO-1 were used to examine interactions between the binding domain of the Kluyveromyces lactis heat shock transcription factor and its specific binding site.
• S YOYO-l dye has been used to study the association of £ coif RNA polymerase with DNA templates d ⁇ r and the binding of a heat-shock transcription factor to its promoter.
• *-® All ten of our spectrally distinct ( Figure 8.1), high-affinity dimeric cyanine dyes (Table 8.2) and the ethidium h ⁇ modimers are potentially useful for multlcomponent analysis in this application.
• Molecular 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. *f ⁇ t The assay is sufficiently accurate to "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 o_-CP 2 , which is part of an RNA-binding complex.*®*-
• the thlol-reactive iodoacetamide of 1,10-phenanthrollne (P-6879, Section 2.3) is a useful adjunct reagent for bandshift assays. Conjugation to thiol-containlng llgands confers the rnetaJ-binding properties of this important complexing agent on the Itgand.
• the covalent copper- phenanthrollne complex of ollgonucleotides or nucleic acid-binding molecules in combination with hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or R A.
• This reagent can also be conjugated to proteins to detect nucleic acid binding and targeted cleavage.
• the comet assay or single-cell gel electrophoresis assay — is used for rapid detection and quantitation of DNA damage from single cells.* ⁇ The comet assay is based on the alkaline lysis of labile DNA at sites of damage. Cells are Immobilized in a thin agarose matrix on slides and gently lysed. 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 tails 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, ⁇ S monitor the sensitivity of tumor cells to radiation damage * " S8- and to assess the sensitivity of molluscan cells to toxins in the environment. ⁇ S ⁇ The comet assay can also be used in combination with FISH to identify specific sequences with damaged D A.*®
• terminal deoxynucleotidyl transferase 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 more 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-7614) to visualize DNA fragment ends are four times more sensitive than TUNEL assays using fluoresceln-labeled dUTP S* (fil).
• Terminal deoxynucleotidyl transferase (TdT)-catalyzed Incorp ' oratlon of bromo dUTP into nucleic acids of apoptotlc cells and detection of the incorporated BrdU with an antibody conjugate Is the basis of the APO-BrdU TUNEL Assay Kit fA-23210 f Section 15.5).
• Indirect TUNEL assays using probes such as biotlnylated dUTP or our ChromaTlde DNP-11- dUTP (C-7610, Section 8.2) allow for amplification of the signal with our fluorophore- or enzyme- conjugated streptavidin conjugates (Section 7.6. Table 7.20) or with antl-DNP antibody (Section 7.4).
• Several additional assays for apoptosls can be found in Section 15.5.
• a quick and sensitive mlcroplate assay for abasic sites can be performed using ARP (A-10550, Figure 8.137), a biotlnylated hydroxylamine that reacts with the exposed aldehyde group at abasic sites.
• Biotlns bound to the abasic sites can be quantltated with our fluorescent- or enzyme-conjugated streptavldin complexes dSfr (Section 7.6, Table 7.20).
• ARP is permeant to cell membranes, permitting detection of abasic sites In living cells. * * * !_ ⁇ >
• the PicoGreen reagent has also been used to simplify denaturation assays for DNA damage.
• Strand breaks in dsDNA that result from DNA damage can be 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 ab ⁇ orbance at 260 n or by separating the two forms, of DNA by alkaline sucrose gradient centrifugation,*6fr filters,*®* or hydroxyapatlte chromatography. ⁇ * E*
• the absorbance-based technique suffers from low sensitivity and thus requires relatively large sample sizes Sfr and separation of ssDNA from dsDNA is laborious.
• the PicoGreen reagent was also used to develop a homogeneous PCR-based genotyping assay.*! ⁇ Because the products do not need to be run on a gel, the assay can be easily adapted for high throughput particularly using the RediPlate 96 version of the PicoGreen dsDNA quantitation assay (R-21495, Section 8.3).
• SYBR Green I stain (S-7563, S-7567, S-7585; SYBR Green I Nucleic Acid Gel Stain) 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 10,000-foId higher sensitivity than the traditional UV hyperchromicity assay.
• a single-length fragment of DNA 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 Kunltz units ( ⁇ 5 pg) of DNase activity.* ⁇ Even greater sensitivity can be achieved using the single radial enzyme diffusion (SRED) method, * ⁇ S!r In which the SYBR Green I stain is mixed with DNA in melted agarose and the mixture is poured Into a 2 mm thick slab. The sample to be tested Is poured into 1.5 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.
