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Polymerase chain reaction with nearby primers
Accepted Manuscript Polymerase chain reaction with nearby primers Ravil R. Garafutdinov, Aizilya A. Galimova, Assol R. Sakhabutdinova PII:
S0003-2697(16)30400-6
DOI:
10.1016/j.ab.2016.11.017
Reference:
YABIO 12563
To appear in:
Analytical Biochemistry
Received Date: 15 August 2016 Revised Date:
25 November 2016
Accepted Date: 26 November 2016
Please cite this article as: R.R. Garafutdinov, A.A. Galimova, A.R. Sakhabutdinova, Polymerase chain reaction with nearby primers, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.11.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
POLYMERASE CHAIN REACTION WITH NEARBY PRIMERS
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Subject category: Enzymatic assays and analysis
Authors:
Ravil R. Garafutdinov
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Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88
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E-mail address: [email protected]
Aizilya A. Galimova
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Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88
E-mail address: [email protected]
Assol R. Sakhabutdinova
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Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88
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E-mail address: [email protected]
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Corresponding author:
Ravil R. Garafutdinov
Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88 E-mail address: [email protected]
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Abstract
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DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention in recent years. DNA extracted from these samples is often inapplicable for conventional polymerase chain reaction (PCR), so for its amplification the nearby primers are commonly used. Here we report the data that clarify the features of PCR with nearby and abutting primers. We have shown that the proximity of primers leads to significant reduction of the reaction time and ensures the successful performance of DNA
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amplification even in the presence of PCR inhibitors. The PCR with abutting primers is usually characterized by the absence of nonspecific amplification products that causes extreme sensitivity with limit of detection on single copy level. The feasibility of PCR with abutting primers was demonstrated
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on species identification of 100 years old rotten wood.
Key words:
PCR sensitivity, degraded DNA.
1. Introduction
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Polymerase chain reaction, amplification, nearby primers, abutting primers, PCR specificity,
Detection of specific DNA fragments by polymerase chain reaction (PCR) is widely used in
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molecular diagnostics of various human [1-3] and animal [4] diseases, in forensics [5, 6], food control [7, 8] and analysis of environmental specimens [9, 10]. PCR is a powerful diagnostic tool with many advantages such as easy operation, high sensitivity, reliability and cost efficiency. However for investigation of biological samples containing degraded DNA (e.g. old/ancient, chemically and/or
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physically destroyed biomaterials) the PCR analysis becomes a complicated task and requires modifications of commonly used protocols. For instance, in molecular archaeology DNA extracted
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from bones is highly fragmented, therefore its amplification would be interrupted [11]. It was shown that all DNA isolated from 4-13000 years old samples had its fragment size in the range of 40-500 bp [12]. In the recent work it has been shown that DNA from the bones of Neanderthal men had average size of just 40-50 bp [13]. More than once it was reported that inaccuracy and amplification mistakes of long DNA fragments cause significant difficulties in human DNA identification, while using biomaterials in poor condition collected from crime or accident scenes [5]. High demand for analysis of severely degraded DNA requires development of unconventional PCR methods that would employ the nearby primers (i.e. resulting in PCR amplicons less than 100120 bp). Thus the mitochondrial genome reconstruction of ancient organisms became possible due to multiplex amplification of ultra short DNA fragments extracted from bones of fossil animals [14-16]. The efficiency of short DNA fragments amplification has been studied using conventional PCR with 2
optimized reaction conditions. The theoretical PCR model has been proposed and applied for detection
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of shortest DNA fragments [17]. It was shown that utilization of nearby primers and small adjustments of reaction mixture provide successful performance of PCR on DNA, affected by chemical treatment [18]. SNP genotyping of degraded human DNA was demonstrated in allele-specific PCR with nearby primers producing 40-67 bp size amplification products (amplicons) [19, 20]. Whole genome amplification with nearby primers was used for accumulation of DNA in the amount enough for comparative genomic hybridization and genotyping [21].
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However, there is a lack of detailed studies of PCR with nearby primers. There are only a few methodical works that show the advantages of such primers. Thus, Ahmad and Ghasemi described the PCR technique wherein FRET (fluorescence resonance energy transfer) phenomenon between abutting forward and reverse primers occurs [22]. At the same time the UFA platform (Universal Fluorescence
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Amplification) has been patented [23]. Recently some features of PCR with abutting primers were examined as well [24]. The aim of this study is to reveal the advantages of PCR with nearby primers
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and to evaluate their preference for amplification of degraded DNA.
2. Materials and Methods
2.1. Reagents
The following reagents were used: phenol, sucrose, RNase A, chloroform, isoamyl alcohol,
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isopropyl alcohol, sodium chloride (Sigma-Aldrich); acrylamide, N,N'-methylenebisacrylamide, Tris, ammonium persulfate, sodium dodecyl sulfate (SDS), N,N,N',N'-ethylenediaminetetraacetic acid disodium salt (EDТА), N,N,N',N'-tetramethylethylenediamine, cetyltrimethylammonium bromide (CTAB), acetic acid (AppliChem); proteinase K, Taq DNA polymerase, deoxynucleoside
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triphosphates (dNTPs) (Fermentas); PCR master mix with EvaGreen intercalating dye. All solutions
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were prepared with highly purified water (>18 MOm) (Millipore).
2.2. Oligonucleotide primers
Primers were designed using an OligoAnalyzer tool (Integrated DNA Technologies, http://eu.idtdna.com/calc/analyzer) and are listed in Table 1. Single copy nucleotide sequences for honeybee, human, mantis, spruce, oak and pine, and high copy number nucleotide sequence for larch are chosen as the targets. Primers were purchased from Syntol (Russia).
2.3. DNA isolation Total DNA of honey bee and mantis were purified from insects' muscles using DNA Extran-2 kit (Syntol, Russia) according to the manufacturer’s instructions. Human DNA was isolated by phenolchloroform extraction from venous blood [25]. 3
DNA of spruce, pine, oak and larch were obtained by the CTAB method [26] with minor
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modifications. About 50 mg of fresh needles (for spruce, pine and larch) or leaves (for oak) were ground in liquid nitrogen, transferred to a 2 mL eppendorf tube, then 400 uL CTAB buffer and 10 uL RNase A (10 mg/mL) were added. The mixtures were vortexed thoroughly and incubated at 65°С for 80 min with stirring. Then 400 uL of chloroform-isoamyl alcohol (v/v 23:2) was added, the mixtures were vortexed for 1 min and centrifuged (1 min, 13000 rpm). The upper phases were carefully transferred into a clean tube and purification with chloroform-isoamyl alcohol was repeated twice.
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Then the water phases were mixed with 350 uL cold isopropanol and vortexed. The samples were incubated for 30 min at -20°С, centrifuged (10 min, 13000 rpm), and supernatants were discarded. DNA pellets were washed with 500 uL of 70% ethanol and centrifuged (5 min, 13000 rpm), after which the alcohol was discarded and the pellets were dried in a vacuum concentrator. Then DNA
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pellets were dissolved in 50 uL of 1× TE-buffer and stored at -20°С. Larch DNA was also extracted from needles with GeneJET Plant Genomic DNA Purification Kit (Thermo Fischer Scientific) and
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used for comparative quantitative PCR studies.
DNA from rotten wood was extracted by the following procedure. Rotten wood was collected from the lowest log of wooden house and previously dried in oven at 65°С for 8 days followed by homogenization in a mortar. DNA from rotten wood was extracted by the salt method with modifications [27]. For this a sample of rotten wood (0.2 g) was mixed with 600 uL salt buffer (0.4 M NaCl, 10 mM Tris-HCl (pH 8.0), 2 mM EDТА), 40 uL 20% SDS, and 8 uL proteinase K (20 mg/mL).
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The sample was mixed thoroughly and incubated for 5 h at 65°С, then 300 uL of 5M NaCl was added. The sample was thoroughly vortexed for 30 s and centrifuged (30 min, 13000 rpm). Supernatant was transferred to a clean tube, mixed with an equal volume of isopropanol and incubated at -70°С for 2 h. Then, the sample was centrifuged for 20 min at 13000 rpm and the yellowish pellet was washed twice
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with 500 uL of 90% ethanol and dried in a vacuum concentrator. The obtained DNA sample was additionally purified by gel extraction kit (Cytokin, Russia) and stored in 1× TE-buffer at -20°С.
nm.
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Nucleic acid concentrations were measured on BioSpec-Mini spectrophotometer (Shimadzu) at 260
2.4. Polymerase chain reaction PCR was performed in a T100 DNA thermocycler (Bio-Rad Laboratories). PCR samples were prepared in UVC/T-M-AR PCR box (Biosan) after the UV pretreatment of the working space, automated dispensers and plastic ware for 20 min. Reaction mixtures with volumes of 10 uL contained DNA, 1 uL of each primer (5.0 pmol), 2.5 a.u. of Taq DNA polymerase, 1.0 uL of dNTP mixture (2.5 nmol) and 1 uL of Taq DNA polymerase buffer. PCR was performed using different programs depending on the experiment: initial denaturation at 94°С (3 min), 25-45 cycles – denaturation at 7594°С (1-20 s), annealing at 55-72°С (1-30 s), elongation at 72°С (1-20 s), and final elongation at 72°С 4
(2 min). DNA from rotten wood was amplified by multiple amplification (re-amplification) as follows:
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initial denaturation at 94°С (3 min), 30-60 cycles of denaturation at 94°С (10 s), annealing at 56°С (20 s), elongation at 72°С (20 s), and final elongation at 72°С (2 min). The reaction mixture for rotten wood DNA amplification contained a fivefold excess of Taq DNA polymerase (12.5 a.u.). PCR results were analyzed by electrophoresis in 10-15% polyacrylamide gels (PAAG) followed by ethidium bromide staining and visualization in Gel Camera System (UVP Inc.).
