Linked linear amplification: a new method for the amplification of DNA.
Ironically, the enormous amplification power of target amplification techniques is also the source of a major drawback, namely susceptibility to "carryover" contamination. Thus, contamination with even a small amount of products from a previous reaction can lead to false-positive results. In PCR, methods currently used to minimize the occurrence of carryover contamination, such as the dUTP-uracil N-glycosylase system (12), ultraviolet irradiation (13), and DNA/RNA hybrid primers (14), have their own disadvantages relating to effectiveness, cost, and convenience (15). An alternative approach is the use of homogeneous systems where amplification and detection occur in the same reaction tube (16-19).
In addition to its application in exponential amplification methods such as PCR, primer extension can be used to amplify target in a linear fashion, as exemplified by cycle sequencing (20). In this variation of the Sanger sequencing technique, primer extension products in each cycle are terminated by incorporation of dideoxynucleotides. Multiple cycles enable accumulation of sufficient amounts of truncated products for gel electrophoretic detection.
Linked linear amplification (LLA)  (21) is a new amplification technique that utilizes nonreplicable element-containing primers and multiple cycles of primer extension reactions. The principles of LLA together with an example of its use in clinical molecular diagnostics are presented here. An unusual feature of LLA, its resistance to carryover contamination, is also demonstrated.
Materials and Methods
[beta]-Globin LLA and PCR primers were synthesized by standard phosphoramidite chemistry using an Ecosyn D300 DNA synthesizer (Eppendorf Scientific). LLA primers contained the 1,3-propanediol moiety ("C3 spacer", purchased from Glen Research) in place of the natural nucleotide at the fourth position from the 3' end. After cleavage from the resin, oligonucleotides were desalted by ethanol precipitation.
Factor V LLA primers substituted with propanediol as described above were purchased from Oligos, Etc. Additionally, the two innermost primers flanking codon 506 were biotinylated at their 5' ends.
Primer sequences were analyzed for melting temperature ([T.sub.m]), secondary structure, and dimer formation, using Oligo 6.0 Primer Analysis Software (Molecular Biology Insights). [T.sub.m] was determined based on the non-propanediol-substituted sequence.
DNA used for [beta]-globin amplification was prepared from EDTA-anticoagulated whole blood using the InstaGene Whole Blood reagent set (Bio-Rad Laboratories, Hercules, CA). DNA yield was estimated using the DNA Dipstick reagent set (Invitrogen).
DNA samples for the factor V Leiden study were collected in accordance with a protocol reviewed and deemed "exempt" by the Oregon Health Sciences University Institutional Review Board.
[beta]-Globin LLA. LLA reactions were performed using 10, 14, or 18 nested primers flanking codon 6 of the [beta]-globin gene. The LLA reaction consisted of LLA primers (each primer present at 10 pmol and containing the propanediol substitution), template DNA (~200, 2000, or 20 000 copies of human genomic DNA), 0.2 mM each dNTP, 10 mM Tris-HCl (pH 9.2), 50 mM KCl, 2.5 mM Mg[Cl.sub.2], and 2 U of AmpliTaq DNA polymerase (PE Biosystems) in a total volume of 50 [micro]L. Samples were placed in a thermal cycler (iCycler from Bio-Rad Laboratories or Perkin-Elmer 9600 from PE Biosystems), heated at 94[degrees]C for 1 min, then subjected to 30, 35, or 40 cycles of heating and cooling as follows: 94[degrees]C for 30 s; 50[degrees]C for 1 min; 72[degrees]C for 30 s. At the end of the last cycle, reactions were heated at 72[degrees]C for 4.5 min.
[beta]-Globin PCR. PCR reactions contained 20 pmol of upstream primer [beta]1-x, 20 pmol of downstream primer [beta]8-x, template DNA (~200, 2000, and 20 000 copies of human genomic DNA), 0.2 mM each dNTP, 10 mM Tris-HCl (pH 9.2), 50 mM KCl, 2.5 mM Mg[Cl.sub.2], and 2 U of AmpliTaq DNA polymerase in a total volume of 50 [micro]L. Samples were placed in the thermal cycler, heated at 94[degrees]C for 1 min, and then subjected to 30, 35, or 40 cycles of heating and cooling as follows: 94[degrees]C for 45 s; 62[degrees]C for 15 s; 72[degrees]C for 30 s. At the end of the last cycle, reactions were heated at 72[degrees]C for 4.5 min.