• SRED single radial enzyme diffusion
• the radius of these dark circles is proportional to the level of DNase activity.
• This method allows detection of as little as 2 x 10' 7 units ( ⁇ 0.1 pg) of DNase I or 2 x 10 -6 ( ⁇ 0.9 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 Identification of heterogeneities in DNase species,*S This method allows the detection of 4 x 10 ⁇ 6 units ( ⁇ 2 pg) DNase I or DNase II.
• Contaminating DNases are often responsible for poor resolution of DNA fragments, degradation of samples and nicking of supercoiled plasmids.
• Conventional DNase assays detect DNase activity by monitoring the increase in UV absorbahce that occurs when the base pairs unstack as the DNA is degraded. This absorbance method, however, is intrinsically insensitive as it requires large sample volumes and relies on small changes in absorbance.
• our dyes for nucleic add 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 ollgonucleotides.
• nuclease activity can be easily and accurately measured by the decrease in fluorescence in the Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage presence of one of these dyes.
• the YOYO-1 nucleic acid stain (Y-3601) has been used in a fluorescence-based microplate assay for nuclease activity.
• Y-3601 has been used in a fluorescence-based microplate assay for nuclease activity.
• This assay takes advantage of the large fluorescence enhancement of the YOYO-1 dye upon binding to nucleic acids and corresponding lack of fluorescence in the presence of released nucleotides and very small nucleic acid fragments.
• the stem sequence in an oligonucleotide 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 cleavage agent breaks the DNA strand, separating the fluorophore from the quencher. sfr
• the EnzChek Reverse Transcrlptase Assay Kit (E-22064) is a convenient, efficient and inexpensive assay for measuring reverse transcriptase activity (Figure 8.138).
• the key to this method is our PicoGreen dsDNA quantitation reagent, which preferentially detects dsDNA or RNA-DNA heteroduplexes over single-stranded nucleic acids or free nucleotides.
• 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 heteroduplexes, which are detected by the PicoGreen reagent at the end of the assay.
• samples can be read in a fluorometer or microplate reader with filter sets appropriate for fluoresceln (FITC).
• FITC fluoresceln
• 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 is ⁇ topic 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 Kit contains: • The PicoGreen dsDNA quantitation reagent • A lambda DNA standard • A poly(A) ribonucleotide template • An oligo(dT)16 primer • TE buffer, polymerization buffer and an EDTA solution • A detailed protocol (EnzChek Reverse Transcriptase Assay Kit)
• telomerase activity the telomerlc repeat amplification protocol or TRAP
• SYBR Green I dye staining was found to be more sensitive than silver staining and gave results comparable to those achieved with a radiolsotope- based TRAP assay. ⁇ & 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 sliver staining In the TRAP assay, and much easier to use. ⁇ f ⁇ - The SYBR Green I stain (S-7567, S-7563, s-7585) has also been used to develop high sensitivity assays to detect topoisomerase activity. *1# Section 8.7 - Analysis of DNA Structure, DNA Binding and DNA Damage
• Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage 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- arnino-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 Polyrnh) P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography.
• yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste.
• 61- fcD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight pell paste.
• the two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCE, and 10- 55 mM KG. The nature of the substrate determines the precise conditions for maximal enzyme activity.
• 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 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity.
• a comparison is made among thermostable DNA polymerases 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.
• tnermocycimg is require ⁇ .
• Taq DNA SurePrfmeTM ⁇ sis DNA polymerase polymerase ArrowTM Q-bioTaq * " Feat ⁇ re/ General PCR Hot Start tipIex'PCR characteristic High Fidelity High Sensitivity/ Mul Long fragment PCR and RAPDs 5' -> 3' exonuclease 3' -> 5' exonuclease + + Error rate (10 6 ) 24 24 0.6 S 24 Thermostabllity (half life at 95°C) 40 min 40 min 18h 40 min 80 min Residual polymerase activity at 25°C yes no yes yes yes yes yes yes yes Longest amplicons Up to 7 kb Up to 7 kb Up to 10 kb Up to 21 kb Up to 7 kb
• thermostable DNA m polymerase is checked for activity, function and purity.
• Each thermostable ⁇ polymerase Is shipped with a lot-specific quality control datasheet.