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2.5. Real-time polymerase chain reaction
Real time PCR was performed in iQ5 DNA thermocycler (Bio-Rad Laboratories). Reaction mixtures with a volume of 10 uL contained mantis or larch DNA along with 4.0 uL of 2.5× PCR master mix containing EvaGreen intercalating dye. Each sample was represented in two repeats. A
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program recommended for real-time PCR with intercalating dyes was used as follows: initial denaturation at 94°С (3 min), >40 cycles of denaturation at 94°С (15 s), annealing at 55 or 60°С (40 s),
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elongation at 72°С (30 s), final elongation at 72°С (2 min).
2.6. Sequencing of amplicons
The nucleotide sequences of PCR products were determined as follows. PCR samples after amplification were previously purified by preparative PAAG electrophoresis [28]. The target amplicons were routinely cloned with pAL2-T vector (Evrogen, Russia) in E. coli competent cells.
Analyzer.
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3. Results and Discussions
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Isolated plasmid DNAs were sequenced using ABI Big Dye Terminator chemistry on 3500 Genetic
When performing PCR analysis of biological specimens, that have been exposed to chemical or
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physical treatment, it is crucial to take into account their DNA integrity. The DNA from such samples is usually fragmented and may contain regions without bases providing failed amplification. The most obvious solution of this problem is the use of nearby primers resulting in small size of PCR amplicons (less than 100-120 bp). However, researchers often avoid the PCR with nearby primers due to the short length of their amplification products that may be confused for primer dimers (PDs), although methods for PDs elimination have been proposed [29-31]. Indeed, in practice sometimes the accumulation of PDs may be observed that is usually a result of long amplification times and inappropriate primer design. Recently several advantages of PCR with abutting primers were shown [24]. To accomplish that study we have designed new species-specific primers for DNA of a honey bee (Apis mellifera), human (Homo sapiens), mantis (Mantis religiosa), larch (Larix sukaczewii), spruce (Picea obovata), 5
pine (Pinus sylvestris) and oak (Quercus Robur). Two primer pairs were chosen for each species:
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conventional forward (F) and reverse (R) primers, and abutting forward (aF) and reverse (aR) primers (Table 1). The abutting primers represent the case of most closely positioned primers with 3’-ends annealing on the adjacent nucleotides of complement DNA strands. For abutting primers the amplicon size is determined by the sum of primer lengths. Three more reverse primers (nR1, nR2, nR3), positioned on some distance from forward primer (aF) were chosen for honey bee and human (Fig. 1). The expected amplicon sizes were in the range of 252-299 bp for F-R pairs, 37-47 bp for aF-aR pairs,
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and 55-121 bp for aF-nR pairs. Examination of all primers demonstrated their high specificity. There was no evidence of PDs formation in negative controls, while cloning and sequencing of reaction products after amplification with abutting primers confirmed their perfect homology to target DNA sequences (data is not shown).
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The proximity of primers gives many advantages for PCR. Firstly, the resulting small amplicon size does not require a high temperature and long denaturation time because strand dissociation of
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short dsDNA molecules may occur rapidly at 75-85°С. Secondly, there is no need for an extended elongation step because a variety of thermostable polymerases is capable for more than a 100 bp chain elongation within several seconds [32]. Ahmad and Ghasemi showed that zero duration of denaturation step at 85°C is sufficient for successful amplification with FRET-primers [22]. Present study also confirmed that the shortest PCR program we earlier found for amplification with abutting primers is sufficient for amplification with nearby primers (aF-nR pairs) as well. For this the series of
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experiments with varying temperature of denaturation, annealing and elongation steps followed by gelelectrophoresis of PCR products were carried out (data is not shown. For details see [24]). PCR was performed on a honey bee DNA at constant number of amplification cycles (30 cycles) and DNA concentration (104 target copies per reaction). The noncoding single copy sequence of A.mellifera
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DNA (LG1 linkage group) was taken as a target. It was found that even 1 second of denaturation is enough for accumulation of the detectable amplicon amount. The same result was obtained for
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elongation step: its reduction to 1 s together with 1 s denaturation step also leaded to formation of the appreciable DNA amount. The detailed study of annealing time reduction had demonstrated that its optimal duration was 5 s, although 7 s duration was earlier shown as optimal for PCR with FRETprimers [22]. At shorter duration time the amplification efficiency decreases, while its extension to 10 s or longer does not affect the PCR. This result indicates the annealing is not an instant process but does require time for primer diffusion and hybridization with target. Therefore the shortened program of thermocycling (denaturation-annealing-elongation as 1-5-1 s) is sufficient for amplification with nearby primers. We evaluated the possibility to decrease the total amplification time by changing the temperature of denaturation and annealing steps in PCR. For this the temperature gradient was used 6
followed by electrophoretic analysis. The threshold temperatures were determined by the
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disappearance of the amplification products on gels (Fig. 2А). The other reaction parameters were kept the same as described in the previous paragraph. When using the shortened program (1-5-1 s) the threshold temperature of denaturation step in PCR with regular primers (F-R) was determined around 90oC, while for PCR with abutting primers it was below 80°C (Fig. 2B). For aF-nR the threshold temperatures of denaturation were in 80-90°C range, raising with the increase of distance between the primers. It was consistent with threshold temperatures
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of the annealing step (Fig. 2C). The duration of both steps did not make a significant difference for temperature threshold values. The increase from 1 to 10 s slowly lowered the threshold of denaturation by 2-3°C, and the threshold of the annealing increased by 3°C. This can be explained by the kinetics of nucleic acids denaturation/annealing: shorter denaturation time requires much higher temperature for
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dissociation of strands, and longer annealing time promotes the complete hybridization at less optimal temperature. Similar pattern was also observed for other primer sets (data is not shown). As a result for
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most primers used in this study the minimal duration of PCR was reduced to 20 min whereas in standard program it was equal to 65-70 min.
To evaluate the sensitivity of PCR with nearby primers the quantitative PCR was performed. Single copy M.religiosa wingless gene was taken as a target since working space and reagents were unlikely to be contaminated with mantis DNA. The size of mantis genome is comparable to that of the human [34]. In amplification with abutting primers (MR aF - MR aR) the negative controls did not rise
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up to the 60th cycle (Fig. 3A), while for conventional primers (MR F - MR R) the fluorescence increase was observed (Fig. 3D) due to nonspecific products formation (Fig. 3E). This proves that PCR with abutting primers is highly sensitive, and its limit of detection (LoD) may be as low as single target copies. LoD parameter for conventional primers was relatively high (102) because the difference
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(CtNC - Ct10) was less than 5 cycles (Table 2). Even without PCR optimization the abutting primers provided better reaction characteristics than conventional primers (Fig. 3C, 3F).
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These results are well-correlated with known fact that for more extended primers the PCR specificity is higher. This rule was proved for abutting primers since they cover entirely the target nucleotide sequence throughout ~40-50 bp, whereas each conventional primer covers only 20-25 bp. Consequently PCR with conventional primers usually lasts less than 40 cycles, because over 40 cycles the accumulation of nonspecific products are often observed even for well designed primers. Melting curves for both abutting and conventional primers indicate the formation of desirable amplicons and presence of nonspecific products in controls only with conventional primers (Fig. 3B, 3E). Since the nearby primers lead to greater PCR sensitivity, this should be kept in mind in some cases. For instance, when single copy human nucleotide sequence is amplified with nearby primers the accumulation of amplicons can be observed in negative controls, if no special decontamination of the 7
reagents and the workspace was performed (Fig. 4А, lines 1 and 2). The sequencing of amplicon from
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line 1 confirmed it to be a specific amplification product, not a primers dimer formation. As a result of kinetic PCR experiments with endpoint detection the amplicons were determined at 23rd PCR cycle for test samples and at 27th cycle for negative control when abutting HS aF – HS aR primers pair and 103 genome copies (3 ng) of human DNA were used. Moreover the qPCR does not show any difference in Ct values of samples vs controls. Therefore, even though the accumulation rate of amplicons varied, by the 30th reaction cycle they can be detected by electrophoresis in both the test
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samples and the negative control. However, when the amplicon size was greater than 80 bp the amplification products in negative control were not detected in electrophoresis in this experiment (Fig. 4A, lines 3 and 4). This phenomenon is valid because the human DNA is highly abundant in the environment, and especially in the labs working with human DNA. On top of that the PCR reagents
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(primers, dNTPs, polymerases and others) can be contaminated with human DNA. More than once the false-positive PCR results have been shown while using the human primers if no special procedures
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for decontamination from human DNA were performed [33]. For reported experiment we intentionally did not perform any additional decontamination of the reagents, plastic ware and workspace from human DNA except those described in "Materials and methods". In practice the researchers (molecular geneticists etc.) usually amplify DNA fragments longer than 100 bp, therefore even when working with human DNA they rarely faces this problem of product accumulation in negative controls. Furthermore for PCR amplification the required amount of DNA starts from 104 copies (~30 ng) per
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reaction; this practice determines much higher rate of product amplification in test samples. The same set of nearby primers for honey bee DNA resulted in specific amplicons only for "positive" samples (Fig. 4B).
