Research Article A Locked Nucleic Acid Probe Based on ...

Research ArticleA Locked Nucleic Acid Probe Based on Selective Salt-InducedEffect Detects Single Nucleotide Polymorphisms

Jing Zhang Huizhe Wu Qiuchen Chen Pengfei ZhaoHaishan Zhao Weifan Yao and Minjie Wei

Department of Pharmacology School of Pharmacy China Medical University Liaoning Key Laboratory of Molecular TargetedAnti-Tumor Drug Development and Evaluation No 77 Puhe Road Shenbei District Shenyang 110013 China

Correspondence should be addressed to Minjie Wei mjweicmuhotmailcom

Received 23 May 2015 Accepted 27 July 2015

Academic Editor Min Zhou

Copyright copy 2015 Jing Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Detection of single based genetic mutation by using oligonucleotide probes is one of the common methods of detecting singlenucleotide polymorphisms at known loci In this paper we demonstrated a hybridization system which included a buffer solutionthat produced selective salt-induced effect and a locked nucleic acidmodified 12 nt oligonucleotide probeThe hybridization systemis suitable for hybridization under room temperature By using magnetic nanoparticles as carriers for PCR products the SNPs(MDR1 C3435TA) from 45 volunteers were analyzed and the results were consistent with the results from pyrophosphoric acidsequencing The method presented in this paper differs from the traditional method of using molecular beacons to detect SNPs inthat it is suitable for research institutions lacking real-time quantitative PCR detecting systems to detect PCR products at roomtemperature

1 Introduction

Single nucleotide polymorphisms (SNPs) are a DNAsequence polymorphism induced by single nucleotidemutation within the genome It is one of the most commontypes of human inheritable mutation [1] Some of the SNPswithin the DNA not only interfere with the occurrence anddevelopment ofmultiple types of disease but also can be usedas potential diagnostic or prognostic molecular markerstherefore they play a more and more important role in theearly diagnosis or prognostic surveillance for personalizedand targeted therapies in patients [2 3] One of the mostcommon methods is to use oligonucleotide probes to detectSNPs at known loci Molecular beacons (MBs) are a typeof oligonucleotide probe with stem-loop structure whichhas demonstrated high specificity in SNPs detection [4 5]Due to the high complexity involved in the design of MBswhich usually requires the aid of a real-time quantitativePCR detecting system the cost of testing is very high A newtesting method with highly specific easy-to-design probeswithout requiring an equipment for temperature control ishighly advantageous

There were reports of ΔTm values reaching 26∘C for thedouble chain formed from the hybridization between LNAmodified 8 nt probes with complementary templates and SMtemplates [6] LNA is an oligonucleotide analog in whichthe 21015840-oxygen and the 41015840-carbon of ribose moiety are joinedvia O-methylene (oxy-LNA) bridge S-methylene (thio-LNA) bridge or amino-methylene (amino-LNA) bridgeThisdouble-loop structure of the ribose severely limits its ability totwist horizontally [7] The LNA-DNA double helix possessesmarkedly higher thermostability than that of the DNA-DNAdouble helix and at the same time LNA is less likely toapproach mismatched nucleotide during hybridization withwrong-pairing DNA therefore LNA demonstrates strongability to recognize wrong base pairings [8] So we proposethat within tailored buffer solutions LNA modified shortchain oligonucleotide probes can rapidly detect SNPs underroom temperature without being affected by temperaturefluctuation

The MDR1 gene is a type of genes that demonstratesmultidrug resistance [9] of which the C3435TA SNP isclosely related to the occurrences of multidrug resistance in

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 391070 6 pageshttpdxdoiorg1011552015391070

2 BioMed Research International

cancer cells [10] and this particular locus has been selectedas the target for analysis in this study