Factor V LLA. A total of 20 nested primers, 10 upstream and 10 downstream of factor V gene codon 506, were used. Factor V LLA was performed under conditions similar to [beta]-globin LLA, with the following modifications: factor V primers were substituted for [beta]-globin primers; the amount of template DNA was ~50-150 ng; annealing time was reduced to 30 s; and a total of 31 cycles were performed.
Factor V PCR for subsequent allele-specific oligonucleotide (ASO) capture. DNA samples were amplified by PCR using the Bio-Rad mDx[R] Factor V Leiden PCR reagent set (22).
Factor V PCR for subsequent restriction fragment length polymorphism (RFLP) analysis. DNA samples were genotyped for the factor V Leiden mutation by PCR-RFLP as described by Liu et al. (23).
DETECTION OF LLA AND PCR PRODUCTS BY ASO CAPTURE
[beta]-Globin. PCR and LLA amplification products were labeled in a single-cycle primer extension reaction using the 5'-biotinylated primer MD792. At the end of the cycling reaction, a 2-[micro]L aliquot of the LLA or PCR reaction was mixed with 18 [micro]L of a mixture containing 10 pmol of MD792, 10 mM Tris-HCl (pH 9.2), 50 mM KCl, 2.5 mM Mg[Cl.sub.2], 0.2 mM each dNTP, and 1 U of AmpliTaq DNA polymerase. Samples were placed in the thermal cycler and subjected to one cycle of heating and cooling as follows: 94[degrees]C for 2 min; 55[degrees]C for 2 min; 72[degrees]C for 5 min.
Biotin-labeled primer extension products were detected using the mDx Variant Gene 1 reagent set (Bio-Rad Laboratories), which detects the presence of the [beta]-globin S and C mutations by ASO capture of biotinylated PCR products (24). MD792 extension products were detected as described in the Variant Gene 1 instruction manual, except that only the "conserved" microwell was used. The conserved well contains an immobilized capture oligonucleotide complementary to a region of the ([beta]-globin gene flanked by [beta]15 and MD792, and it captures MD792 extension products. The labeling reaction (20 [micro]L) was mixed with 20 [micro]L of Denaturation Solution, and then incubated for 10 min at room temperature. A 10-[micro]L aliquot of the denatured primer extension product and 40 [micro]L of Hybridization Solution were loaded into a conserved well. The well was incubated for 1 h at 37[degrees]C, and then washed five times with Well Wash Buffer. Streptavidin-horseradish peroxidase conjugate (50 [micro]L) was then added to the well. The well was incubated for 30 min at 37[degrees]C, and then washed five times with Well Wash Buffer. Finally, 50 [micro]L of a tetramethylbenzidine/hydrogen peroxide solution was added to the well. After 10 min at room temperature, the colorimetric reaction was stopped by the addition of 50 [micro]L of Stop Solution. The absorbance was measured at 450 nm with 595 nm as reference wavelength.
[FIGURE 1 OMITTED]
Factor V. LLA and PCR products were detected using the Bio-Rad mDx Factor V Leiden PCR reagent set (22), which provides microwells coated with either an ASO specific for the wild type (N) sequence at codon 506, or one specific for the mutant (M) sequence. Because two LLA and both PCR primers were biotinylated, amplification products could be detected directly after thermal cycling. A 1-[micro]L aliquot of the LLA or PCR product was mixed with 4 [micro]L of water and 5 [micro]L of Denaturation Solution and added to the N or M well. The rest of the procedure was identical to ([beta]-globin amplicon detection, except that color development time was reduced to 5 min for LLA products. The presence or absence of mutation was determined by the signal ratio of wild type to mutant (N/M).