We also found that the amplification with abutting primers maintains lower sensitivity to PCR
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inhibitors. qPCR with larch DNA, extracted with CTAB method, demonstrates that abutting primers provide more efficient amplification of high copy number sequence of internal transcribe spacer 1
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(ITS1) comparing to conventional primers (Fig. 5A). Larch is a conifer tree loaded with high amount of PCR inhibitors such as phenolic (humic and fulvic acids, polyphenols etc.) and other compounds that interrupt the amplification with DNA from the plants, soil and similar sources. Amplification of DNA extracted from insects (honey bee and mantis) does not encounter this problem, when performed in same conditions. When larch DNA was isolated by special kit, the PCR inhibition was not observed (data is not shown). The abutting primers allowed to detect the larch DNA for almost the entire range of 10-fold serial DNA dilutions (100 – 10-3) however the linearity of Ct values was not observed (Table 3). For conventional primers PCR occurred only with slightly diluted DNA samples (10-1 and 10-2) and their Ct values were much greater than for the same DNA samples with abutting primers. PCR with LS F LS R pair was completely inhibited when using samples with highest DNA concentration, even 8
nonspecific products were not accumulated. For samples with low DNA concentrations Ct values were
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similar to negative controls but specific amplification did not occur as well (Fig. 5B). Taking into account high copy number of ITS1 sequence the obtained data show very low PCR efficiency for conventional primers, obviously due to inhibition. Therefore the abutting primers more preferable while working with difficult DNA samples. In order to demonstrate the capability of degraded DNA amplification with nearby primers, DNA from rotten wood was extracted. Rotten wood was obtained from the lowest log of wooden
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house built in 1914 (Fig. 6A). The age of wood was around 60 years old, judging by the log’s diameter. The origin of this wood was unknown, therefore we selected primers for the most commonly used building woods in Russia – pine, larch, spruce and oak. Primers for pine, spruce and oak were designed to unique (single copy) genes whereas for larch the primers to ITS1 were used. Obviously the
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wood was incessantly affected by environment influence (sunlight, air oxidation, rainfall wash and others). The first attempt to determine the origin of rotten wood by PCR was unsuccessful, although
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pre-amplification demonstrated the applicability and specificity of all primers used. No results were obtained even when the prolonged amplification (>60 cycles) with abutting primers and excess of Taq DNA polymerase was exploited.
The absence of PCR products (even nonspecific) indicated strong inhibition of the reaction. This problem can often be solved by multiple re-amplification. Indeed the specific product was obtained by double amplification only when the abutting primers for spruce were used (Fig. 6B, line
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3). At the same time, 3-fold re-amplification with other species specific primers was not successful. To validate the plant species and to exclude the primers dimer formation we sequenced an amplicon and
4. Conclusion
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confirmed its identity as a spruce DNA fragment (Fig. 6C).
Thus, 19 pairs of species specific conventional and nearby primers were designed. For all
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primer pairs only specific amplification products were detected, not primer dimers formation. We have proved that due to the short length of PCR products obtained with nearby primers there is no need to set conventional temperature and duration of annealing and denaturation steps. When using abutting primers, one can choose a short thermocycling program (e.g. “1-5-1 s”) that will provide reliable product amplification. For abutting primers denaturation temperature can be lowered to 80°C and annealing temperature can be raised for 5-6°C from its calculated value as well. Consequently for primers used in this study the minimal duration of PCR with sustained efficiency was reduced to 20 min whereas in standard program it was equal to 65-70 min. Such decrease of PCR duration is due to reduction of time spent on warming up the heating block and cooling it down. This advantage is especially valuable in clinical diagnostics or in field studies. The proximity of nearby primers ensures the successful DNA amplification even in the presence of PCR inhibitors such as phenolic substances. 9
For abutting primers the reaction is characterized by the complete absence of nonspecific amplification
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products providing high specificity and high sensitivity of PCR even in complicated cases (LoD parameter about 10 target copies). There is no doubt that each PCR experiment requires consideration of key thermodynamic parameters of primers, quality and origin of DNA samples. However, the features of PCR with nearby primers, that we have shown in this study, are fundamental and can be taken into account when analyzing fragmented DNA.
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Author Contributions
R.G. and A.S. coordinated the project. R.G. designed the experiments. A.G. and A.S did the experiments. A.S. drafted the manuscript. R.G. edited the manuscript.
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Acknowledgments
This work was supported by Russian State Federal budget (No. АААА-А16-116020350030-7).
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The laboratory equipment of the "Biomics" collective use center was exploited.
Conflict of interest
The authors declare that there are no conflicts of interest.
References
Burtis, C. A., Ashwood, E. R., Bruns, D.E. (2013) Tietz Textbook of Clinical Chemistry and
TE D
1.
Molecular Diagnostics (5th ed.), Saunders Elsevier, St. Louis. 2.
Song, Y., Huang, Y. Y., Liu, X., Zhang, X., Ferrari, M., Qin, L. (2014) Point-of-care technologies for molecular diagnostics using a drop of blood. Trends Biotechnol. 32, 132-9. Agrawal, M., Biswas, A. (2015) Molecular diagnostics of neurodegenerative disorders. Front.
EP
3.
Mol. Biosci. doi: 10.3389/fmolb.2015.00054. Aranaz, A. (2015) Significance and integration of molecular diagnostics in the framework of
AC C
4.
veterinary practice. Methods in Molecular Biology. 1247, 19-30. 5.
Brettell, T. A., Butler, J. M., Almirall, J. R. (2011) Forensic science. Anal. Chem. 83, 4539-4556.
6.
Johnson, R. N., Wilson-Wilde, L., Linacre, A. (2014) Current and future directions of DNA in wildlife forensic science. Forensic Sci. Int. Genet. 10, 1-11.
7.
Fraiture, M. A., Herman, P., Taverniers, I., De Loose, M., Deforce, D., Roosens, N. H. (2015) Current and New Approaches in GMO Detection: Challenges and Solutions. Biomed. Res. Int. doi: 10.1155/2015/392872.
8.
Milavec, M., Dobnik, D., Yang, L., Zhang, D., Gruden, K., Zel, J. (2014) GMO quantification: valuable experience and insights for the future. Anal. Bioanal. Chem. 406, 6485−97. 10
9.
Marangi, M., Giangaspero, A., Lacasella, V., Lonigro, A., Gasser, R. B. (2015) Multiplex PCR
ACCEPTED MANUSCRIPT
for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels. Mol. Cell Probes. 29, 122−5. 10.
Saito, M., Miyahara, S., Villanueva, S. Y., Aramaki, N., Ikejiri, M., Kobayashi, Y., Guevarra, J. P., Masuzawa, T., Gloriani, N. G., Yanagihara, Y., Yoshida, S. (2014) PCR and culture identification of pathogenic Leptospira spp. from coastal soil in Leyte, Philippines, after a storm surge during Super Typhoon Haiyan (Yolanda). Appl. Environ. Microbiol. 80, 6926−6932. Dabney, J., Meyer, M., Pääbo, S. (2013) Ancient DNA damage. Cold Spring Harb. Perspect. Biol. doi: 10.1101/cshperspect.a012567.
12.
Pääbo, S. (1989) Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc. Natl. Acad. Sci. USA. 86, 1939–1943.
Green, R. E., Briggs, A. W., Krause, J., Prüfer, K., Burbano, H. A., Siebauer, M., Lachmann, M.,
SC
13.
RI PT
11.
Pääbo, S. (2009) The Neandertal genome and ancient DNA authenticity. EMBO J. 28, 2494−502. Dabney, J., Knapp, M., Glocke, I., Gansauge, M. T., Weihmann, A., Nickel, B., Valdiosera, C.,
M AN U
14.
García, N., Pääbo, S., Arsuaga, J. L., Meyer, M. (2013) Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl. Acad. Sci. USA. 110, 15758−63. 15.
Krause, J., Dear, P. H., Pollack, J. L., Slatkin, M., Spriggs, H., Barnes, I., Lister, A. M., Ebersberger, I., Pääbo, S., Hofreiter, M. (2006) Multiplex amplification of the mammoth
16.
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mitochondrial genome and the evolution of Elephantidae. Nature. 439, 724−7. Templeton, J. E., Brotherton, P. M., Llamas, B., Soubrier, J., Haak, W., Cooper, A., Austin, J. J. (2013) DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification. Investig. Genet. 4, 26. Colotte, M., Couallier, V., Tuffet, S., Bonnet, J. (2009) Simultaneous assessment of average
EP
17.
fragment size and amount in minute samples of degraded DNA. Anal. Biochem. 388, 345−347. Dietrich, D., Uhl, B., Sailer, V., Holmes, E. E., Jung, M., Meller, S., Kristiansen, G. (2013)
AC C
18.
Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition. PLoS ONE. 8, e77771. 19.
Asari, M., Watanabe, S., Matsubara, K., Shiono, H., Shimizu, K. (2009) Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal. Biochem. 386, 85−90.
20.
Mead, S., Poulter, M., Beck, J., Uphill, J., Jones, C., Ang, C. E., Mein, C. A., Collinge, J. (2008). Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms. Hum. Mutat. 29, 1452−1458.
11
21.
Lee, C. I., Leong, S. H., Png, A. E., Choo, K. W., Syn, C., Lim, D. T., Law, H. Y., Kon, O. L.
ACCEPTED MANUSCRIPT
(2006) An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection. DNA Res. 13, 77−88. 22.
Ahmad, A. I., Ghasemi, J. B. (2007) New FRET primers for quantitative real-time PCR. Anal. Bioanal. Chem. 387, 2737−2743.
23.