2 Materials and Methods

21 High Resolution Melting (HRM) The length of LNAmodified oligonucleotide probe was 12 nt purchased fromEXIQON (Holland) The probe C had the sequence of51015840-CTCACGL ATCTCT-31015840 and the probe A had the sequenceof 51015840-CTCACAL ATCTCT-31015840 The bases of upper right Llabelled are LNA in the probesThe templates were purchasedfrom Sangon bioengineering Co Ltd (Shanghai) withequal lengths of 107 nt The template sequence was obtainedfrom NCBI-SNP (httpwwwncbinlmnihgovsnp)which was 51015840-ACATTGCCTATGGAGACAACAGCC-GGGTGGTGTCACAGGAAGAGAT[CTA]GTGAGG-GCAGCAAAGGAGGCCAACATACATGCCTTCATC-GAGTCACTGCCTAATGTAAGT-31015840 with the SNP locusunderlined LC Green was purchased from Idaho (USA)Formamide was purchased from SIGMA The real-timePCR with HRM (Model LightCycler 48 II) was purchasedfrom ROCHE (Switzerland) Four types of PBS bufferwith Na+ concentration of 1M 05M 025M and 0125Mwere selected within which the LNA probes were allowedto hybridize with the complementary template and thesingle-base mismatch template (MS template) under eachconcentration The saturated fluorochrome LC green wasadded and temperature range was set between 20 and 90∘CHRM curves were produced by real-time PCR with HRMIn the comparator arm the LNA probe was replaced withDNA probe during hybridization with all other conditionsbeing the same as mentioned above When the optimal Na+concentration was confirmed different amounts of deionisedformamide (10VV 20VV 30VV and 40VV)were added to the hybridization process and HRM curveswere drawn

22 Coupling of Template and Magnetic Nanoparticles ThexMag Streptavidin magnetic nanoparticles and 8-hole mag-netic separator were purchased from GoldMag NanobiotechCo Ltd (Xirsquoan) Binding buffer (TE 1M NaCl and pH 74)was prepared PBST (PBS with Na+ concentration of 015Mand 005 Tween20) was prepared For detailed couplingprocedures please refer to Zhan et alrsquos work [11] Due to thefact that the allele from gene MDR1 C3435TA was triallelic3 templates were used CCTTAA CCTTAA A portion ofeach template was taken out to be mixed with a portion ofone other template until three heterozygous templates wereobtained CTCATA These six templates represent all thesubtypes of geneMDR1 C3435TA SNP

23 Simulated Hybridization The sequence and modifica-tion for Fluorescence Probe C were as follows 51015840-6FAM-CTCACGL ATCTCT-31015840 the sequence and modification forFluorescence Probe T were as follows 51015840-ROX-CTCACAL

ATCTCT-31015840 and the sequence and modification for Fluores-cence ProbeAwere as follows 51015840-ROX-CTCACTL ATCTCT-31015840 Nikon florescent microscope was purchased from JapanFluorescence Probe C was mixed with Fluorescence Probe

T with equal portions allowed to hybrid with 6 types oftemplates under 20 and 30 degrees hybridization time was 5minutes the purpose was to detect the allele C and T in geneMDR1 C3435TA To prevent the excitation wavelength andthe emission wavelength interfering with each other betweendifferent types of florescent dyes Probe A was not modifiedwith a third type of the fluorescence radical instead paralleltesting was performed in a separate vessel After hybridiza-tion was completed PBST was used to elute any unbindingprobes magnetic separator was used to separate and discardsupernatant nanoparticles were resuspended and 05 120583L ofthe mixed suspension solution was dropped onto a slide andanalyzed under the florescent microscope The 6FAM wasthe green florescent radical with the excitation wavelengthof 494 nm and ROX was the red florescent radical withthe excitation wavelength of 584 nm Fluorescent intensitywas read and linear range of concentration-intensity wasestablished based on 119883 (concentration) and 119884 (fluorescentintensity)

24 Testing on Real-Life Samples The sequence and modifi-cation for the sense primer were as follows Biotin-51015840ACA-TTGCCTATGGAGACAACA-31015840 and the sequence for thereverse primer was as follows 51015840-ACTTACATTAGGCAG-TGACTCG-31015840 Pfu DNA Polymerase expansion lab kitswere purchased from Sangon Biotech (Shanghai) Co Ltd(Shanghai) DNA denaturing liquid was prepared GradientPCR machine was purchased from Takara BIO Co Ltd(Japan) Gene TAC 2000Model CCD scanner was purchasedfromGenemic Solutions (USA)The sample typewas genomeDNA extracted from peripheral blood of human subjects atotal of 45 cases were supplied by the First Affiliated Hospitalof the China Medical University Firstly PCR reactionsoccurred within the following PCR reaction system 2 PfuDNA polymerase 25 120583L genomic DNA sample 1 120583L andsense primer and reverse primer with the concentration of200 nmoLL 2120583L each Ultrapure water 20120583L The PCRreaction steps were as follows denaturing 94∘C 4 minutesannealing 94∘C 30 seconds rarr 55∘C 30 seconds rarr 72∘C30 seconds repeated 30 times extension 72∘C 30 secondsThe PCR product had length of 107 bp and the sequence wasconsistent with the template

