Download Poly-linker primer system for detection of single nucleotide

Acta Biochim Biophys Sin, 2015, 47(2), 139–141
doi: 10.1093/abbs/gmu115
Advance Access Publication Date: 17 December 2014
Lab Note
Lab Note
Poly-linker primer system for detection of single
nucleotide polymorphisms and its application
in genotyping hepatitis B virus
Yongzhong Wang1,*,†, Bo-Yu Xue2,†, Longgen Liu2, Zhen Zhu1, Xin Xu1,
Hongyu Zhang1, Xiangke Pu1, and Xiaoqin Liu1
1
Laboratory Centre, The Third People’s Hospital of Changzhou, Changzhou 213001, China, and 2University of Nanjing
Traditional Chinese Medicine, Nanjing 210023, China
†
These authors contributed equally to this work.
*Correspondence address. Tel: +86-519-83016186; Fax: +86-519-83016007; E-mail: [email protected]
Single nucleotide polymorphisms (SNPs) and single-base mismatch
in genome are closely associated with disease development, progress,
treatment, and prognosis [1]. Many methods have been developed to
detect SNPs. Some assays, such as Taqman MGB probe and high
resolution melting, amplify and detect both wild-type and mutant
genomic DNA by specific primers and probes [2], while some other
assays, such as ARMS and PNA-LNA clamp PCR, only amplify and
detect mutant templates [2,3]. Amplification Refractory Mutation
System is widely used to detect SNP and mutations; however, the detection limit is ∼1% to 5% of mutants in wild-type DNA content [4].
PNA-LNA clamp PCR is very sensitive and specific, but it is based on
nest PCR and just a lab assay [5]. For the detection of drug-resistant
genotypes of virus, such as hepatitis B virus (HBV), it is necessary to
detect a few mutant templates at high level of wild-type DNA background. Recently, a dual priming oligonucleotide system (DPO) has
been reported that poly-linker inserted into a primer formed a
bubble-like structure, which would block the extension of nonspecifically primed templates, and generate consistently high PCR
specificity [6]. It greatly improves the distinguishing ability of primers for the detection of SNPs.
In this study, we reported a new real-time PCR-based assay for the
detection of SNPs and single-base mismatch. The method is based on
poly-linker primer system (PLPS) which is an improvement of DPO.
The poly-linker primer contains four parts: (i) 5′-segment tail: 2–
3 nt natural bases added to 5′-segment terminal, and not complementary with target sequence at the same position; (ii) anchor: 5′-segment
16–20 nt in length; (iii) 3 nt poly-linker (5′-III-3′), and (iv) distinguishing part: 3′-segment 6–7 nt in length. We hypothesize that the inserted
3 nt deoxyinosines (dI) which locates at primer 3′ segment n-6–7 position without bases mapping in the target sequence endows the SNP
distinguishing ability of the primers. We used this improved assay
for genotyping of HBV, which validated the ability of the assay to distinguish SNPs or single-base mismatch.
Serum samples from 159 patients were randomly retrieved from
the Third People’s Hospital of Changzhou. This study was approved
by the Ethics Committee of The Third People’s Hospital of Changzhou. All participants provided their written informed consent to participate in this study. HBV viral DNA was isolated from 200 μl of
serum samples using the QIAamp DNA blood mini kit (Qiagen,
Hilden, Germany) and eluted into 50 μl of buffer according to manufacturer’s instructions. Full-length HBV genotype B and C genomes
were cloned according to the method of Parekh et al. [7]. The plasmids
were serially diluted from 1 × 109 to 1 × 102 IU/ml by using NanoDrop
2000 (Thermal Fisher Scientific, New Hampshire, USA) and used as
control. Poly-linker primers and TaqMan probes for genotyping
HBV are listed in Table 1. The RT-PLPS was performed in 50 μl of
reaction mixture containing 0.15 μM PLPS primers, 0.1 μM probe,
2.5 mM Mg2+, 0.2 μM dNTP, 20 mM Tris–HCl ( pH 8.0), 0.1 mM
EDTA, 1 mM DTT, 50 mM KCl, 50% glycerol, and 2U Hotstart
Taq DNA polymerase (BiOSCI, Shanghai, China). The amplification
conditions were as follows: 94°C for 10 min, 10 cycles of 94°C for
20 s, 58°C for 30 s, 72°C for 30 s, and then 30 cycles of 95°C for
20 s, 58°C for 40 s, 72°C for 20 s. The PCR was carried out on a
7500 real-time PCR (Applied Biosystems, Foster City, USA). DNA sequencing and analysis of HBV genotypes were described as previously
reported [8]. PCR products were separated by electrophoresis, purified
by using a Qiagen gel extraction kit, and sequencing was done with the
BigDye terminator cycle-sequencing reaction kit and Prism 3730 DNA
analyzer (Applied Biosystems).