PRINCIPLES OF LLA
LLA utilizes primers that contain nonreplicable elements. Examples of such elements include 1,3-propanediol (25), 1,4-anhydro-2-deoxy-n-ribitol (26), and 2'-deoxyribofuranosyl naphthalene (27). A nonreplicable element can be situated along a primer such that it will allow effective primer-template binding and subsequent DNA synthesis by DNA polymerase. In contrast, when incorporated into a template strand, a nonreplicable element blocks the DNA polymerase-mediated extension of the opposite strand (25).
LLA requires thermal cycling to effect template denaturation, primer annealing, and extension. Fig. 1 illustrates the LLA principle for a two-primer reaction. In the first cycle, annealing of primers 1 and 2 to the template and subsequent extension produce first-generation product molecules 1-0 and 2-0. (Each product molecule is derived from a primer and a template. Product molecules are designated x-y, where x is the primer that is extended to form the product molecule, and y is the primer that in a preceding cycle had been incorporated at the 5' end of the template molecule. Because the 5' ends of the original template strands do not incorporate LLA primer sequence, first-generation products are designated x-0.) In the second cycle, molecules 1-0 and 2-0 are again produced by the priming of the original template by primers 1 and 2. In addition, because molecules 1-0 and 2-0 are themselves templates for primers 2 and 1, respectively, the synthesis of second-generation products 1-2 and 2-1 occurs. Note however, that these second-generation products do not incorporate binding sites for primers and therefore are not templates for further primer extension. Hence, first- and second-generation products will accumulate linearly in the course of the cycling reaction (Table 1).
Clearly, second-generation molecules can be used as templates for additional amplification if a new pair of primers that is nested with respect to the first pair is added to the reaction. A second round of temperature cycling would then lead to additional amplification of second-generation molecules. The process of adding nested primers and cycling could be repeated until the desired amplification is achieved. However, the sequential linking of linear amplification rounds in this manner is impractical because of the number of cycles required and the need to add a new set of primers at the end of each amplification round.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
An alternative to sequential LLA is concerted LLA, in which multiple sets of nested primers are added at the start of the reaction. Amplification reactions are still linked because the only means by which any product molecule can participate as template in additional primer extension reactions would be through the annealing of a nested primer.
In concerted LLA, each primer in the reaction mixture is potentially capable of annealing to the template and priming DNA polymerization. However, if the DNA polymerase used has an associated 5'-3' exonuclease activity (e.g., the enzyme isolated from Thermus aquaticus, T. thermophilus, or T. flavus), a simple amplification model can be proposed. The 5'-3' exonuclease activity has been shown to be structure specific, the preferred substrate being a forked structure consisting of a template strand that is duplexed to two daughter strands. In particular, the 5' end of the downstream daughter strand has been displaced by the upstream daughter strand to create a "flap" (16, 27, 28). The exonuclease activity cleaves between the first two bases of the downstream duplex to leave a nick. In vivo the nick is sealed by DNA ligase; in vitro, the combined polymerase and 5'-3' exonuclease activities of Taq DNA polymerase mediate DNA synthesis by nick translation, a property that has been exploited in the TaqMan assay (16).
In view of these observations, it is reasonable to hypothesize that in a primer extension reaction where for any given template molecule multiple primers are capable of binding (such as occurs in concerted LLA), the major, if not exclusive, extension product would be the one derived from the outermost (most 5') primer capable of annealing to that template. Product synthesis in concerted LLA would then proceed as an orderly cascade: in any cycle, a primer can be extended only if the template molecule for which that primer is the outermost primer present, said template having been synthesized in the preceding cycle(s). This is illustrated for four-primer LLA in Fig. 2. In the first cycle, although primers 1 and 3 can anneal to the same template strand and prime polymerization, only the extension product of primer 1 (molecule 1-0) is produced. Likewise, in cycle 2, molecule 1-2 but not 3-2 is synthesized off of template 2-0. At the start of cycle 3, molecule 2-1 is available as template. Primer 3 but not primer 1 can bind to 2-1; hence, the synthesis of molecule 3-2 proceeds. Progressively shorter molecules are thus produced and accumulate as additional cycles are performed.