For the PCR products tagged with biotins the couplingprocess was the same as the coupling process of the templatesDue to the high amount of samples involved a CCD scannerwith the capability of high volume testing was used

3 Results and Discussion

31 Salt-Induced Effect The phosphate backbone of thenucleotide is negatively charged preventing any two singlechain DNAs getting too close together and the hybridizationreally relies on the base pairing principal hence reducingthe rate of pairing mismatch Though Na+ can neutralisethe negative charges of the phosphoric acid skeleton andpromote the single chain DNAs to get close to each otherit can improve the rate of hybridization in turn [12] Whenthe Na+ concentration gets even higher its ability to promotehybridization was able to overcome the repulsion effect

BioMed Research International 3Fl

uore

scen

ce (465

ndash510

)

652928

592928

532928

472928

412928

352928

292928

232928

112928

172928

52928

minus7072

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

[Na+] 0125M[Na+] 025M

[Na+] 05M[Na+] 1M

I

II

III

IV

V VII

VIII

VI

(a)

Fluo

resc

ence

(465

ndash510

)

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

[Na+] 0125M[Na+] 025M

[Na+] 05M[Na+] 1M

I

II

III

IV

V

VII

VIII

VI

886793

801793

716793

631793

546793

461793

291793

376793

206793

121793

36793

(b)

Figure 1 (a) The figure is showing the HRM curve for LNA probes with complementary template (complementary-curve) and the HRMcurve for LNA probes with single-basemismatch template (the SM-curve) Complementary-curve (I) and SM-curve (II) in buffer with [Na+]0125M complementary-curve (III) and SM-curve (IV) in buffer with [Na+] 025M complementary-curve (V) and SM-curve (VI) in bufferwith [Na+] 05M complementary-curve (VII) and SM-curve (VIII) in buffer with [Na+] 1M (b) The figure is showing the complementarycurves and SM curves for probes without the LNA modification and naming methods for these curves were the same as that of (a)

between the phosphoric acid skeletons and it significantlyimproves the rate of single chain DNA hybridization how-ever in the meantime it increases the rate of wrong basepairing When Na+ concentration was lower or even absentthe repulsion between phosphoric acid skeletons would besignificant so that the rate of wrong base pairing would berelatively low though the rate of hybridization between singlechain DNAs would also be very slow [13] In this study eithercomplementary template or single-base mismatch templatewith the same probe was hybridized in the same bufferrespectively We prepared 4 types of buffer for hybridiza-tion which had the different concentration of sodium only(0125M to 10M) Then the HRM curves were drew by real-time PCR with HRM Furthermore selective salt-inducedeffect was observed when the concentration of sodium was025M in the buffer which can induce hybridization reactionbetween two complementary single-strand DNAs howeverwithout the above special reaction between two single-basemismatch single-strand DNAs

In Figure 1(a) we could see that in the buffer with differ-ent Na+ concentrations two types of formation of the DNAfusion curves occurred The variations observed between thetwo types of fusion curves obtained depend on whether thebase at the mutation positions was complementary to theprobes [14] In the buffer with suitable Na+ concentrationthe complementary fusion curve and the SM curve fromthe same group demonstrated apparent ldquobranchingrdquo effectsThe complementary curves were affected by the salt-inducedeffect hence showing the same formation as the fusioncurves formed under high Na+ concentration (curves IIIand V) However SM-curve was not affected by the salt-induced effect under the same Na+ concentration henceshowing the same formation as the fusion curves formedunder low Na+ concentration (curves IV and VI) Thereforewe established that when Na+ concentration was between025 and 05M the salt-induced effect was selective and was

able to improve the ability of the LNA modified probes todetect SM-templates When we replaced LNA probes withDNA probes under the same conditions we obtained thefusion curves shown in Figure 1(b) and the selective salt-induced effect had a significant effect on LNA modifiedoligonucleotide probes but did not produce evident effectson the standard oligonucleotide probes without the LNAmodification Comparing with lower concentrations of Na+it was more suitable for low temperature hybridizationtherefore the reaction buffer with 025M [Na+] was used forthe subsequent study