• m m Absence of nic ases Confirmed by incubating increasing amounts of enzyme with supercoiled plasmid ci DNA (pBR322). The maximum number of units that results In no relaxation of the m supercoiled DNA, as visualized on an agarose gel, is stated on the lot-specific data r sheet.
• ⁇ 2 PCR assay 2 oo Carried out using phage DNA as a template ( DNA) with decreasing amounts of ff ⁇ template DNA and decreasing units of DNA polymerase. A specific PCR product of 500 bp must be obtained. c m Purity
• this reagent binds to nucleic acids, it should The unopened vial Is stable at -1 S to -25° C through be treated as a potential mutagen and used with the expiration date printed on the label. appropriate care, the DMSO stock solution shoutd be iltot ⁇ Allquote the stock solution In 50 ⁇ l allquots, handled with particular caution as DMSO is known to brown tubes should be used. facilitate the entry of organic molecules into tissues, When handling the DM SO stock solution, double gloves, protective clothing and eyewear should be worn and safe laboratory practices should be followed.
• each vial should be allowed to warm up to 15-25° C and briefly centrif ⁇ ged in a microfuge Test principle
• the dye exhibits a preferential affinity for nucleic acids to deposit the DMSO solution at the bottom of the vial. and its fluorescent signal Is largely enhanced when bound to DNA .(more than one-order of magnitude store aqueous stain solutions In polypropylene larger than the fluorescent enhancement or bound rather than glass, as the stain may adsorb to glass ethidium bromide). surfaces, dye Is not stable In water alone.
• SYBR Green I dye Is a highly sensitive fluorescent stain for detecting nucleic acids In agarose and polyacrylamide gels (1,2). 2. Product characteristics The exceptional sensitivity of SYBR Green I stain makes it useful for those applications where the amount of DNA is limiting, Including the detection Sensitivity The detection limit using SYBR Green I is as low as of low-cycle number and low-target number DNA 100 pg per band of ds DNA usln ⁇ 312 nm trans- amplification products; the detection and restriction illumination with the Luml Image?' F Instrument analysis of low-copy number of DNA and RNA vectors; from Roche Diagnostics (Cat No.2015170).
• translllumlnatlon (Luml-lmager Fl) or BteJg: SYBR Green I stain can also be applied as 25 nmeplillumlnatlon detection format In the UghtCycl ⁇ r Instrument But the provided concentration in DMSO Is not standardized Toxlclty The Ames mammalian mlcrosome reverse mutation for the precise quantification and detection of nucleic assay shews significantly less mutagenlclty of SYBR acids with the LlghtCycler. Please refer to specialized Green I than ethidium bromide (3,4) kits and reagents for this instrument Spectral SYBR Green I Is maximally excited at 497 nm and has
• Sample material SYBR Green I can detect: characteristics secondary broad excitation peaks at 284 nm and 382 nm.
• RNA (with lower sensitivity); for RNA staining we recommend to use SYBR Green II Detection The spectral characteristics of SYBR Green I makes • Ollgonucleotldes it compatible with a wide variety of gel Imaging Instruments: ⁇ fej ⁇ .
• the detection limit for ollgonucleotldes stained with SYBR Green I is 1-2 ng with 254 nm * UVtra ⁇ s-llluml ⁇ ators epl-illumlnator or 312 nm translllumlnatlon, * UVepi-lllumlnators * argon Ion lasers.
• Number of stains 500 ⁇ l stock solution Is sufficient to prepare a total of that contain DNA with single stranded regions may 5 liters of working solution, which can be used to stain show fluorescence that Is more orange tt»n green. more than 100 agarose or potyacryl ml ⁇ lgels.
• Advantage 3.2 Preparation of working solution Caution Since SYBR Green I binds to nucleic acids, It should be treated as a potential mutagen and used with appropriate care.
• the DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules Into tissues. When handling the DMSO stock solution double ' gloves, protective clothing and eyewear should be worn and safe laboratory practices should be followed.
• Procedures and required materials solution at the temperature used for staining Is • between 7.5 and 8 (preferably pH 8.0).