Chemeris, A. V., Nikonorov, Yu. M., Romanenkova, M. L., Chemeris, D. A., Garafutdinov, R. R., Magazova, R. A., Maleev, G. V., Vakhitov, V. A., Vasilov, R. G. (2012) Novel methods of
24.
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amplifying DNA or RNA with real-time PCR, US Patent No. 8,198,026 B2.
Garafutdinov, R. R., Galimova, A. A., Sakhabutdinova, A. R., Vakhitov, V. A., Chemeris, A. V. (2015) DNA Amplification Using PCR with Abutting Primers. Mol. Biol. 49, 628−37. Mathew, C. G. (1985) The isolation of high molecular weight eucariotic DNA. Methods Mol. Biol. 2, 31−34.
26.
Doyle, J. J., Doyle, J. L. (1987) A rapid DNA isolation procedure for small quantities of fresh
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leaf tissue. Phytochem. Bull. 19, 11−15. 27.
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25.
Miller, S. A., Dykes, D. D., Polesky, H. F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215.
28.
Sambrook, J., Russell D.W. (2006) Isolation of DNA fragments from polyacrylamide gels by the crush and soak method // CSH Protoc. doi: 10.1101/pdb.prot2936.
29.
Brownie, J., Shawcross, S., Theaker, J., Whitcombe, D., Ferrie, R., Newton, C., Little, S. (1997)
30.
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The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 25, 3235−3241. Chen, F., Zhang, D., Zhang, Q., Zuo, X., Fan, C., Zhao, Y. (2016) Zero-Background HelicaseDependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts. Chembiochem. 17, 1171-1176. Vandesompele, J., De Paepe, A., Speleman, F. (2002) Elimination of primer-dimer artifacts and
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31.
genomic coamplification using a two-step SYBR green I real-time RT-PCR. Anal. Biochem.
32.
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303, 95-98.
Perler, F. B., Kumar, S., Kong, H. (1996) Thermostable DNA polymerases. Adv. Protein Chem. 48, 377−435.
33.
Champlot, S., Berthelot, C., Pruvost, M., Bennett, E. A., Grange, T., Geigl, E.-M. (2010) An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications. PLoS ONE. 5, e13042.
34.
Koshikawa, S., Miyazaki, S., Cornette, R., Matsumoto, T., Miura, T. (2008) Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae). Naturwissenschaften. 95, 859−867.
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Table 1. Characteristics of primers.
Primer
Sequence, 5’→3’
Size, nt
Organism / target
°С*
gene or genome region
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№
Тanneal.,
PCR product size (with appropriate forward primer), b.p.
AM F
ATGTCCACAACCCGATCAAGGC
22
59.9
2
AM R
CTCCCTCTGCTACCACGTTTACG
23
59.2
3
AM aF
CGTTCCAAAGCGAGAGCGAC
20
58.8
Honeybee (Apis mellifera)/
-
4
AM aR
CGGTGGTTACGGGAACGC
18
59.1
noncoding region, strain DH4,
38
5
AM nR1
GGGCACCTGTGCATCGG
linkage group LG1 (Amel 4.5)
55
6
AM nR2
CCTGGCACCCTTACGAGAGC
7
AM nR3
GGACACGATCATTCCAGGCGTTAG
8
HS aF
GGTGTGGTCTCAGGCAGACAT
9
HS aR
GCCACATCTGACATAGCTTGCTGTTC
10
HS nR1
GGCCAATGGTGGGCCA
11
HS nR2
TGGTGCATCCTCTGTCATTCAGCTA
12
HS nR3
13
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1
255
59.7
20
60.2
75
24
59.6
99
21
59.3
Human (Homo sapiens)/
-
26
59.9
noncoding flanking region of
47
16
59.2
rs2312154 (chr. 1, pos. 66607293),
59
25
59.8
start 66607360 - end 66607480
86
GCCATCAATTTCCAGCCAAACCC
23
59.4
(assembly GRCh38.p7)
121
MR F
ACAGCGGAGGAAATGGTGGTAG
22
59.1
14
MR R
GTATCTCCTGCGTCTTGTAGCCTC
24
58.7
Mantis (Mantis religiosa) /
252
15
MR aF
TCCAGCTCAAACCCTACAACCC
22
59.2
wingless gene (Wnt-1)
-
16
MR aR
ATGACTCCTGGAGGTTTGTGATCC
24
58.5
17
PO F
AGTTCAAGAGACCCTCGATAAGA
23
54.9
Spruce (Picea obovata) /
-
18
PO R
GTCTGTGTCTGACTATCGCC
20
54.9
dehydrin 7 gene (Dhn7)
268
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17
-
46
13
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19
PO aF
GCAGAATCAGGCTACGCTTA
20
54.8
-
20
PO aR
GCCTTCTCAGCTAGATTTGGAT
22
54.6
42
21
QR F
CAGACTAGGAAAGAGCAGATTGGA
24
56.1
-
22
QR R
CTAATGGAACACAAGCACAAACCAG
25
56.7
23
QR aF
AAAGATCAGAACTTGACCCCAGG
23
56.8
24
QR aR
AATAACAGTCTCTCCTTGCCTTGC
24
57.2
25
PS F
GTCTTCAGGTCTCTTCCTCCC
21
56.5
26
PS R
CTGTTCCACTTCTACCTCTGTTC
23
54.8
Pine (Pinus sylvestris) /
299
27
PS aF
TACCATGACCCAAGAGGAGT
20
55.0
constans-like 1 gene (Col1)
-
28
PS aR
CCCGAGGAGGAATCTCGAAT
20
56.1
40
29
LS F
TTAGCCGCTTGCATCCTC
18
55.3
-
30
LS R
CAAGAAGATGAGCGAAACAACAAC
24
54.9
Larch (Larix sukaczewii) /
253
31
LS aF
TTGACGGGAGAGTGGTTG
18
54.5
internal transcribed spacer 1 (ITS1)
-
32
LS aR
CGAACGGTTGCACATCCTG
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56.8
47 -
37
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19
Em protein gene (Emp1)
299
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* calculated by OligoAnalyzer
Oak (Quercus robur) /
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Ct mean
SD
Ct mean
SD
10000
28.76
0.235
26.37
0.463
1000
32.86
0.096
30.93
0.394
100
36.49
0.445
34.25
10
39.96
0.253
37.85
0
-
-
40.48
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Abutting primers
Genome copies
0.160 0.496
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SD - standard deviation.
0.689
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Table 3. Threshold cycle (Ct) mean values.
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DNA amount (ng)
Dilution
Approximate number of genome copies
Ct mean (curve number) LS aF - LS aR
LS F - LS R
100
1000
19.0 (1)
- (2)
2
10-1
100
27.5 (3)
36.0 (4)
0.2
10-2
10
31.0 (5)
40.0 (6)
0.02
10
-3
1
38.0 (7)
44.5 (8)
0.002
10-4
0.1
- (9)
45.5 (10)
0
- (11)
45.5 (12)
-
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0
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20
16
Figure legends
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Fig. 1. The scheme of primer annealing positions.
Fig. 2. Temperature limits of PCR (DNA from A. mellifera). A – electrophoretic determination of denaturation temperature limits (1 s duration of denaturation step, PCR samples had all five primer pairs: AM F – AM R, AM aF – AM aR, AM aF – AM nR1, AM aF – AM nR2, AM aF – AM nR3; М
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– pUC19/MspI ladder). B – temperature profiles of denaturation, and C – temperature profiles of annealing: 1 - AM F – AM R primers pair, 2 - AM aF – AM nR3, 3 - AM aF – AM nR2, 4 - AM aF – AM nR1, 5- AM aF – AM aR.
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Fig. 3. qPCR sensitivity (DNA from M. religiosa). A-C - abutting primers (MR aF - MR aR), D-F conventional primers (MR F - MR R). A, D - amplification curves (number of genome copies are
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indicated (104, 103, 102, 101), NC - negative control). B, E - melting curves. C, F - standard plots.
Fig. 4. PCR with nearby primers: A – H. sapiens primers, B – A. mellifera primers. Lines: 1-4 and 912 – negative controls; 5-8 – "positive" samples ("+" DNA of H. sapiens); 13-16 – "positive" samples ("+" DNA of A. mellifera); 1, 5 - HS aF - HS aR primer pair; 2, 6 - HS aF - HS nR1; 3, 7 - HS aF - HS nR2; 4, 8 - HS aF - HS nR3 (М1 – 100 bp ladder); 9, 13 - AM aF - AM aR primer pair; 10, 14 - AM
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aF - AM nR1; 11, 15 - AM aF - AM nR2; 12, 16 - AM aF - AM nR3 (M2 - pUC19/MspI ladder).
Fig. 5. Inhibition of qPCR with DNA from L. sukaczewii (each curve represents one of two replicates for 10-fold serial dilutions of a standard DNA sample). A: curves 1, 3, 5, 7, 9, 11 - abutting primers
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(LS aF - LS aR); curves 2, 4, 6, 8, 10, 12 – conventional primers (LS F - LS R). B: electrophoretic
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analysis of qPCR results; number of lines coincide with curve numbers.
Fig. 6. Determination of rotten wood origin. A - rotten wood sample. B - re-amplification results (lines: 1 - P. sylvestris primers PS aF - PS aR, 2 - Q. robur primers QR aF - QR aR, 3 - P. obovata primers PO aF - PO aR, 4 - L. sukaczewii primers LS aF - LS aR, M - pUC19/MspI ladder); C sequence of cloned amplicon.