32 Effects of Formamide in Buffer With the addition offormamide the Tm value of the probes was reduced andthe irregular secondary structures of the ssDNA templateswere opened up improving the chances of the probes toapproach targeted sequences to complete hybridization [15]By adding different concentration of formamide in the [Na+]025M buffer we observed that the HRM curves for boththe probes and templates were shifted horizontally towardslower temperature range the amount shifted horizontally wasdirectly proportional to the amount of formamide added andthe addition of formamide did not interfere with theNa+ salt-induced effect as shown in Figure 2(a) When formamideconcentration was 20 the Tm value for the LNA-probe andSM template was 3470∘C as shown in Figure 2(b) curveIV It was commonly believed that when Tm value of theprobes was lower than 15∘C it was more suitable for completehybridization therefore 20 (vv) formamide was selectedto be used in follow-up testing

33 Analytical Results from Fluorescent Probes Figures 3(a)and 3(b) are showing the testing results from fluorescentprobes under 20∘C and 30∘C respectively It could be seenthat the testing results for fluorescent probes to each typesof template are identical to the known sequences The


40 formamide30 formamide


Fluo

resc

ence

(465

ndash510

)

508100

463100

418100

373100

328100

283100

238100

193100

148100

103100

58100

13100

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIIIVI

(a)



Fluo

resc

ence

(465

ndash510

) 1756916069

14569

13069

11569

10069

8569

7069

5569

4069

2569

1069

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIII

VI

(b)

Figure 2 (a) The figure is showing the HRM curve for LNA probes with complementary template and SM template when different VVratios of formamide were added during hybridization complementary-curve (I) and SM-curve (II) in buffer containing 10 formamidecomplementary-curve (III) and SM-curve (IV) in buffer containing 20 formamide complementary-curve (V) and SM-curve (VI) in buffercontaining 30 formamide complementary-curve (VII) and SM-curve (VIII) in buffer containing 40 formamide (b)The figure is showingthe first-order derivative curves of (a) and naming methods for these curves were the same as that of (a)

Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T

A

Probe C Probe T Probe A

(a)

Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T

AProbe C Probe T Probe A

(b)

Figure 3 (a) Testing results for fluorescent probe under 20∘C The three pictures on each row are showing the results from the three probesto each known template In the first row if green fluorescent was detected it showed that the SNP locus ofMDR1 C3435TA gene containedallele C in the tested sample In the middle row if red fluorescent was detected it showed that the SNP locus contained allele T in the testedsample in the third row if red fluorescent was detected it showed that the SNP locus contained allele A in the tested sample (b) Testingresults for fluorescent probe under 30∘CThe method of analysis was the same as that described in (a)

BioMed Research International 5

Fluo

resc

ence

inte

nsity

700

500

300

100

The concentration of template (nM)10

minus110

110

310

5

Figure 4 On this linear graph coordinate axis 119883 is the log of the template concentration coordinate axis 119884 is the intensity of the greenfluorescent

(a) (b) (c)

Figure 5 (a) The graphic results from CCD scanner under excitation wavelength of 494 nm samples with green fluorescent carry allele C(b) the graphic results from CCD scanner under excitation wavelength of 584 nm samples with red fluorescent carry allele T (c) mergedgraph of (a) and (b)

fluorescent intensity was read and any reading higher thanbackground level was considered positive linear range was1 nMsim10000 nM and testing limit was 01 nmoLL as shownin Figure 4

Lastly we performed MDR1 C3435TA SNP testing ongenetic samples exacted from the peripheral blood from 45volunteers High throughput was achieved by using fluores-cent microarray scanners From Figure 5(a) we could seethat allele C was not detected in three samples and fromFigure 5(b) we could see that allele T was not detected in4 samples and the results were more evident in Figure 5(c)which was a merged diagram of (a) and (b) Allele A was notdetected in any of the above-mentioned samples The PCRproducts from all sampleswere sent to Shanghai Bioengineer-ing for pyrophsphoric acid sequencing to compare with theresults listed above and conclusions were consistent Allele A

in geneMDR1 C3435TA in human population is rather rare[16 17] even though this subtype was not detected in the reallife samples used in this study based on Figure 5 it could bedetected by the method described in this study