[FIGURE 4 OMITTED]
Table 2 shows the predicted accumulation of the different LLA product molecules in a 14-primer LLA reaction and compares the yield to that of PCR after 20 cycles. It is interesting to note that after 14 cycles, the ratio of LLA yield (total number of product molecules) to PCR yield is 1. However, beginning at cycle 15, LLA yield starts to lag behind PCR, falling to 15% of PCR after 35 cycles (Fig. 3) The deficiency in LLA yield can be overcome by the use of more primers. For example, the ratio of LLA yield to PCR yield for 20-primer LLA is 1 after 20 cycles, 0.98 after 30 cycles, and 0.84 after 35 cycles (Fig. 3).
In the examples shown above, primer configuration is symmetric and the product pool should have equal amounts of both strands of the target region. To increase the yield of one strand over the other, an asymmetric n/n+1 configuration (where n is the number of primers located on each side of the target) could be used. However, the LLA model predicts that n/n+1 will give the same yield as n/n+2, n/n+3, and so forth. The smallest template generated in the course of the reaction will bind all "unpaired" primers, and only the outermost primer in this group will be extended efficiently. This assumes that no premature termination of primer extension occurs and that the 5'-exonuclease activity of the DNA polymerase is able to degrade all downstream extension products.
[FIGURE 5 OMITTED]
LLA products consist of molecules of different lengths (Table 2). The model described above can be used to determine the composition of product molecules as a function of cycle number. In the case of 20-primer LLA (Fig. 4), at lower cycle numbers there is nearly symmetric distribution of products with respect to length, such that the longest and shortest molecules are least abundant. However, as cycle number increases, the distribution becomes more skewed toward shorter molecules.
[FIGURE 6 OMITTED]
The LLA model also predicts that because of the cascading nature of product synthesis, shorter molecules will be amplified to a lesser extent than longer molecules. This is in contrast to PCR where each product molecule can be used as template for further exponential amplification and essentially all products have the same length.
For these reasons, carryover amplification yield should be less in LLA than in PCR. Fig. 5 shows the amount of amplification in a hypothetical case where after an initial amplification of one double-stranded target molecule in 20-primer, 35-cycle LLA, a [10.sup.-9] dilution of product molecules is introduced into a fresh 20-primer LLA reaction. The amplification yield is calculated after various cycle numbers and compared with the case where the same number of PCR product molecules is added to a fresh PCR reaction. Depending on the number of cycles performed in the second round of amplification, LLA carryover amplification is between two and four orders of magnitude lower than in PCR.
[FIGURE 7 OMITTED]
[beta]-GLOBIN GENE AMPLIFICATION BY LLA
Propanediol-substituted LLA primers were designed to flank codon 6 of the ([beta]-globin gene (Table 3 and Fig. 6). Primers were designed to abut each other head-to-tail with little or no gap between except between [beta]6 and [beta]8, where an ~250-nucleotide gap exists. The reason for that gap is that the primer cluster comprising [beta]2, [beta]4, and [beta]6 was originally used to amplify the [beta]-thalassemia mutation site, IVS 2-1, located just downstream of [beta]6 (data not shown). Smaller gaps were introduced among the rest of the primers to avoid sequences that could potentially form stable secondary structures or dimers. Calculated [T.sub.m]s based on the non-propanediol-substituted sequences were in the 64-80[degrees]C range. Because the presence of the propanediol was expected to lower the actual [T.sub.m], annealing temperature was set at 50[degrees]C. Ten-, 14-, and 18-primer LLA reactions were compared with PCR using different starting template amounts and cycle numbers. At the end of the reaction, LLA and PCR products were labeled by the addition of a 5'-biotinylated primer complementary to a region of the gene between the two innermost LLA primers, followed by one additional cycle of primer extension. Specific amplification of the ([beta]-globin gene was detected colorimetrically by ASO capture (24).