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S0003-2697(16)30400-6
DOI:
10.1016/j.ab.2016.11.017
Reference:
YABIO 12563
To appear in:
Analytical Biochemistry
Received Date: 15 August 2016 Revised Date:
25 November 2016
Accepted Date: 26 November 2016
Please cite this article as: R.R. Garafutdinov, A.A. Galimova, A.R. Sakhabutdinova, Polymerase chain reaction with nearby primers, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.11.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
POLYMERASE CHAIN REACTION WITH NEARBY PRIMERS
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Subject category: Enzymatic assays and analysis
Authors:
Ravil R. Garafutdinov
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Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88
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E-mail address: [email protected]
Aizilya A. Galimova
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Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88
E-mail address: [email protected]
Assol R. Sakhabutdinova
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Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88
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E-mail address: [email protected]
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Corresponding author:
Ravil R. Garafutdinov
Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences Address: 450054, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia Tel./fax: +7 347 235-60-88 E-mail address: [email protected]
1
Abstract
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DNA analysis of biological specimens containing degraded nucleic acids such as mortal remains, archaeological artefacts, forensic samples etc. has gained more attention in recent years. DNA extracted from these samples is often inapplicable for conventional polymerase chain reaction (PCR), so for its amplification the nearby primers are commonly used. Here we report the data that clarify the features of PCR with nearby and abutting primers. We have shown that the proximity of primers leads to significant reduction of the reaction time and ensures the successful performance of DNA
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amplification even in the presence of PCR inhibitors. The PCR with abutting primers is usually characterized by the absence of nonspecific amplification products that causes extreme sensitivity with limit of detection on single copy level. The feasibility of PCR with abutting primers was demonstrated
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on species identification of 100 years old rotten wood.
Key words:
PCR sensitivity, degraded DNA.
1. Introduction
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Polymerase chain reaction, amplification, nearby primers, abutting primers, PCR specificity,
Detection of specific DNA fragments by polymerase chain reaction (PCR) is widely used in
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molecular diagnostics of various human [1-3] and animal [4] diseases, in forensics [5, 6], food control [7, 8] and analysis of environmental specimens [9, 10]. PCR is a powerful diagnostic tool with many advantages such as easy operation, high sensitivity, reliability and cost efficiency. However for investigation of biological samples containing degraded DNA (e.g. old/ancient, chemically and/or
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physically destroyed biomaterials) the PCR analysis becomes a complicated task and requires modifications of commonly used protocols. For instance, in molecular archaeology DNA extracted
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from bones is highly fragmented, therefore its amplification would be interrupted [11]. It was shown that all DNA isolated from 4-13000 years old samples had its fragment size in the range of 40-500 bp [12]. In the recent work it has been shown that DNA from the bones of Neanderthal men had average size of just 40-50 bp [13]. More than once it was reported that inaccuracy and amplification mistakes of long DNA fragments cause significant difficulties in human DNA identification, while using biomaterials in poor condition collected from crime or accident scenes [5]. High demand for analysis of severely degraded DNA requires development of unconventional PCR methods that would employ the nearby primers (i.e. resulting in PCR amplicons less than 100120 bp). Thus the mitochondrial genome reconstruction of ancient organisms became possible due to multiplex amplification of ultra short DNA fragments extracted from bones of fossil animals [14-16]. The efficiency of short DNA fragments amplification has been studied using conventional PCR with 2
optimized reaction conditions. The theoretical PCR model has been proposed and applied for detection
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of shortest DNA fragments [17]. It was shown that utilization of nearby primers and small adjustments of reaction mixture provide successful performance of PCR on DNA, affected by chemical treatment [18]. SNP genotyping of degraded human DNA was demonstrated in allele-specific PCR with nearby primers producing 40-67 bp size amplification products (amplicons) [19, 20]. Whole genome amplification with nearby primers was used for accumulation of DNA in the amount enough for comparative genomic hybridization and genotyping [21].
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However, there is a lack of detailed studies of PCR with nearby primers. There are only a few methodical works that show the advantages of such primers. Thus, Ahmad and Ghasemi described the PCR technique wherein FRET (fluorescence resonance energy transfer) phenomenon between abutting forward and reverse primers occurs [22]. At the same time the UFA platform (Universal Fluorescence
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Amplification) has been patented [23]. Recently some features of PCR with abutting primers were examined as well [24]. The aim of this study is to reveal the advantages of PCR with nearby primers
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and to evaluate their preference for amplification of degraded DNA.
2. Materials and Methods
2.1. Reagents
The following reagents were used: phenol, sucrose, RNase A, chloroform, isoamyl alcohol,
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isopropyl alcohol, sodium chloride (Sigma-Aldrich); acrylamide, N,N'-methylenebisacrylamide, Tris, ammonium persulfate, sodium dodecyl sulfate (SDS), N,N,N',N'-ethylenediaminetetraacetic acid disodium salt (EDТА), N,N,N',N'-tetramethylethylenediamine, cetyltrimethylammonium bromide (CTAB), acetic acid (AppliChem); proteinase K, Taq DNA polymerase, deoxynucleoside
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triphosphates (dNTPs) (Fermentas); PCR master mix with EvaGreen intercalating dye. All solutions
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were prepared with highly purified water (>18 MOm) (Millipore).
2.2. Oligonucleotide primers
Primers were designed using an OligoAnalyzer tool (Integrated DNA Technologies, http://eu.idtdna.com/calc/analyzer) and are listed in Table 1. Single copy nucleotide sequences for honeybee, human, mantis, spruce, oak and pine, and high copy number nucleotide sequence for larch are chosen as the targets. Primers were purchased from Syntol (Russia).
2.3. DNA isolation Total DNA of honey bee and mantis were purified from insects' muscles using DNA Extran-2 kit (Syntol, Russia) according to the manufacturer’s instructions. Human DNA was isolated by phenolchloroform extraction from venous blood [25]. 3
DNA of spruce, pine, oak and larch were obtained by the CTAB method [26] with minor
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modifications. About 50 mg of fresh needles (for spruce, pine and larch) or leaves (for oak) were ground in liquid nitrogen, transferred to a 2 mL eppendorf tube, then 400 uL CTAB buffer and 10 uL RNase A (10 mg/mL) were added. The mixtures were vortexed thoroughly and incubated at 65°С for 80 min with stirring. Then 400 uL of chloroform-isoamyl alcohol (v/v 23:2) was added, the mixtures were vortexed for 1 min and centrifuged (1 min, 13000 rpm). The upper phases were carefully transferred into a clean tube and purification with chloroform-isoamyl alcohol was repeated twice.
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Then the water phases were mixed with 350 uL cold isopropanol and vortexed. The samples were incubated for 30 min at -20°С, centrifuged (10 min, 13000 rpm), and supernatants were discarded. DNA pellets were washed with 500 uL of 70% ethanol and centrifuged (5 min, 13000 rpm), after which the alcohol was discarded and the pellets were dried in a vacuum concentrator. Then DNA
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pellets were dissolved in 50 uL of 1× TE-buffer and stored at -20°С. Larch DNA was also extracted from needles with GeneJET Plant Genomic DNA Purification Kit (Thermo Fischer Scientific) and
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used for comparative quantitative PCR studies.
DNA from rotten wood was extracted by the following procedure. Rotten wood was collected from the lowest log of wooden house and previously dried in oven at 65°С for 8 days followed by homogenization in a mortar. DNA from rotten wood was extracted by the salt method with modifications [27]. For this a sample of rotten wood (0.2 g) was mixed with 600 uL salt buffer (0.4 M NaCl, 10 mM Tris-HCl (pH 8.0), 2 mM EDТА), 40 uL 20% SDS, and 8 uL proteinase K (20 mg/mL).
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The sample was mixed thoroughly and incubated for 5 h at 65°С, then 300 uL of 5M NaCl was added. The sample was thoroughly vortexed for 30 s and centrifuged (30 min, 13000 rpm). Supernatant was transferred to a clean tube, mixed with an equal volume of isopropanol and incubated at -70°С for 2 h. Then, the sample was centrifuged for 20 min at 13000 rpm and the yellowish pellet was washed twice
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with 500 uL of 90% ethanol and dried in a vacuum concentrator. The obtained DNA sample was additionally purified by gel extraction kit (Cytokin, Russia) and stored in 1× TE-buffer at -20°С.
nm.
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Nucleic acid concentrations were measured on BioSpec-Mini spectrophotometer (Shimadzu) at 260
2.4. Polymerase chain reaction PCR was performed in a T100 DNA thermocycler (Bio-Rad Laboratories). PCR samples were prepared in UVC/T-M-AR PCR box (Biosan) after the UV pretreatment of the working space, automated dispensers and plastic ware for 20 min. Reaction mixtures with volumes of 10 uL contained DNA, 1 uL of each primer (5.0 pmol), 2.5 a.u. of Taq DNA polymerase, 1.0 uL of dNTP mixture (2.5 nmol) and 1 uL of Taq DNA polymerase buffer. PCR was performed using different programs depending on the experiment: initial denaturation at 94°С (3 min), 25-45 cycles – denaturation at 7594°С (1-20 s), annealing at 55-72°С (1-30 s), elongation at 72°С (1-20 s), and final elongation at 72°С 4
(2 min). DNA from rotten wood was amplified by multiple amplification (re-amplification) as follows:
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initial denaturation at 94°С (3 min), 30-60 cycles of denaturation at 94°С (10 s), annealing at 56°С (20 s), elongation at 72°С (20 s), and final elongation at 72°С (2 min). The reaction mixture for rotten wood DNA amplification contained a fivefold excess of Taq DNA polymerase (12.5 a.u.). PCR results were analyzed by electrophoresis in 10-15% polyacrylamide gels (PAAG) followed by ethidium bromide staining and visualization in Gel Camera System (UVP Inc.).