34 Advantages of Short-Chain LNA Probes LNA has aunique double-loop structure When hybridization occurredbetween LNA and wrong base pairings its double-loopstructure markedly restricted the spatial twist of the LNAribosome comparing with DNA probes this noncomple-mentary spatial formation resulted in hybrids with relativelyhigh thermodynamic instability hence LNAprobe hasmuchhigher specificity against single wrong base pairings If moremodified LNA were added in the probes the Tm value willbe significantly increased [18] The elute process can alsoproduce great effects on the testing results After mismatched


hybridization of short chain probes because the amount ofcomplementary bases was relatively low noncomplementaryspatial formation produces even higher effects on the stabilityof hybrids and mismatched probes could be removed withsimple elution This is one of the advantages of short chainprobes as long as specificity could be maintained the shorterthe chain the better the probes

4 Conclusion

For this hybrid system that we developed in this study forthe rapid detection of SNPs under room temperature therewere three key elements involved first a buffer solutionthat was suitable for hybridization under room temperatureand had the salt-induced effect that enhanced the abilityof the LNA probes to detect wrong single-base pairingssecond the LNA modification of the oligonucleotide probeswhich significantly increased the specificity of the probes andwith the aid of the buffer was able to detect SNPs withoutbeing affected by room temperature fluctuations third therelatively short oligonucleotide chain which made testingunder room conditions much easier to perform enabled theconformation of the wrongly paired LNA to produce greatereffect on thermodynamic stability of the hybrids and alsoutilized the characteristic of the magnetic nanoparticles toelute noncomplementary probes easily under room temper-ature Single-base mismatch can be detected under roomtemperature of 20∘Cndash30∘C with high specificity

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

Thisworkwas supported by Program for Liaoning InnovativeResearch Team in University (LT2014016) and SampT PlanedProjects in Shenyang City (F14-232-6-05) (no F12-148-9-00)

References

[1] D C Crawford D T Akey and D A Nickerson ldquoThe patternsof natural variation in human genesrdquo Annual Review ofGenomics and Human Genetics vol 6 pp 287ndash312 2005

[2] D J Jonker C J OrsquoCallaghan C S Karapetis et al ldquoCetuximabfor the treatment of colorectal cancerrdquoTheNew England Journalof Medicine vol 357 no 20 pp 2040ndash2048 2007

[3] T E Meyer E Boerwinkle A C Morrison et al ldquoDiabetesgenes and prostate cancer in the atherosclerosis risk in commu-nities studyrdquo Cancer Epidemiology Biomarkers and Preventionvol 19 no 2 pp 558ndash565 2010

[4] J Guo J Ju and N J Turro ldquoFluorescent hybridization probesfor nucleic acid detectionrdquo Analytical and Bioanalytical Chem-istry vol 402 no 10 pp 3115ndash3125 2012

[5] K Wang Z Tang C J Yang et al ldquoMolecular engineering ofDNA molecular beaconsrdquo Angewandte Chemie InternationalEdition vol 48 pp 856ndash870 2009

[6] P Mouritzen A T Nielsen H M Pfundheller Y Choleva LKongsbak and S Moslashller ldquoSingle nucleotide polymorphismgenotyping using locked nucleic acid (LNA)rdquo Expert Review ofMolecular Diagnostics vol 3 no 1 pp 27ndash38 2003

[7] L Wang C J Yang C D Medley S A Benner and WTan ldquoLocked nucleic acid molecular beaconsrdquo Journal of theAmerican Chemical Society vol 127 no 45 pp 15664ndash156652005

[8] M Petersen and J Wengel ldquoLNA a versatile tool for therapeu-tics and genomicsrdquo Trends in Biotechnology vol 21 no 2 pp74ndash81 2003

[9] A Johne K Kopke T Gerloff et al ldquoModulation of steady-statekinetics of digoxin by haplotypes of the P-glyeoProtein MDR1generdquo Clinical Pharmacology amp Therapeutics vol 72 no 5 pp584ndash594 2002

[10] B Chowbay H Li M David Y B Cheung and E J D LeeldquoMeta-analysis of the influence of MDR1 C3435T polymor-phism on digoxin pharmacokinetics and MDR1 gene expres-sionrdquo British Journal of Clinical Pharmacology vol 60 no 2 pp159ndash171 2005

[11] Z Zhan X Ma C Cao and S J Sim ldquoGold-based opticalbiosensor for single-mismatched DNA detection using salt-induced hybridizationrdquo Biosensors and Bioelectronics vol 32no 1 pp 127ndash132 2012