[FIGURE 8 OMITTED]
The use of a nested set of LLA primers, in which each primer contains a nonreplicable element, produced specific amplification of the target sequence (Fig. 7). The extent of amplification increased with the number of primers present and the number of cycles performed. In agreement with the proposed LLA model, 18-primer LLA gave an amplification yield similar to that of PCR when 200 molecules of starting template were used. However, with higher amounts of starting template, 18-primer or even 14-primer LLA amplified the target to a greater extent than PCR.
[beta]-GLOBIN LLA CARRYOVER CONTAMINATION
The LLA model predicts that carryover amplification will be several orders of magnitude lower in LLA than in PCR. To test this hypothesis, equivalent amounts (as judged from absorbance values) of 14-primer LLA and PCR products from an initial 40-cycle amplification of the ([beta]-globin gene were serially diluted and then added to freshly made, template-free LLA and PCR mixtures, respectively, for a second round of amplification. After 40 cycles, amplification products were detected as described above. For LLA, a [10.sup.-5]-[10.sup.-6] dilution of the carryover amplicon gave signals that were not above background; for PCR, the required dilution was [10.sup.-10]-[10.sup.-11], or at least four orders of magnitude greater (Fig. 8).
[FIGURE 9 OMITTED]
FACTOR V LEIDEN GENOTYPING BY LLA
The factor V Leiden mutation, R506Q, destroys an activated protein C cleavage site in the factor Va molecule (29) and is the most common genetic factor for predisposition to venous thrombosis (30, 31). To investigate whether LLA can be used to detect the factor V Leiden mutation, we used an assay consisting of LLA amplification followed by ASO capture to test a DNA panel that had been genotyped previously by two other methods, PCR-RFLP (23) and PCR-ASO capture (22) (data not shown). Twenty LLA primers were designed to flank codon 506 (Table 4). Because codon 506 is located near the 39 end of a 215-bp exon, most of the primer sequences were complementary to intronic regions. Factor V primers were designed to abut each other head-to-tail if possible, but gaps were introduced as necessary to avoid A-T-rich intronic regions or sequences with potential for forming stable secondary structures or dimers. Primers were selected such that calculated [T.sub.m]s (based on the non-propanediol-substituted sequences) were in the 57-75[degrees]C range, and annealing was performed at 50[degrees]C. To simultaneously amplify the target and label amplification products, the two innermost primers flanking the target region were 5' biotinylated. The LLA model predicts that after 31 cycles, ~15% of product molecules would have incorporated the biotinylated primer (for example, molecules 19-18 and 19-20 in Fig. 4). The presence of the factor V Leiden mutation was then detected by capture with AS[O.sub.s] complementary to the wild-type or mutant sequence at codon 506, followed by colorimetric detection of the biotin tag. The wild-type-to-mutant signal ratios (N/M) obtained produced unequivocal assignment of sample genotype, with N/M >10 for normals (88 samples), ~1 for heterozygotes (21 samples), and <0.1 for homozygous mutants (2 samples; Fig. 9). There was 100% agreement among LLA-ASO, PCR-ASO, and PCR-RFLP genotypes in all 111 samples.
The LLA model predicts stepwise synthesis of progressively shorter products as the cycling reaction proceeds. The associated 5'-3' exonuclease activity of the DNA polymerase makes possible the synthesis of extension products derived from upstream primers even in the presence of downstream primers. The accumulation of these longer products is critical to the success of the amplification scheme because they "feed" the stepwise synthesis cascade. A corollary to this is that LLA efficiency could be improved by use of a DNA polymerase that not only mediates DNA synthesis from outer primers but also does not degrade downstream extension products, e.g., via a strand displacement mechanism that leaves downstream products intact.
The observation that [beta]-globin 18-primer LLA has a markedly higher yield than PCR, at least in cases where the amount of starting template is high, suggests that the simple LLA model might not hold true for all situations. For example, degradation of downstream primer extension products might not be complete at each cycle because of premature termination of primer extension or <100% efficiency of the polymerase 5'-3' exonuclease activity. The latter situation could lead to the generation of shorter amplification products earlier than predicted in the cycling process. Alternatively, LLA might be inherently more efficient per cycle than PCR, e.g., primer-dimers are less likely to form in LLA because of the destabilizing effect of the propanediol moiety on duplex formation.