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2.5. Real-time polymerase chain reaction
Real time PCR was performed in iQ5 DNA thermocycler (Bio-Rad Laboratories). Reaction mixtures with a volume of 10 uL contained mantis or larch DNA along with 4.0 uL of 2.5× PCR master mix containing EvaGreen intercalating dye. Each sample was represented in two repeats. A
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program recommended for real-time PCR with intercalating dyes was used as follows: initial denaturation at 94°С (3 min), >40 cycles of denaturation at 94°С (15 s), annealing at 55 or 60°С (40 s),
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elongation at 72°С (30 s), final elongation at 72°С (2 min).
2.6. Sequencing of amplicons
The nucleotide sequences of PCR products were determined as follows. PCR samples after amplification were previously purified by preparative PAAG electrophoresis [28]. The target amplicons were routinely cloned with pAL2-T vector (Evrogen, Russia) in E. coli competent cells.
Analyzer.
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3. Results and Discussions
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Isolated plasmid DNAs were sequenced using ABI Big Dye Terminator chemistry on 3500 Genetic
When performing PCR analysis of biological specimens, that have been exposed to chemical or
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physical treatment, it is crucial to take into account their DNA integrity. The DNA from such samples is usually fragmented and may contain regions without bases providing failed amplification. The most obvious solution of this problem is the use of nearby primers resulting in small size of PCR amplicons (less than 100-120 bp). However, researchers often avoid the PCR with nearby primers due to the short length of their amplification products that may be confused for primer dimers (PDs), although methods for PDs elimination have been proposed [29-31]. Indeed, in practice sometimes the accumulation of PDs may be observed that is usually a result of long amplification times and inappropriate primer design. Recently several advantages of PCR with abutting primers were shown [24]. To accomplish that study we have designed new species-specific primers for DNA of a honey bee (Apis mellifera), human (Homo sapiens), mantis (Mantis religiosa), larch (Larix sukaczewii), spruce (Picea obovata), 5
pine (Pinus sylvestris) and oak (Quercus Robur). Two primer pairs were chosen for each species:
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conventional forward (F) and reverse (R) primers, and abutting forward (aF) and reverse (aR) primers (Table 1). The abutting primers represent the case of most closely positioned primers with 3’-ends annealing on the adjacent nucleotides of complement DNA strands. For abutting primers the amplicon size is determined by the sum of primer lengths. Three more reverse primers (nR1, nR2, nR3), positioned on some distance from forward primer (aF) were chosen for honey bee and human (Fig. 1). The expected amplicon sizes were in the range of 252-299 bp for F-R pairs, 37-47 bp for aF-aR pairs,
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and 55-121 bp for aF-nR pairs. Examination of all primers demonstrated their high specificity. There was no evidence of PDs formation in negative controls, while cloning and sequencing of reaction products after amplification with abutting primers confirmed their perfect homology to target DNA sequences (data is not shown).
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The proximity of primers gives many advantages for PCR. Firstly, the resulting small amplicon size does not require a high temperature and long denaturation time because strand dissociation of
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short dsDNA molecules may occur rapidly at 75-85°С. Secondly, there is no need for an extended elongation step because a variety of thermostable polymerases is capable for more than a 100 bp chain elongation within several seconds [32]. Ahmad and Ghasemi showed that zero duration of denaturation step at 85°C is sufficient for successful amplification with FRET-primers [22]. Present study also confirmed that the shortest PCR program we earlier found for amplification with abutting primers is sufficient for amplification with nearby primers (aF-nR pairs) as well. For this the series of
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experiments with varying temperature of denaturation, annealing and elongation steps followed by gelelectrophoresis of PCR products were carried out (data is not shown. For details see [24]). PCR was performed on a honey bee DNA at constant number of amplification cycles (30 cycles) and DNA concentration (104 target copies per reaction). The noncoding single copy sequence of A.mellifera
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DNA (LG1 linkage group) was taken as a target. It was found that even 1 second of denaturation is enough for accumulation of the detectable amplicon amount. The same result was obtained for
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elongation step: its reduction to 1 s together with 1 s denaturation step also leaded to formation of the appreciable DNA amount. The detailed study of annealing time reduction had demonstrated that its optimal duration was 5 s, although 7 s duration was earlier shown as optimal for PCR with FRETprimers [22]. At shorter duration time the amplification efficiency decreases, while its extension to 10 s or longer does not affect the PCR. This result indicates the annealing is not an instant process but does require time for primer diffusion and hybridization with target. Therefore the shortened program of thermocycling (denaturation-annealing-elongation as 1-5-1 s) is sufficient for amplification with nearby primers. We evaluated the possibility to decrease the total amplification time by changing the temperature of denaturation and annealing steps in PCR. For this the temperature gradient was used 6
followed by electrophoretic analysis. The threshold temperatures were determined by the
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disappearance of the amplification products on gels (Fig. 2А). The other reaction parameters were kept the same as described in the previous paragraph. When using the shortened program (1-5-1 s) the threshold temperature of denaturation step in PCR with regular primers (F-R) was determined around 90oC, while for PCR with abutting primers it was below 80°C (Fig. 2B). For aF-nR the threshold temperatures of denaturation were in 80-90°C range, raising with the increase of distance between the primers. It was consistent with threshold temperatures
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of the annealing step (Fig. 2C). The duration of both steps did not make a significant difference for temperature threshold values. The increase from 1 to 10 s slowly lowered the threshold of denaturation by 2-3°C, and the threshold of the annealing increased by 3°C. This can be explained by the kinetics of nucleic acids denaturation/annealing: shorter denaturation time requires much higher temperature for
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dissociation of strands, and longer annealing time promotes the complete hybridization at less optimal temperature. Similar pattern was also observed for other primer sets (data is not shown). As a result for
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most primers used in this study the minimal duration of PCR was reduced to 20 min whereas in standard program it was equal to 65-70 min.
To evaluate the sensitivity of PCR with nearby primers the quantitative PCR was performed. Single copy M.religiosa wingless gene was taken as a target since working space and reagents were unlikely to be contaminated with mantis DNA. The size of mantis genome is comparable to that of the human [34]. In amplification with abutting primers (MR aF - MR aR) the negative controls did not rise
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up to the 60th cycle (Fig. 3A), while for conventional primers (MR F - MR R) the fluorescence increase was observed (Fig. 3D) due to nonspecific products formation (Fig. 3E). This proves that PCR with abutting primers is highly sensitive, and its limit of detection (LoD) may be as low as single target copies. LoD parameter for conventional primers was relatively high (102) because the difference
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(CtNC - Ct10) was less than 5 cycles (Table 2). Even without PCR optimization the abutting primers provided better reaction characteristics than conventional primers (Fig. 3C, 3F).
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These results are well-correlated with known fact that for more extended primers the PCR specificity is higher. This rule was proved for abutting primers since they cover entirely the target nucleotide sequence throughout ~40-50 bp, whereas each conventional primer covers only 20-25 bp. Consequently PCR with conventional primers usually lasts less than 40 cycles, because over 40 cycles the accumulation of nonspecific products are often observed even for well designed primers. Melting curves for both abutting and conventional primers indicate the formation of desirable amplicons and presence of nonspecific products in controls only with conventional primers (Fig. 3B, 3E). Since the nearby primers lead to greater PCR sensitivity, this should be kept in mind in some cases. For instance, when single copy human nucleotide sequence is amplified with nearby primers the accumulation of amplicons can be observed in negative controls, if no special decontamination of the 7
reagents and the workspace was performed (Fig. 4А, lines 1 and 2). The sequencing of amplicon from
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line 1 confirmed it to be a specific amplification product, not a primers dimer formation. As a result of kinetic PCR experiments with endpoint detection the amplicons were determined at 23rd PCR cycle for test samples and at 27th cycle for negative control when abutting HS aF – HS aR primers pair and 103 genome copies (3 ng) of human DNA were used. Moreover the qPCR does not show any difference in Ct values of samples vs controls. Therefore, even though the accumulation rate of amplicons varied, by the 30th reaction cycle they can be detected by electrophoresis in both the test
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samples and the negative control. However, when the amplicon size was greater than 80 bp the amplification products in negative control were not detected in electrophoresis in this experiment (Fig. 4A, lines 3 and 4). This phenomenon is valid because the human DNA is highly abundant in the environment, and especially in the labs working with human DNA. On top of that the PCR reagents
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(primers, dNTPs, polymerases and others) can be contaminated with human DNA. More than once the false-positive PCR results have been shown while using the human primers if no special procedures
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for decontamination from human DNA were performed [33]. For reported experiment we intentionally did not perform any additional decontamination of the reagents, plastic ware and workspace from human DNA except those described in "Materials and methods". In practice the researchers (molecular geneticists etc.) usually amplify DNA fragments longer than 100 bp, therefore even when working with human DNA they rarely faces this problem of product accumulation in negative controls. Furthermore for PCR amplification the required amount of DNA starts from 104 copies (~30 ng) per
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reaction; this practice determines much higher rate of product amplification in test samples. The same set of nearby primers for honey bee DNA resulted in specific amplicons only for "positive" samples (Fig. 4B).