• Prestained gels We do not recommend preparing prestained gels with should be poured through activated charcoal before SYBR Green I stain more than 1-2 days in advance. disposal. The charcoal must then be Incinerated to destroy the dye. One gram of activated charcoal easily Gels previously stained with ethidium bromide can absorbs the ye from 10 liters of freshly prepared subsequently be stained with SYBR Green I following working solution.- the standard protocol for poststainlng. There may be some decreases in sensitivity when compared to a gel stained only with SYBR Green I. 3.3 Staining DNA following electrophoresis
• Electrophoresis Perform electrophoresis on an agarose gel or denaturing polyacrylamide gel using: Additional UV trans- or epi-llluml ⁇ ators, or respective Imaging TBE [89 M Trls base, 89 M boric acid, 1 mM EDTA, equipment and instruments, such as Luml-lmager F1, argon ion pH 8] or reagents required lasers or respective Imaging instrument TAE [40 mM Trls-acetate, 1 M EDTA, pH 8] buffer.
• • clear polyproylene container for staining Note: Do not add any SDS to the electrophoresis • TBE buffer or buffer as this will dramatically reduce staining • TAE buffer efficiency. Procedure Please refer to the following table for the protocol,
• Procedure The final dilution of the SYBR Green I is best determined empirically, as there may be some non linear ef ects on the migration of different fragment size.
• Step Action Dilute SYBR Green I stock reagent 1:10000 into the gel solution Just prior to pouring the gel. 3.6 Removing SYBR Green I stain from double-stranded DNA The liquid should be as cool as possible when the dye Is added.
• Boiling and near boiling temperatures At least 99.9% of SYBR Green I can be removed from destroy the ability of SYBR Green I to stain double-stranded DNA by simple ethanol precipitation. nucleic acid. Do not heat SYBR Green 1 In the microwave.
• Gel electrophoresis as usual. Illumination of the stained gel:
• Fluorophores and Quencher The use of fluorescent tags as an alternative to radiolabels in DNA probes and primers has blossomed over the years. Fluorescence is safely measured with inexpensive instrumentation and it is very straightforward to multiplex assays for exceptionally high throughput. Molecular beacon and fluorescence resonance energy transfer (FRET) probes can be used in assays, which can be carried out in closedjybe formats with less sample handling at higher
• Molecular beacon and FRET probes require efficient quenching until the probe is hybridized to the target.
• Molecular beacon probes are hairpin structures wherein the fluorescence is quenched by the proximity of the fluorophore to the quencher molecule. When the probe hybridizes to the target, it becomes linear, quenching is disrupted, and the probe fluoresces ready for detection.
• FRET assays when the probe is hybridized to the target, it is digested by nuclease activity in the polymerase being used for amplification of target copies. The fluorophore, released from the target and separated from the quencher, is now highly fluorescent and ready for detection " .
• Fluorophore/quencher pairs can be chosen based on spectral properties - the emission of the fluorophore should overlap the absorption of the quencher.
• the quencher may absorb its partner's fluorescence and emit the fluorescence at a new wavelength or, in the case of a non-fluorescent quencher, as heat.
• the new Epoch products offer two new fluorescent dyes, available immediately as phosphoramidites and supports, as shown in Figure 1 ; Their absorbance and emission characteristics are shown in Figure 2.
• Yakima Yellow has an absorption maximum at 530 nm and emission maximum at 549 nm, while Redmond Red's absorption and emission maxima are at 579 nm and 595 nm, respectively.
• dabcyl is a very stable molecule and synthesis of doubly-labelled probes containing a dabcyl quencher is quite straightforward.
• the mechanism of quenching relies on the close proximity of the fluorophore to the dabcyl group, generally called static quenching, which is independent of spectral overlap between fluorophore and quencher.
• static quenching which is independent of spectral overlap between fluorophore and quencher.
• dabcyl's ability to act as a dark quencher is limited by its absorption spectrum to use with dyes emitting at 400 - 550 nm.
• the Eclipse Quencher from Epoch solves most of the problems inherent in the synthesis of molecular beacon and FRET probes.
• the Eclipse molecule is highly stable and can be used safely in all common oligo deprotection schemes.
• the absorption maximum for Eclipse Quencher is at 522 n , compared to 479 nm for dabcyl.
• the structure of the Eclipse Quencher (Figure 1) is substantially more electron deficient than that of dabcyl and this lead m to better quenching over a wider range of dyes, especially those with emission maxima at longer wavelength (red shifted) such as Redmond Red and Cy5.
• PCR Real-time quantitative PCR is a powerful tool that can be used for gene expression analysis, genotyping, pathogen detection/quantitation, mutation screening and DNA quantitation.