RT-PLPS is an improvement of DPO system. Three nt dI instead of
5 nt in DPO are inserted into the natural base primer, located at
3′-segment n-6–8 position. Due to weak hydrogen bonding, 3 nt polylinker easily forms a ‘bubble-linker’ structure. There are three forms of
‘bubbles’ when the poly-linker primer binds to the template, called
‘small’, ‘middle’, and ‘large’ bubbles. When the3 nt natural bases at
targets complements with 3 nt dI poly-linker (3 vs. 3), a ‘small’ bubble
© The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences.
139
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Poly-linker primer system for detection of SNPs
Table 1. Primers and probes for genotyping HBV genotypes B and C
Name
Primers and probe for HBV genotype B
Forward
Reverse
Probe
Primers and probe for HBV genotype C
Forward 1
Forward 2
Forward 3
Forward 4
Forward 5
Forward 6
Reverse
Probe
Sequences
5′-CCCGCAGTCCCAAATCTIIICCAGTC-3′
5′-AGATGAGGCATAGCAGCAGGAT-3′
FAM-5′-AGGAAGATGATAAAACGCCGCAGA-3′-TAMRA
5′-CCTTGCCCGTTTGTCCTIIICTACTT-3′
5′-CCTGTTGCCCGTTTGTCCIIICTACTT-3′
5′-CCTGTTGCCCGTTTGTCIIICTACTT-3′
5′-CCTGTTGCCCGTTTGTIIICTACTT-3′
5′-CCTTGCCCGTTTGTCCTIIIICTACTT-3′
5′-CCTTGCCCGTTTGTCCTIIIIITCTACTT-3′
5′-CCAAGATGATGGGATGGGAAT-3′
Hex-5′-ACAGCAACAAGAGGGAAACATAGAGGT-3′-TAMRA
Figure 1. Principle of poly-linker primer system (A) A draft of the bubble formed in the PLPS. (B) Examples of the small, middle, and the big bubbles. (C) The
process of the PLPS amplification.
is formed. The ‘3 vs. 1 or 3 vs. 2’ forms middle bubbles, and ‘3 vs. 0’
forms ‘large’ bubbles (Fig. 1A). We hypothesize that poly-linker primer carried ‘large’ bubble linker has more excellent ‘single-base mismatch’ distinguishing ability than others.
To demonstrate the sensitivity and specificity of different types of
poly-linker primer, HBV genotype C was used as an example. In
China, the most prevalent HBV genotypes are Genotypes B and
C. By comparison of genotype A–H complete genome, HBV could
be genotyped by detection of single-base mismatch (Supplementary
Fig. S1). Poly-linker primers 1–4 with 3 nt dI insertion forming
‘small’, ‘middle’ and ‘large’ bubble-linker were designed to detect
HBV genotype C. Primers were used to detect 1 × 102 IU/ml−1 × 106
IU/ml full-length plasmids of HBV genotype C. All primers ( p1–p4)
could detect 1 × 102 IU/ml HBV genotype C plasmid. For the same
concentration of the plasmids, Ct value was almost the same, with
<0.5 cycle variation.
To evaluate the specificity of the four primers, experiments were
carried out to detect 1 × 108 and 1 × 107 IU/ml Genotype B plasmids, respectively. The results proved our hypothesis that Primer
1 (forms large bubble) was more specific than the others. The Ct values were 26.45 + 0.34 and 27.94 + 0.30, respectively, for the detection of 1 × 108 and 1 × 107 IU/ml Genotype B plasmid. It was about
two cycles delay compared with detection of 2 × 102 IU/ml Genotype C plasmids (Supplementary Fig. S2A). When Primers 2–4 (carrying ‘small’ and ‘middle’ bubble-linker) were used to detect
1 × 108 IU/ml Genotype B plasmids, the Ct values were 19.9 + 1.01,
22 + 0.49, and 20.75 + 0.99, respectively. When Primers 2–4 were
used to detect 1 × 10 7 IU/ml Genotype B plasmid, the C t value
were almost the same as that in the detection of 1 × 102 IU/ml Genotype C plasmids (Supplementary Fig. S2B–D). So the poly-linker
primer that formed ‘large’ bubble-linker has better single-base mismatch detection ability.