We also found that the amplification with abutting primers maintains lower sensitivity to PCR
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inhibitors. qPCR with larch DNA, extracted with CTAB method, demonstrates that abutting primers provide more efficient amplification of high copy number sequence of internal transcribe spacer 1
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(ITS1) comparing to conventional primers (Fig. 5A). Larch is a conifer tree loaded with high amount of PCR inhibitors such as phenolic (humic and fulvic acids, polyphenols etc.) and other compounds that interrupt the amplification with DNA from the plants, soil and similar sources. Amplification of DNA extracted from insects (honey bee and mantis) does not encounter this problem, when performed in same conditions. When larch DNA was isolated by special kit, the PCR inhibition was not observed (data is not shown). The abutting primers allowed to detect the larch DNA for almost the entire range of 10-fold serial DNA dilutions (100 – 10-3) however the linearity of Ct values was not observed (Table 3). For conventional primers PCR occurred only with slightly diluted DNA samples (10-1 and 10-2) and their Ct values were much greater than for the same DNA samples with abutting primers. PCR with LS F LS R pair was completely inhibited when using samples with highest DNA concentration, even 8
nonspecific products were not accumulated. For samples with low DNA concentrations Ct values were
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similar to negative controls but specific amplification did not occur as well (Fig. 5B). Taking into account high copy number of ITS1 sequence the obtained data show very low PCR efficiency for conventional primers, obviously due to inhibition. Therefore the abutting primers more preferable while working with difficult DNA samples. In order to demonstrate the capability of degraded DNA amplification with nearby primers, DNA from rotten wood was extracted. Rotten wood was obtained from the lowest log of wooden
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house built in 1914 (Fig. 6A). The age of wood was around 60 years old, judging by the log’s diameter. The origin of this wood was unknown, therefore we selected primers for the most commonly used building woods in Russia – pine, larch, spruce and oak. Primers for pine, spruce and oak were designed to unique (single copy) genes whereas for larch the primers to ITS1 were used. Obviously the
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wood was incessantly affected by environment influence (sunlight, air oxidation, rainfall wash and others). The first attempt to determine the origin of rotten wood by PCR was unsuccessful, although
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pre-amplification demonstrated the applicability and specificity of all primers used. No results were obtained even when the prolonged amplification (>60 cycles) with abutting primers and excess of Taq DNA polymerase was exploited.
The absence of PCR products (even nonspecific) indicated strong inhibition of the reaction. This problem can often be solved by multiple re-amplification. Indeed the specific product was obtained by double amplification only when the abutting primers for spruce were used (Fig. 6B, line
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3). At the same time, 3-fold re-amplification with other species specific primers was not successful. To validate the plant species and to exclude the primers dimer formation we sequenced an amplicon and
4. Conclusion
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confirmed its identity as a spruce DNA fragment (Fig. 6C).
Thus, 19 pairs of species specific conventional and nearby primers were designed. For all
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primer pairs only specific amplification products were detected, not primer dimers formation. We have proved that due to the short length of PCR products obtained with nearby primers there is no need to set conventional temperature and duration of annealing and denaturation steps. When using abutting primers, one can choose a short thermocycling program (e.g. “1-5-1 s”) that will provide reliable product amplification. For abutting primers denaturation temperature can be lowered to 80°C and annealing temperature can be raised for 5-6°C from its calculated value as well. Consequently for primers used in this study the minimal duration of PCR with sustained efficiency was reduced to 20 min whereas in standard program it was equal to 65-70 min. Such decrease of PCR duration is due to reduction of time spent on warming up the heating block and cooling it down. This advantage is especially valuable in clinical diagnostics or in field studies. The proximity of nearby primers ensures the successful DNA amplification even in the presence of PCR inhibitors such as phenolic substances. 9
For abutting primers the reaction is characterized by the complete absence of nonspecific amplification
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products providing high specificity and high sensitivity of PCR even in complicated cases (LoD parameter about 10 target copies). There is no doubt that each PCR experiment requires consideration of key thermodynamic parameters of primers, quality and origin of DNA samples. However, the features of PCR with nearby primers, that we have shown in this study, are fundamental and can be taken into account when analyzing fragmented DNA.
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Author Contributions
R.G. and A.S. coordinated the project. R.G. designed the experiments. A.G. and A.S did the experiments. A.S. drafted the manuscript. R.G. edited the manuscript.
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Acknowledgments
This work was supported by Russian State Federal budget (No. АААА-А16-116020350030-7).
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The laboratory equipment of the "Biomics" collective use center was exploited.
Conflict of interest
The authors declare that there are no conflicts of interest.
References
Burtis, C. A., Ashwood, E. R., Bruns, D.E. (2013) Tietz Textbook of Clinical Chemistry and
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1.
Molecular Diagnostics (5th ed.), Saunders Elsevier, St. Louis. 2.
Song, Y., Huang, Y. Y., Liu, X., Zhang, X., Ferrari, M., Qin, L. (2014) Point-of-care technologies for molecular diagnostics using a drop of blood. Trends Biotechnol. 32, 132-9. Agrawal, M., Biswas, A. (2015) Molecular diagnostics of neurodegenerative disorders. Front.
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3.
Mol. Biosci. doi: 10.3389/fmolb.2015.00054. Aranaz, A. (2015) Significance and integration of molecular diagnostics in the framework of
AC C
4.
veterinary practice. Methods in Molecular Biology. 1247, 19-30. 5.
Brettell, T. A., Butler, J. M., Almirall, J. R. (2011) Forensic science. Anal. Chem. 83, 4539-4556.
6.
Johnson, R. N., Wilson-Wilde, L., Linacre, A. (2014) Current and future directions of DNA in wildlife forensic science. Forensic Sci. Int. Genet. 10, 1-11.
7.
Fraiture, M. A., Herman, P., Taverniers, I., De Loose, M., Deforce, D., Roosens, N. H. (2015) Current and New Approaches in GMO Detection: Challenges and Solutions. Biomed. Res. Int. doi: 10.1155/2015/392872.
8.
Milavec, M., Dobnik, D., Yang, L., Zhang, D., Gruden, K., Zel, J. (2014) GMO quantification: valuable experience and insights for the future. Anal. Bioanal. Chem. 406, 6485−97. 10
9.
Marangi, M., Giangaspero, A., Lacasella, V., Lonigro, A., Gasser, R. B. (2015) Multiplex PCR
ACCEPTED MANUSCRIPT
for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels. Mol. Cell Probes. 29, 122−5. 10.
Saito, M., Miyahara, S., Villanueva, S. Y., Aramaki, N., Ikejiri, M., Kobayashi, Y., Guevarra, J. P., Masuzawa, T., Gloriani, N. G., Yanagihara, Y., Yoshida, S. (2014) PCR and culture identification of pathogenic Leptospira spp. from coastal soil in Leyte, Philippines, after a storm surge during Super Typhoon Haiyan (Yolanda). Appl. Environ. Microbiol. 80, 6926−6932. Dabney, J., Meyer, M., Pääbo, S. (2013) Ancient DNA damage. Cold Spring Harb. Perspect. Biol. doi: 10.1101/cshperspect.a012567.
12.
Pääbo, S. (1989) Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc. Natl. Acad. Sci. USA. 86, 1939–1943.
Green, R. E., Briggs, A. W., Krause, J., Prüfer, K., Burbano, H. A., Siebauer, M., Lachmann, M.,
SC
13.
RI PT
11.
Pääbo, S. (2009) The Neandertal genome and ancient DNA authenticity. EMBO J. 28, 2494−502. Dabney, J., Knapp, M., Glocke, I., Gansauge, M. T., Weihmann, A., Nickel, B., Valdiosera, C.,
M AN U
14.
García, N., Pääbo, S., Arsuaga, J. L., Meyer, M. (2013) Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl. Acad. Sci. USA. 110, 15758−63. 15.
Krause, J., Dear, P. H., Pollack, J. L., Slatkin, M., Spriggs, H., Barnes, I., Lister, A. M., Ebersberger, I., Pääbo, S., Hofreiter, M. (2006) Multiplex amplification of the mammoth
16.
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mitochondrial genome and the evolution of Elephantidae. Nature. 439, 724−7. Templeton, J. E., Brotherton, P. M., Llamas, B., Soubrier, J., Haak, W., Cooper, A., Austin, J. J. (2013) DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification. Investig. Genet. 4, 26. Colotte, M., Couallier, V., Tuffet, S., Bonnet, J. (2009) Simultaneous assessment of average
EP
17.
fragment size and amount in minute samples of degraded DNA. Anal. Biochem. 388, 345−347. Dietrich, D., Uhl, B., Sailer, V., Holmes, E. E., Jung, M., Meller, S., Kristiansen, G. (2013)
AC C
18.
Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition. PLoS ONE. 8, e77771. 19.
Asari, M., Watanabe, S., Matsubara, K., Shiono, H., Shimizu, K. (2009) Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal. Biochem. 386, 85−90.
20.
Mead, S., Poulter, M., Beck, J., Uphill, J., Jones, C., Ang, C. E., Mein, C. A., Collinge, J. (2008). Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms. Hum. Mutat. 29, 1452−1458.
11
21.
Lee, C. I., Leong, S. H., Png, A. E., Choo, K. W., Syn, C., Lim, D. T., Law, H. Y., Kon, O. L.
ACCEPTED MANUSCRIPT
(2006) An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection. DNA Res. 13, 77−88. 22.
Ahmad, A. I., Ghasemi, J. B. (2007) New FRET primers for quantitative real-time PCR. Anal. Bioanal. Chem. 387, 2737−2743.
23.
