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Articles |
1
Molecular Systems Division, Bio-Rad Laboratories, 5500 East Second St., Benicia, CA 94510.
2
Department of Pathology, Oregon Health Sciences
University, Portland, OR 97201.
a Author for correspondence. Fax 510-741-4650;
craig_hixson{at}bio-rad.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: The LLA model predicts (a) that amplification yield will approach that of PCR as the number of primers increases and (b) that the unique composition of LLA products will give lower carryover amplification efficiency compared with PCR. To test these hypotheses, the human ß-globin gene was amplified by 10-, 14-, or 18-primer LLA and the yield was compared with PCR. Carryover contamination was simulated by reamplifying a dilution series of LLA or PCR products. To demonstrate the clinical utility of the method, LLA coupled with allele-specific oligonucleotide (ASO) capture was used to detect the factor V Leiden mutation in a panel of 111 DNA samples.
Results: Fourteen- and 18-primer LLA gave amplification yields comparable to PCR. However, LLA carryover amplification efficiency was four orders of magnitude lower than that of PCR. The LLA-ASO assay detected the correct factor V Leiden genotype in all 111 samples.
Conclusions: LLA is a robust target amplification method that is comparable to PCR in yield. However, LLA is more resistant to false results caused by carryover amplicon contamination.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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)(17)(18)(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)2 (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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (Tm), secondary structure, and dimer formation, using Oligo 6.0 Primer Analysis Software (Molecular Biology Insights). Tm was determined based on the non-propanediol-substituted sequence.
dna samples
DNA used for ß-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.
amplification
ß-Globin LLA.
LLA reactions were performed using 10, 14, or
18 nested primers flanking codon 6 of the ß-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 MgCl2, and 2
U of AmpliTaq DNA polymerase (PE Biosystems) in a total volume of 50
µL. Samples were placed in a thermal cycler (iCycler from Bio-Rad
Laboratories or Perkin-Elmer 9600 from PE Biosystems), heated at
94 °C for 1 min, then subjected to 30, 35, or 40 cycles of heating
and cooling as follows: 94 °C for 30 s; 50 °C for 1 min;
72 °C for 30 s. At the end of the last cycle, reactions were
heated at 72 °C for 4.5 min.
ß-Globin PCR.
PCR reactions contained 20 pmol of upstream
primer ß1-x, 20 pmol of downstream primer ß8-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
MgCl2, and 2 U of AmpliTaq DNA polymerase in a
total volume of 50 µL. Samples were placed in the thermal cycler,
heated at 94 °C for 1 min, and then subjected to 30, 35, or 40
cycles of heating and cooling as follows: 94 °C for 45 s;
62 °C for 15 s; 72 °C for 30 s. At the end of the last
cycle, reactions were heated at 72 °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 ß-globin LLA, with the
following modifications: factor V primers were substituted for
ß-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® 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
ß-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-µL aliquot of
the LLA or PCR reaction was mixed with 18 µL of a mixture containing
10 pmol of MD792, 10 mM Tris-HCl (pH 9.2), 50 mM KCl, 2.5 mM
MgCl2, 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 °C for 2 min;
55 °C for 2 min; 72 °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 ß-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 ß-globin gene flanked by ß15 and MD792, and it captures MD792 extension products. The labeling reaction (20 µL) was mixed with 20 µL of Denaturation Solution, and then incubated for 10 min at room temperature. A 10-µL aliquot of the denatured primer extension product and 40 µL of Hybridization Solution were loaded into a conserved well. The well was incubated for 1 h at 37 °C, and then washed five times with Well Wash Buffer. Streptavidin-horseradish peroxidase conjugate (50 µL) was then added to the well. The well was incubated for 30 min at 37 °C, and then washed five times with Well Wash Buffer. Finally, 50 µL 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 µL of Stop Solution. The absorbance was measured at 450 nm with 595 nm as reference wavelength.
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-µL aliquot of the LLA or PCR product was mixed with 4
µL of water and 5 µL of Denaturation Solution and added to the N or
M well. The rest of the procedure was identical to ß-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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
).
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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.
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.
lla model
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.
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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
).
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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.
carryover contamination
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.
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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 10-9 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.
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ß-globin gene amplification by lla
Propanediol-substituted LLA primers were designed to flank codon 6
of the ß-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 ß6 and ß8, where an
250-nucleotide gap exists. The reason for that gap is that the
primer cluster comprising ß2, ß4, and ß6 was originally used to
amplify the ß-thalassemia mutation site, IVS 2-1, located just
downstream of ß6 (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
Tms based on the
non-propanediol-substituted sequences were in the 64–80 °C range.
Because the presence of the propanediol was expected to lower the
actual Tm, annealing temperature was
set at 50 °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 ß-globin
gene was detected colorimetrically by ASO capture (24).
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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.
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ß-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 ß-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
10-5–10-6 dilution of
the carryover amplicon gave signals that were not above background; for
PCR, the required dilution was
10-10–10-11, or at least
four orders of magnitude greater (Fig. 8
).
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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 3' 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 Tms (based on the
non-propanediol-substituted sequences) were in the 57–75 °C range,
and annealing was performed at 50 °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 ASOs 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that ß-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 ß-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.
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Acknowledgments |
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Footnotes |
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2 Nonstandard abbreviations: LLA, linked linear amplification; Tm, melting temperature; ASO, allele-specific oligonucleotide; and RFLP, restriction fragment length polymorphism.
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References |
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The following articles in journals at HighWire Press have cited this article:
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  J. Aslanzadeh Preventing PCR Amplification Carryover Contamination in a Clinical Laboratory Ann. Clin. Lab. Sci., October 1, 2004; 34(4): 389 - 396. [Abstract] [Full Text] [PDF]
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 A. A. Killeen, J. W. Breneman III, A. R. Carillo, J. Liu, and C. S. Hixson Linked Linear Amplification for Simultaneous Analysis of the Two Most Common Hemochromatosis Mutations Clin. Chem., July 1, 2003; 49(7): 1050 - 1057. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and the
same number of PCR product molecules reamplified by PCR (
).