To further evaluate the number of dI inserted, Primers 5 and 6 contained 4 and 5 nt dI forming ‘large’ bubble-linker were applied to detect serially diluted plasmids (1 × 107–1 × 102 IU/ml) of Genotype C
plasmids and 1 × 108 IU/ml∼1 × 107 IU/ml Genotype B plasmids, respectively. It was found that the more dI inserted, the higher the specificity was acquired, but some sensibility was lost. Only 1 × 103 IU/ml
HBV Genotype C could be stably amplified by Primers 5 and 6 (Supplementary Fig. S3A,B). Poly-linker primer with 3 nt dI inserted and
formed ‘large (3 vs 0)’ bubble-linker had excellent capability of distinguishing single-base mismatch.
To evaluate the distinguishing ability of HBV genotypes B and C
mixed population, 1 × 107 IU/ml mixed population T1 and T2 were
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Poly-linker primer system for detection of SNPs
used. T1 mixtures contained: 50%, 10%, 1%, 0.1, 0.01%, and
0.001% Genotype C full-length genome DNA and 1 × 107 IU/ml Genotype B, respectively; T2 mixtures included 0.001% to 50% Genotype B
genome DNA and 1 × 107 IU/ml Genotype C. RT-PLPS could detect
0.01% Genotype B or C contained in mixed populations. A total of
159 clinical samples got from HBV infected patients were detected by
PLPS and sequencing. The results are summarized in Supplementary
Table S1. Among the 159 samples, 48 were Genotype B, 108 were
Genotype C, and 3 were B/C mixture. The results in 95.5% of the samples were completely consistent with the sequencing results.
In summary, we have developed a new, sensitivity and specificity
SNP detection system, RT-PLPS, which is an improvement of the
DPO system. First, the DPO system was originally developed for multiple PCR. To block non-specific amplification, 5 nt dI are inserted
into natural base primers and there are five bases in target sequence
complementary with 5 nt poly-linker, forming analogously a ‘small’
bubble-linker. As for PLPS, 3 nt dI insertion at n-6–7 position at
3′-segment formed a ‘large’ bubble-linker (3 vs. 0). Second, in PLPS,
two bases are added to primer 5′-segment terminal to promote sensitivity and specificity of poly-linker primer (data not shown). Third, the
distinguishing part of PLPS is composed of six to seven base sequence,
and single-base mismatch or SNP detection position is located at the
3′-segment n-3 position. Due to these improvements, RT-PLPS exhibits excellent SNP detection ability. The RT-PLPS could detect 1 × 102
IU/ml HBV, and detect 0.01% mutants in 1 × 107 IU/ml wild-type
templates. When RT-PLPS was used to genotype HBV, 159 clinical
samples were genotyped, and the results are almost completely consistent with those by sequencing.
Many reports have shown that HBV could be genotyped by detecting single base or two bases disparity [8,9]. Accumulated data demonstrated that HBV genotypes influence the clinical profile, anti-virus
drug-response, the long-term prognosis, and seroconversion [10]. In
China, Genotypes B and C take great dominance compared with
other genotypes, and Genotype C is more prevalent than Genotype
B. In addition, HBV genotype C can cause more danger for liver cirrhosis and hepatocellular carcinoma [11], lower response rate to antivirus drugs, and higher rate of A1762T/G1764A double mutation
in the basal core promoter region [12]. So HBV genotyping is very
important for the adjustment of the treatment program and long-term
prognosis.
Several assays have been developed to genotype HBV by detection
of single- or two-base disparity. However, those assays either need an
additional support from melting-curve analyses [9], or are based on
Taqman probe [8]. Although these methods are very sensitive, nonspecific amplification is still an unsolved issue. The RT-PLPS improves
DPO system. By using TaqMan probe as signal system, RT-PLPS
effectively avoids non-specific amplification, and realizes real-time
detection. In conclusion, the RT-PLPS is a novel SNP and single-base
mismatch detection assay which can be widely used to develop
molecular assays useful in clinical labs.
Supplementary Data
Supplementary data are available at ABBS online.
Funding
This study was supported by a grant from the Key Program of the
Board of Health of Changzhou (No. ZD201312).
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