Chemeris, A. V., Nikonorov, Yu. M., Romanenkova, M. L., Chemeris, D. A., Garafutdinov, R. R., Magazova, R. A., Maleev, G. V., Vakhitov, V. A., Vasilov, R. G. (2012) Novel methods of
24.
RI PT
amplifying DNA or RNA with real-time PCR, US Patent No. 8,198,026 B2.
Garafutdinov, R. R., Galimova, A. A., Sakhabutdinova, A. R., Vakhitov, V. A., Chemeris, A. V. (2015) DNA Amplification Using PCR with Abutting Primers. Mol. Biol. 49, 628−37. Mathew, C. G. (1985) The isolation of high molecular weight eucariotic DNA. Methods Mol. Biol. 2, 31−34.
26.
Doyle, J. J., Doyle, J. L. (1987) A rapid DNA isolation procedure for small quantities of fresh
M AN U
leaf tissue. Phytochem. Bull. 19, 11−15. 27.
SC
25.
Miller, S. A., Dykes, D. D., Polesky, H. F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215.
28.
Sambrook, J., Russell D.W. (2006) Isolation of DNA fragments from polyacrylamide gels by the crush and soak method // CSH Protoc. doi: 10.1101/pdb.prot2936.
29.
Brownie, J., Shawcross, S., Theaker, J., Whitcombe, D., Ferrie, R., Newton, C., Little, S. (1997)
30.
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The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 25, 3235−3241. Chen, F., Zhang, D., Zhang, Q., Zuo, X., Fan, C., Zhao, Y. (2016) Zero-Background HelicaseDependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts. Chembiochem. 17, 1171-1176. Vandesompele, J., De Paepe, A., Speleman, F. (2002) Elimination of primer-dimer artifacts and
EP
31.
genomic coamplification using a two-step SYBR green I real-time RT-PCR. Anal. Biochem.
32.
AC C
303, 95-98.
Perler, F. B., Kumar, S., Kong, H. (1996) Thermostable DNA polymerases. Adv. Protein Chem. 48, 377−435.
33.
Champlot, S., Berthelot, C., Pruvost, M., Bennett, E. A., Grange, T., Geigl, E.-M. (2010) An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications. PLoS ONE. 5, e13042.
34.
Koshikawa, S., Miyazaki, S., Cornette, R., Matsumoto, T., Miura, T. (2008) Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae). Naturwissenschaften. 95, 859−867.
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Table 1. Characteristics of primers.
Primer
Sequence, 5’→3’
Size, nt
Organism / target
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gene or genome region
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№
Тanneal.,
PCR product size (with appropriate forward primer), b.p.
AM F
ATGTCCACAACCCGATCAAGGC
22
59.9
2
AM R
CTCCCTCTGCTACCACGTTTACG
23
59.2
3
AM aF
CGTTCCAAAGCGAGAGCGAC
20
58.8
Honeybee (Apis mellifera)/
-
4
AM aR
CGGTGGTTACGGGAACGC
18
59.1
noncoding region, strain DH4,
38
5
AM nR1
GGGCACCTGTGCATCGG
linkage group LG1 (Amel 4.5)
55
6
AM nR2
CCTGGCACCCTTACGAGAGC
7
AM nR3
GGACACGATCATTCCAGGCGTTAG
8
HS aF
GGTGTGGTCTCAGGCAGACAT
9
HS aR
GCCACATCTGACATAGCTTGCTGTTC
10
HS nR1
GGCCAATGGTGGGCCA
11
HS nR2
TGGTGCATCCTCTGTCATTCAGCTA
12
HS nR3
13
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1
255
59.7
20
60.2
75
24
59.6
99
21
59.3
Human (Homo sapiens)/
-
26
59.9
noncoding flanking region of
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16
59.2
rs2312154 (chr. 1, pos. 66607293),
59
25
59.8
start 66607360 - end 66607480
86
GCCATCAATTTCCAGCCAAACCC
23
59.4
(assembly GRCh38.p7)
121
MR F
ACAGCGGAGGAAATGGTGGTAG
22
59.1
14
MR R
GTATCTCCTGCGTCTTGTAGCCTC
24
58.7
Mantis (Mantis religiosa) /
252
15
MR aF
TCCAGCTCAAACCCTACAACCC
22
59.2
wingless gene (Wnt-1)
-
16
MR aR
ATGACTCCTGGAGGTTTGTGATCC
24
58.5
17
PO F
AGTTCAAGAGACCCTCGATAAGA
23
54.9
Spruce (Picea obovata) /
-
18
PO R
GTCTGTGTCTGACTATCGCC
20
54.9
dehydrin 7 gene (Dhn7)
268
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-
46
13
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19
PO aF
GCAGAATCAGGCTACGCTTA
20
54.8
-
20
PO aR
GCCTTCTCAGCTAGATTTGGAT
22
54.6
42
21
QR F
CAGACTAGGAAAGAGCAGATTGGA
24
56.1
-
22
QR R
CTAATGGAACACAAGCACAAACCAG
25
56.7
23
QR aF
AAAGATCAGAACTTGACCCCAGG
23
56.8
24
QR aR
AATAACAGTCTCTCCTTGCCTTGC
24
57.2
25
PS F
GTCTTCAGGTCTCTTCCTCCC
21
56.5
26
PS R
CTGTTCCACTTCTACCTCTGTTC
23
54.8
Pine (Pinus sylvestris) /
299
27
PS aF
TACCATGACCCAAGAGGAGT
20
55.0
constans-like 1 gene (Col1)
-
28
PS aR
CCCGAGGAGGAATCTCGAAT
20
56.1
40
29
LS F
TTAGCCGCTTGCATCCTC
18
55.3
-
30
LS R
CAAGAAGATGAGCGAAACAACAAC
24
54.9
Larch (Larix sukaczewii) /
253
31
LS aF
TTGACGGGAGAGTGGTTG
18
54.5
internal transcribed spacer 1 (ITS1)
-
32
LS aR
CGAACGGTTGCACATCCTG
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56.8
47 -
37
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19
Em protein gene (Emp1)
299
AC C
* calculated by OligoAnalyzer
Oak (Quercus robur) /
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Ct mean
SD
Ct mean
SD
10000
28.76
0.235
26.37
0.463
1000
32.86
0.096
30.93
0.394
100
36.49
0.445
34.25
10
39.96
0.253
37.85
0
-
-
40.48
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Abutting primers
Genome copies
0.160 0.496
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SD - standard deviation.
0.689
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Table 3. Threshold cycle (Ct) mean values.
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DNA amount (ng)
Dilution
Approximate number of genome copies
Ct mean (curve number) LS aF - LS aR
LS F - LS R
100
1000
19.0 (1)
- (2)
2
10-1
100
27.5 (3)
36.0 (4)
0.2
10-2
10
31.0 (5)
40.0 (6)
0.02
10
-3
1
38.0 (7)
44.5 (8)
0.002
10-4
0.1
- (9)
45.5 (10)
0
- (11)
45.5 (12)
-
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0
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20
16
Figure legends
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Fig. 1. The scheme of primer annealing positions.
Fig. 2. Temperature limits of PCR (DNA from A. mellifera). A – electrophoretic determination of denaturation temperature limits (1 s duration of denaturation step, PCR samples had all five primer pairs: AM F – AM R, AM aF – AM aR, AM aF – AM nR1, AM aF – AM nR2, AM aF – AM nR3; М
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– pUC19/MspI ladder). B – temperature profiles of denaturation, and C – temperature profiles of annealing: 1 - AM F – AM R primers pair, 2 - AM aF – AM nR3, 3 - AM aF – AM nR2, 4 - AM aF – AM nR1, 5- AM aF – AM aR.
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Fig. 3. qPCR sensitivity (DNA from M. religiosa). A-C - abutting primers (MR aF - MR aR), D-F conventional primers (MR F - MR R). A, D - amplification curves (number of genome copies are
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indicated (104, 103, 102, 101), NC - negative control). B, E - melting curves. C, F - standard plots.
Fig. 4. PCR with nearby primers: A – H. sapiens primers, B – A. mellifera primers. Lines: 1-4 and 912 – negative controls; 5-8 – "positive" samples ("+" DNA of H. sapiens); 13-16 – "positive" samples ("+" DNA of A. mellifera); 1, 5 - HS aF - HS aR primer pair; 2, 6 - HS aF - HS nR1; 3, 7 - HS aF - HS nR2; 4, 8 - HS aF - HS nR3 (М1 – 100 bp ladder); 9, 13 - AM aF - AM aR primer pair; 10, 14 - AM
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aF - AM nR1; 11, 15 - AM aF - AM nR2; 12, 16 - AM aF - AM nR3 (M2 - pUC19/MspI ladder).
Fig. 5. Inhibition of qPCR with DNA from L. sukaczewii (each curve represents one of two replicates for 10-fold serial dilutions of a standard DNA sample). A: curves 1, 3, 5, 7, 9, 11 - abutting primers
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(LS aF - LS aR); curves 2, 4, 6, 8, 10, 12 – conventional primers (LS F - LS R). B: electrophoretic
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analysis of qPCR results; number of lines coincide with curve numbers.
Fig. 6. Determination of rotten wood origin. A - rotten wood sample. B - re-amplification results (lines: 1 - P. sylvestris primers PS aF - PS aR, 2 - Q. robur primers QR aF - QR aR, 3 - P. obovata primers PO aF - PO aR, 4 - L. sukaczewii primers LS aF - LS aR, M - pUC19/MspI ladder); C sequence of cloned amplicon.
17
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