[12] I Turel and J Kljun ldquoInteractions of metal ions with DNAits constituents and derivatives which may be relevant foranticancer researchrdquoCurrent Topics inMedicinal Chemistry vol11 no 21 pp 2661ndash2687 2011

[13] S Mishra S Ghosh and R Mukhopadhyay ldquoRegulating theon-surface lna probe density for the highest target recognitionefficiencyrdquo Langmuir vol 30 no 34 pp 10389ndash10397 2014

[14] C T Wittwer ldquoHigh-resolution DNA melting analysisadvancements and limitationsrdquo Human Mutation vol 30 no6 pp 857ndash859 2009

[15] M Carbonari M Cibati N Sette A Catizone and M FiorillildquoMeasurement of telomere length using PNA probe by cytome-tryrdquoMethods in Cell Biology vol 103 pp 189ndash202 2011

[16] E Jazwinska-Tarnawska I Jęskowiak E Waszczuk et alldquoGenetic polymorphism of ABCB1 gene (C3435T) in patientswith inflammatory bowel diseases Is there any gender depen-dencyrdquo Pharmacological Reports vol 67 no 2 pp 294ndash2982015

[17] H Chai J Pan X Zhang et al ldquoERCC1 C118T associates withresponse to FOLFOX4 chemotherapy in colorectal cancerpatients in Han Chineserdquo International Journal of Clinical andExperimental Medicine vol 5 no 2 pp 186ndash194 2012

[18] Q Wang L Chen Y Long H Tian and J Wu ldquoMolecularbeacons of xeno-nucleic acid for detecting nucleic acidrdquoThera-nostics vol 3 no 6 pp 395ndash408 2013

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cancer cells [10] and this particular locus has been selectedas the target for analysis in this study

2 Materials and Methods

21 High Resolution Melting (HRM) The length of LNAmodified oligonucleotide probe was 12 nt purchased fromEXIQON (Holland) The probe C had the sequence of51015840-CTCACGL ATCTCT-31015840 and the probe A had the sequenceof 51015840-CTCACAL ATCTCT-31015840 The bases of upper right Llabelled are LNA in the probesThe templates were purchasedfrom Sangon bioengineering Co Ltd (Shanghai) withequal lengths of 107 nt The template sequence was obtainedfrom NCBI-SNP (httpwwwncbinlmnihgovsnp)which was 51015840-ACATTGCCTATGGAGACAACAGCC-GGGTGGTGTCACAGGAAGAGAT[CTA]GTGAGG-GCAGCAAAGGAGGCCAACATACATGCCTTCATC-GAGTCACTGCCTAATGTAAGT-31015840 with the SNP locusunderlined LC Green was purchased from Idaho (USA)Formamide was purchased from SIGMA The real-timePCR with HRM (Model LightCycler 48 II) was purchasedfrom ROCHE (Switzerland) Four types of PBS bufferwith Na+ concentration of 1M 05M 025M and 0125Mwere selected within which the LNA probes were allowedto hybridize with the complementary template and thesingle-base mismatch template (MS template) under eachconcentration The saturated fluorochrome LC green wasadded and temperature range was set between 20 and 90∘CHRM curves were produced by real-time PCR with HRMIn the comparator arm the LNA probe was replaced withDNA probe during hybridization with all other conditionsbeing the same as mentioned above When the optimal Na+concentration was confirmed different amounts of deionisedformamide (10VV 20VV 30VV and 40VV)were added to the hybridization process and HRM curveswere drawn

22 Coupling of Template and Magnetic Nanoparticles ThexMag Streptavidin magnetic nanoparticles and 8-hole mag-netic separator were purchased from GoldMag NanobiotechCo Ltd (Xirsquoan) Binding buffer (TE 1M NaCl and pH 74)was prepared PBST (PBS with Na+ concentration of 015Mand 005 Tween20) was prepared For detailed couplingprocedures please refer to Zhan et alrsquos work [11] Due to thefact that the allele from gene MDR1 C3435TA was triallelic3 templates were used CCTTAA CCTTAA A portion ofeach template was taken out to be mixed with a portion ofone other template until three heterozygous templates wereobtained CTCATA These six templates represent all thesubtypes of geneMDR1 C3435TA SNP

23 Simulated Hybridization The sequence and modifica-tion for Fluorescence Probe C were as follows 51015840-6FAM-CTCACGL ATCTCT-31015840 the sequence and modification forFluorescence Probe T were as follows 51015840-ROX-CTCACAL