With respect to the asymmetry of the 18-primer [beta]-globin LLA, the model predicts that the 8/10 configuration used will give the same yield as an 8/9 or 8/11 arrangement. This hypothesis remains to be tested.
In PCR, carryover contamination is an important problem, but none of the methods currently used to minimize its occurrence is ideal (15). Results of a simulated amplicon contamination experiment showed that LLA carryover amplification efficiency was substantially lower than that seen in PCR. In practical terms, this suggests that LLA is less susceptible than PCR to false-positive results that are attributable to amplicon contamination.
We demonstrated the clinical utility of LLA by genotyping 111 samples for the factor V Leiden mutation. The factor V gene was simultaneously amplified and labeled by concerted LLA, and the mutation was subsequently detected by ASO capture. Ease of use and assay times for the factor V LLA-ASO and PCR-ASO assays were similar. Genotypes obtained by LLA-ASO, PCR-ASO, and PCR-RFLP were concordant in 111 of 111 samples.
In the factor V LLA assay, only the two innermost primers were biotinylated. Theoretically, these two primers are incorporated into only a small fraction of the final LLA products. More product molecules could be labeled and analytical sensitivity of LLA improved by increasing the number of biotinylated primers present during the reaction. Preferably, these would be the inner primers because they would contribute more to the final product pool than the outer ones.
Although the presence of multiple primers provides a potential for mispriming in LLA, extension products resulting from such events are not likely to be further amplified because they would not contain binding sites for the succeeding nested primers. The use of nested primers is a standard PCR strategy to increase target specificity, and it has the same effect in LLA.
The use of multiple primers in LLA offers another potential advantage over PCR. The presence of even a single nucleotide polymorphism in a primer binding site could cause PCR failure (32), a phenomenon that is the basis of allele-specific PCR (33, 34). Because LLA uses multiple primers, the ineffective binding of any one primer should affect final amplification yield to a lesser extent than it would PCR.
In conclusion, LLA has the same robustness as PCR but offers additional features that make it potentially useful in molecular diagnostics: lower carryover contamination, less likelihood of amplification failure in the presence of single nucleotide polymorphisms in the priming region, and high specificity through the use of nested primers.
We thank Thuan Tran for technical assistance in factor V Leiden genotyping.
Received September 26, 2000; accepted October 13, 2000.
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 Nonstandard abbreviations: LLA, linked linear amplification; [T.sub.m], melting temperature; ASO, allele-specific oligonucleotide; and RFLP, restriction fragment length polymorphism.
Antonio A. Reyes,  Luis A. Ugozzoli,  Jimmie D. Lowery,  John W. Breneman III,  Craig S. Hixson,  * Richard D. Press,  and R. Bruce Wallace  [[dagger]]
 Molecular Systems Division, Bio-Rad Laboratories, 5500 East Second St., Benicia, CA 94510.
 Depar[T.sub.m]ent of Pathology, Oregon Health Sciences University, Portland, OR 97201.
[[dagger]] Current address: Nanogen, Inc., 10398 Pacific Center Court, San Diego, CA 92121.
* Author for correspondence. Fax 510-741-4650; e-mail craig_hixson@biorad. com.