• ABI Prism 7900 Real Time Quantitative PCR instrument (TaqMan®) to detect accumulation 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® prbbe. 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.
• the probe When the probe is intact, energy transfer occurs between the two flourophors, and the fluorescent emission is quenched.
• 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.
• the ABI 7900 Prism software examines 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.
• the amplification plot is examined at a point during the early log phase of product accumulation above background (defined as the threshold cycle number or CT). 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 essential to have a well thought out experimental design for Real Time PCR. 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. (www.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.
• the Taqman® facility requests an acknowledgement 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.”
• LUXTM Light Upon extension
• Primers are an easy to use, highly sensitive, and efficient method for performing real-time quantitative PCR (qPCR) and RT-FCR (qRT-PCR).
• LUXTM Primers combine high specificity and multiplexing capability with simple design and streamlined protocols.
• LUXTM Primers require no special probes or quenchers, and are compatible with melting curve analysis of real-time qPCR products, allowing the dif erentiation of amplicons and primer di er artifacts by their melting temperatures.
• Each primer pair in the LUXTM system includes a fluorogenic primer with a fluorophore attached to its 3' end, as well as a corresponding unlabeled primer.
• the fluorogenic 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).
• Each fluorogenic LUXTM primer is labeled with one of two reporter dyes — FAM (6-carboxy-fluorescein) orfOE (6-carboxy-4', 5'-dichIoro-2', 7'-dimethoxy- fluorescein). Additional reporter dyes will be available in the future.
• LUXTM Primers can be used in real-time PCR and RT-PCR to quantify 100 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 7-8 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 LUXTM Primer Set would be used to detect a housekeeping gene as an internal control.
• Instrument LUXTM Primers are compatible with a wide variety of real-time PCR Compatibility instruments, including but not limited to the ABI PRISM ® 7700/7000/7900 and GeneAmp ® 5700, the Bio-Rad iCyclerTM, the Stratagene Mx4000TM, the Stratagene Mx3000TM, the Cepheid Smart Cycler ® , the Corbett Research Rotor-Gene, and the Roche LightCycler ® .
• ABI PRBM is a registered trademark of Applera Corporation.
• GeneAmp is a registered trademark of Roche Molecular Systems, Inc.
• LightCycler is a registered trademark of Idaho Technologies, Inc.
• iCycler, Mx 000, Mx300O, Rotor-Gene, and Smart Cycler are trademarks of their respective companies.
• LUX Designer To design and order custom LUXTM Primers for your genes of interest, visit the Primer Design I vitrogen LUXTM Web site at www.invitrogen.com/LUX and follow the link to Software the LUXTM Designer software.
• the software is available as either a Web-based application or a Microsoft* Windows ® -compatible download.
• LUXTM 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.
• Target Sequence The optimal amplicon length for real-time PCR ranges from 80 to 200 bases. You can specify a n ⁇ nimum, 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 lUPAC (International Union of Pure and Applied Chemistry) letter abbreviations. • When you select the design parameters, the default melting temperature range is 60-68°C. Do not change this default unless the design engine finds no primers in this range.
• PCR annealing temperatures from 55° to 6i C are appropriate.
• 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 4.0 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 higher score, the efficiency of the reaction may be less than optimal. See the LUXTM Designer Help for additional guidance.
• LUXTM Designer will first generate one or Design more designs for the labeled primer.
• the labeled primer can be either the forward or the reverse primer. 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 Designing and Ordering Custom LUX TM Primers, Continued
• Custom LUXTM 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 oligonucleotide 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 for 15 seconds. We recommend that you rehydrate primers at concentrations greater than 10 ⁇ M.
• To prepare a 100 ⁇ M primer stock solution multiply the primer amount in nmoles by ten to determine the volume of diluent in ul. After reconstitution, store the primer stock at-20°C in the dark, where it will be stable for 6 months or more.
• Certified LUXTM Certified LUXTM Primer Sets for Housekeeping Genes are predesigned primer Primer Sets for sets for genes that are commonly used as internal controls for normalizing Housekeeping real-time RT-PCR experiments. These primer sets have been optimized and Genes functionally validated to provide accurate, reproducible results using standard LUXTM protocols. They are supplied ready to use in TE buffer.
• Each Certified LUXTM Primer Set includes a FAM- or JOE-labeled LUX * " primer and a corresponding unlabeled primer. Each primer (labeled and unlabeled is supplied at 100 ⁇ l and a concentration of 10 ⁇ M. Available sets are listed below. For additional information, visit www.mvittogen.com/LUX.