Table 1. Accumulation of products in two-primer LLA. (a) 1st Cumulative 1st 2nd generation generation generation Cycle (molecule 1-0) (molecule 1-0) (molecule 1-2) 1 1 1 0 2 1 2 1 3 1 3 2 4 1 4 3 5 1 5 4 6 1 6 5 7 1 7 6 8 1 8 7 9 1 9 8 10 1 10 9 100 1 100 99 Cumulative 1st and 2nd Cumulative 2nd generations generation (molecules Cycle (molecule 1-2) 1-0 and 1-2) 1 0 1 2 1 3 3 3 6 4 6 10 5 10 15 6 15 21 7 21 28 8 28 36 9 36 45 10 45 55 100 4950 5050 (a) Only product molecules complementary to one of two original template strands are shown. Table 2. Accumulation of LLA products in 14-primer LLA. (a) Cycle number Product molecule (b) 1 2 3 4 1-0 1 1 1 1 1-2 1 2 3 3-2 1 3 3-4 1 5-4 5-6 7-6 7-8 9-8 9-10 11-10 11-12 13-12 13-14 Total LLA 1 2 4 8 Total PCR 1 2 4 8 Cycle number Product molecule (b) 5 6 7 8 1-0 1 1 1 1 1-2 4 5 6 7 3-2 6 10 15 21 3-4 4 10 20 35 5-4 1 5 15 35 5-6 1 6 21 7-6 1 7 7-8 1 9-8 9-10 11-10 11-12 13-12 13-14 Total LLA 16 32 64 128 Total PCR 16 32 64 128 Cycle number Product molecule (b) 9 10 11 12 1-0 1 1 1 1 1-2 8 9 10 11 3-2 28 36 45 55 3-4 56 84 120 165 5-4 70 126 210 330 5-6 56 126 252 462 7-6 28 84 210 462 7-8 8 36 120 330 9-8 1 9 45 165 9-10 1 10 55 11-10 1 11 11-12 1 13-12 13-14 Total LLA 256 512 1024 2048 Total PCR 256 512 1024 2048 Cycle number Product molecule (b) 13 14 15 16 1-0 1 1 1 1 1-2 12 13 14 15 3-2 66 78 91 105 3-4 220 286 364 455 5-4 495 715 1001 1365 5-6 792 1287 2002 3003 7-6 924 1716 3003 5005 7-8 792 1716 3432 6435 9-8 495 1287 3003 6435 9-10 220 715 2002 5005 11-10 66 286 1001 3003 11-12 12 78 364 1365 13-12 1 13 91 455 13-14 1 14 105 Total LLA 4096 8192 16383 32752 Total PCR 4096 8192 16384 32768 Cycle number Product molecule (b) 17 18 19 1-0 1 1 1 1-2 16 17 18 3-2 120 136 153 3-4 560 680 816 5-4 1820 2380 3060 5-6 4368 6188 8568 7-6 8008 12376 18564 7-8 11440 19448 31824 9-8 12870 24310 43758 9-10 11440 24310 48620 11-10 8008 19448 43758 11-12 4368 12376 31824 13-12 1820 6188 18564 13-14 560 2380 8568 Total LLA 65399 130238 258096 Total PCR 65536 131072 262144 Cycle number Product molecule (b) 20 Total 1-0 1 2.0 x [10.sup.1] 1-2 19 1.9 x [10.sup.2] 3-2 171 1.1 x [10.sup.3] 3-4 969 4.8 x [10.sup.3] 5-4 3876 1.6 x [10.sup.4] 5-6 11628 3.9 x [10.sup.4] 7-6 27132 7.8 x [10.sup.4] 7-8 50388 1.3 x [10.sup.5] 9-8 75582 1.7 x [10.sup.5] 9-10 92378 1.8 x [10.sup.5] 11-10 92378 1.7 x [10.sup.5] 11-12 75582 1.3 x [10.sup.5] 13-12 50388 7.8 x [10.sup.4] 13-14 27132 3.9 x [10.sup.4] Total LLA 507624 1.0 x [10.sup.6] Total PCR 524288 1.0 x [10.sup.6] (a) Primer configuration: 1-3-5-7-9-11-13-(target region)-14-12-10-8-6-4-2. (b) Only product molecules complementary to one of two original template strands are shown. Table 3. [beta]-Globin primer sequences. Location, (c) Primera 5'-3' Sequence (b) nucleotides LLA primers [beta]1 TAAGCCAGTGCCAGAAGAGCCAAXGAC 61979-62005 [beta]3 TACGGCTGTCATCACTTAGACXTCA 62009-62033 [beta]5 CCCTGTGGAGCCACACCCTAGXGTT 62034-62058 [beta]7 AATCTACTCCCAGGAGCAGGGXGGG 62063-62087 [beta]9 GAGCCAGGGCTGGGCATAAAAXTCA 62091-62115 [beta]11 GGCAGAGCCATCTATTGCTTAXATT 62117-62141 [beta]13 TGCTTCTGACACAACTGTGTTXACT 62142-62166 [beta]15 AGCAACCTCAAACAGACACCAXGGT 62167-62191 [beta]18 CCTCACCACCAACTTCATCCAXGTT 62244-62268 [beta]17 AACCTTGATACCAACCTGCCCXGGG 62269-62293 [beta]16 TATTGGTCTCCTTAAACCTGTXTTG 62295-62319 [beta]14 TTCTCTGTCTCCACATGCCCAXTTT 62321-62345 [beta]12 CAGTGCCTATCAGAAACCCAAXAGT 62347-62371 [beta]10 AAATAGACCAATAGGCAGAGAXAGT 62372-62396 [beta]8 GACCACCAGCAGCCTAAGGGTXGGA 62397-62421 [beta]6 TTCCTATGACATGAACTTAACXATA 62675-62699 [beta]4 AAACTGTACCCTGTTACTTCTXCCC 62700-62724 [beta]2 CAATCATTCGTCTGTTTCCCAXTCT 62725-62749 PCR primers [beta]1-x TAAGCCAGTGCCAGAAGAGCCAAGGAC 61979-62005 [beta]8-x GACCACCAGCAGCCTAAGGGTGGGA 62397-62421 Labeling primer MD792 biotin-CACCTTGCCCCACAGGGCAGTAACG 62219-62243 (a) Ten-, 14-, and 18-primer LLA configurations are shown in Fig. 6. (b) x, propanediol. (c) Numbering of the human [beta]-globin gene sequence is taken from locus HUMHBB, GenBank Accession No. U01317.1. Table 4. Factor V Leiden LLA primer sequences. Location, (c) Primer (a) 5'-3' Sequence (b) nucleotides FV1 AGCACACCAACATGACACATGXATA 63317-63341 FV3 CACATGTACCCTAGAACTTAAAGXATA 63264-63290 FV5 AATTGGTTCCAGCGAAAGCXTAT 63199-63221 FV7 CAGGCAGGAACAACACCATXATC 63114-63136 FV9 AGAGCAGTTCAACCAGGGGXAAC 63091-63113 FV11 TCTTAGAGTTTGATGAACCCAXAGA 63046-63070 FV13 AAATGATGCCCAGTGCTTAACXAGA 63021-63045 FV15 CCATACTACAGTGACGTGGACXTCA 62996-63020 FV17 GAGAGACATCGCCTCTGGGXTAA 62972-62994 FV19 biotin-TAGGACTACTTCTAATCTGTAAXAGC 62946-62971 FV20 biotin-AAAGGTTACTTCAAGGACAAAAXACC 62894-62919 FV18 TTCTAGCCAGAAGAAATTCTCXGAA 62863-62887 FV16 TTATTTAGCCAGGAGACCTAAXATG 62838-62862 FV14 CTGTTCTCTTGAAGGAAATGCXCCA 62813-62837 FV12 GGTGCTAAAAAGGACTACTTGXCAA 62785-62809 FV10 CTTCGGCAGTGATGGTACTGAXAAA 62716-62730 FV8 AACAGACCTGGAATTTGAAACXAAG 62661-62685 FV6 CAACTTGCTCAACACATCCAAXACC 62636-62660 FV4 AAGGAAGAAATTAGGAAAGGCXAAT 62611-62635 FV2 CATTTTTAGGAGGGTTATTACCXATT 62579-62604 (a) Primer configuration: FV1-FV3-FV5-FV7-FV9-FV11-FV13-FV15- FV17-FV19-(codon 506)-FV20-FV18-FV16-FV14-FV12-FV10-FV8-FV6-FV4-FV2. (b) X, propanediol. (c) Numbering of the human factor V gene sequence is taken from locus HS86F14, GenBank Accession No. Z99572.
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|Title Annotation:||Molecular Diagnostics and Genetics|
|Author:||Reyes, Antonio A.; Ugozzoli, Luis A.; Lowery, Jimmie D.; Breneman, John, W., III; Hixson, Craig S.;|
|Date:||Jan 1, 2001|
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