• 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 10 2 to 10 7 copies or 1 pg to 10 ⁇ g of template. Seepage 12 for instructions on generating cDNA using reverse transcription as part of two-step real-time RT-PCR.
• Primer For optimal PCR conditions, primer titrations 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 ⁇ l of a 10 ⁇ M primer solution.
• Magnesium The optimal M " * 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 ⁇ M each. If Concentration dUTP is used in place of dTTP, its optimal concentration is 400 ⁇ M.
• Enzyme We recommend using a "hot-start" DNA polymerase, preferably one that has Specifications been optimized for real-time PCR.
• Platinum ® Quantitative PCR SuperMix UDG (Catalog no. 11730-017) is a 2X-concentrated, ready-to-use mixture containing all components except primers and template. It uses Platinum *81 Taq DNA polymerase and has been specifically formulated to provide optimal performance in real-time PCR systems.
• Instrument LUXTM 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 ininimum, the instrument used to perform real-time PCR with LUXTM.
• Protocol for The following protocol uses Platinum 18 Quantitative PCR SuperMix-UDG with Instruments 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 Plates LightCycler ® is provided on the following page. Mote: The following protocol uses a 50- ⁇ l 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 variation, prepare a Master Mix of all the reaction ingredients except template.
• Protocol for the Hie following protocol uses Platinum ® Quantitative PCR SuperMix-UDG and
• Roche LightCycler ® has been optimized for the Roche LightCycler ® . Consult the LightCycler ® documentation for detailed instructions on preparing the capillary tubes and operating the instrument.
• FAM-labeled LUXTM Primers are also compatible with Roche enzyme mixes. Note JOE-labeled LUXTM Primers are not compatible with the current version of the LightCycler ® ; use FAM-labeled primers only.
• the following protocol uses a 20- ⁇ l 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.
• Multiplex 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 LUXTM Primers offers simplified kinetics when compared with probe-based technologies, because only two oligos are used per target.
• LUXTM 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 LUXTM Primer Sets for Housekeeping Genes for the internal control.
• RNA usually total cellular RNA Specifications 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 RNase inhibitors and DNase digestion to minimize these.
• High-quality total RNA can be purified from as little as 100 cells up to 10 7 cells or 200 mg of tissue.
• To isolate mRNA we recommend using the FastTrack ® 2.0 mRNA Isolation Kit (Catalog no. K1593-02).
• RNA Genomic DNA from 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 ⁇ g of RNA; for larger amounts of RNA, increase volumes accordingly.
• Component Cone " Volume RNA template — x ⁇ l DNase reaction buffer 10X l ⁇ l DNase I, Amplification Grade l U/ ⁇ l l ⁇ l DEPC-treated ddHaO to lO ⁇ l 1. Incubate at room temperature for 15 min. 2. Add 1 ⁇ l of 25-mM EDTA solution to the reaction mixture and incubate at 65°C for 10 min to inactivate the DNase I.
• Two-Step Real-Time RT-PCR continued
• Protocol First-Strand Synthesis System for RT-PCR (with SuperscriptTM HI RT). The protocol has been optimized for LUXTM Primers. Follow this protocol to generate cDNA, which can then be used in real-time PCR (see pages 7-10). 1. Combine the following kit components in a tube on ice.
• a master mix without RNA may be prepared: 01igo(dT)i2-i8 (0.5 ⁇ g/ ⁇ l) or 01igo(dT)2o (50 M)* 0.5 ⁇ l Random hexamers (50 ng/ ⁇ l) 0.5 ⁇ l RNA (up to 1 ⁇ g) x ⁇ l lOx Buffer 2 ⁇ l 25 m MgCl2 4 ⁇ l lOmM dNTP i ⁇ l 0.1 M DTT 2 ⁇ l RNaseOUTTM (40 U/ ⁇ l) l ⁇ l SuperscriptTM II RT (50 U/ ⁇ l) or SuperscriptTM HI RT (200 U/ ⁇ l) l ⁇ l DEPC-treated ddH-O to 20 ⁇ l *01igo(dT) ⁇ -u is recommended for use with Superscript *" ' ⁇ RT; oligo(dT)2o is recommended for use with Superscript' "' Id RT 2.
• 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 SuperscriptTM HI Platinum ® One-Step Quantitative RT-PCR System for superior specificity and sensitivity with LUXTM Primers.
• Primer For optimal PCR, primer titrations of 50-500 nM per primer are recommended. Concentration The 50- ⁇ l sample reaction on page 16 uses 200 nM of each primer, equivalent to 1 ⁇ l of a 10 ⁇ M primer solution. See also the Important note below.
• the reverse primer drives the reverse transcription reaction.
• 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 for guidance on primer design.
• RNA sually total Specifications cellular 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 direct impact on results. RNase and genomic DNA contamination are the most common problems, and purification methods should be designed to avoid these.
• High-quality total RNA can be purified from as little as 100 cells up to 10 7 cells or 200 mg of tissue.
• To isolate mRNA we recommend using the FastTrack ® 2.0 mRNA Isolation Kit (Catalog no. K1593-02).
• the one-step RT-PCR enzyme mix should be optimized for real-time PCR.
• We Specifications recommend using the SuperscriptTM HI Platinum ® One-Step Quantitative RT- PCR System (Catalog nos.11732-020 and -088), which uses a SuperscriptTM HI RT/Platinum ® Taq enzyme mix. It has been optimized for use in real-time fluorescent PCR systems. See the sample reactions on pages 16-17.
• Magnesium The optimal MgCl. 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 reaction on page 16).
• dNTP The optimal concentration of dATP, dCTP, dGTP, and dTTP is 200 ⁇ M each. If Concentration dUTP is used in place of dTTP, its optimal concentration is 400 ⁇ M.
• Instrument LUXTM 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 LightCycler ® is provided on page 17.
• the instrument used to perform one-step real-time RT-PCR with LUXTM Primers must be able to: » Detect fluorescence at each PCR cycle • Excite and detect FAM-labeled LUXTM Primers near their excitation/emission wavelengths of 490/520 nm, and/or • Excite and detect JOE-labeled LUXTM Primers near their excitation/emission wavelengths of 520/550 nm
• the DNase I digest is designed for up to 1 ⁇ g of RNA; for larger amounts of RNA, increase volumes accordingly.
• Component Cone Volume RNA template ⁇ ⁇ l DNase reaction buffer 10X I ⁇ l DNase I, Amplification Grade l U/ ⁇ l l ⁇ l DEPC-treated ddH 2 0 to lO ⁇ l 1. Incubate at room temperature for 15 min. 2. Add 1 ⁇ l of 25-mM EDTA solution to the reaction mixture and incubate at 65°C for 10 min to inactivate the DNase I.
• RNase inhibitor proteins such as RNaseOUTTM (Catalog no.10777-019), maybe added to the reaction to safeguard against degradation of RNA. 1.
• the following table provides Master Mix volumes for a standard 20- ⁇ l reaction size. Note that preparation of a master mix is crucial in quantitative applications to reduce pipetting errors.
• UDG prevents reampUfication 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 cycling, 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 nunimize 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 LUXTM primer.
• the purchase of this product conveys to the buyer the non-transferable right to use the License No. 114: purchased amount of the product and components of the product in research LUXTM Fluorogenic conducted by the buyer (whether the buyer is an academic or for-profit entity).
• the buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) Primer 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 for 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 conunercial 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 information 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: (1) 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 its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not 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 Invitrogen based upon the manufacture, use or 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 manuf acture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the products with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.
• MSDS Requests To request an MSDS please visit our Web site (www.invitrogen.com) and follow the instructions below. 1. On the home page, go to the left-hand column under 'Technical Resources' and select 'MSDS Requests'. 2. Follow instructions on the page and fill out all the required fields. 3. To request additional MSDSs, click the 'Add Another' button. 4. All requests will be faxed unless another method is selected. 5. When you are finished entering information, click the 'Submit' button. Your MSDS will be sent within 24 hours. Continued on next page Technical Service, Continued
• Invitrogen is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, please contact our Technical Service Representatives. Invitrogen warrants that all of its products will perform according to the specifications stated on the certificate of analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen Cprporatjon's liability only to the cost of the product No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions.
• Invitrogen reserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Service Representatives. Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness or a particular purpose.
• Nazarenko I., Lowe, B., Darfler, M., Ikonomi, P., Schuster, D., and Rashtchian, A. (2002) Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore.
• Japan 03 a ⁇ ss 7974 The Netherlands 08000993310 New Zealand 0800600200 Norway 00800545654S8

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