ATCTCT-31015840 and the sequence and modification for Fluores-cence ProbeAwere as follows 51015840-ROX-CTCACTL ATCTCT-31015840 Nikon florescent microscope was purchased from JapanFluorescence Probe C was mixed with Fluorescence Probe

T with equal portions allowed to hybrid with 6 types oftemplates under 20 and 30 degrees hybridization time was 5minutes the purpose was to detect the allele C and T in geneMDR1 C3435TA To prevent the excitation wavelength andthe emission wavelength interfering with each other betweendifferent types of florescent dyes Probe A was not modifiedwith a third type of the fluorescence radical instead paralleltesting was performed in a separate vessel After hybridiza-tion was completed PBST was used to elute any unbindingprobes magnetic separator was used to separate and discardsupernatant nanoparticles were resuspended and 05 120583L ofthe mixed suspension solution was dropped onto a slide andanalyzed under the florescent microscope The 6FAM wasthe green florescent radical with the excitation wavelengthof 494 nm and ROX was the red florescent radical withthe excitation wavelength of 584 nm Fluorescent intensitywas read and linear range of concentration-intensity wasestablished based on 119883 (concentration) and 119884 (fluorescentintensity)

24 Testing on Real-Life Samples The sequence and modifi-cation for the sense primer were as follows Biotin-51015840ACA-TTGCCTATGGAGACAACA-31015840 and the sequence for thereverse primer was as follows 51015840-ACTTACATTAGGCAG-TGACTCG-31015840 Pfu DNA Polymerase expansion lab kitswere purchased from Sangon Biotech (Shanghai) Co Ltd(Shanghai) DNA denaturing liquid was prepared GradientPCR machine was purchased from Takara BIO Co Ltd(Japan) Gene TAC 2000Model CCD scanner was purchasedfromGenemic Solutions (USA)The sample typewas genomeDNA extracted from peripheral blood of human subjects atotal of 45 cases were supplied by the First Affiliated Hospitalof the China Medical University Firstly PCR reactionsoccurred within the following PCR reaction system 2 PfuDNA polymerase 25 120583L genomic DNA sample 1 120583L andsense primer and reverse primer with the concentration of200 nmoLL 2120583L each Ultrapure water 20120583L The PCRreaction steps were as follows denaturing 94∘C 4 minutesannealing 94∘C 30 seconds rarr 55∘C 30 seconds rarr 72∘C30 seconds repeated 30 times extension 72∘C 30 secondsThe PCR product had length of 107 bp and the sequence wasconsistent with the template

For the PCR products tagged with biotins the couplingprocess was the same as the coupling process of the templatesDue to the high amount of samples involved a CCD scannerwith the capability of high volume testing was used

3 Results and Discussion

31 Salt-Induced Effect The phosphate backbone of thenucleotide is negatively charged preventing any two singlechain DNAs getting too close together and the hybridizationreally relies on the base pairing principal hence reducingthe rate of pairing mismatch Though Na+ can neutralisethe negative charges of the phosphoric acid skeleton andpromote the single chain DNAs to get close to each otherit can improve the rate of hybridization in turn [12] Whenthe Na+ concentration gets even higher its ability to promotehybridization was able to overcome the repulsion effect


uore

scen

ce (465

ndash510

)

652928

592928

532928

472928

412928

352928

292928

232928

112928

172928

52928

minus7072

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

[Na+] 0125M[Na+] 025M

[Na+] 05M[Na+] 1M

I

II

III

IV

V VII

VIII

VI

(a)

Fluo

resc

ence

(465

ndash510

)

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

[Na+] 0125M[Na+] 025M

[Na+] 05M[Na+] 1M

I

II

III

IV

V

VII

VIII

VI

886793

801793

716793

631793

546793

461793

291793

376793

206793

121793

36793

(b)










Fluo

resc

ence

(465

ndash510

)

508100

463100

418100

373100

328100

283100

238100

193100

148100

103100

58100

13100

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIIIVI

(a)



Fluo

resc

ence

(465

ndash510

) 1756916069

14569

13069

11569

10069

8569

7069

5569

4069

2569

1069

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIII

VI

(b)


Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T

A


(a)

Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T


(b)



Fluo

resc

ence

inte

nsity

700

500

300

100


minus110

110

310

5


(a) (b) (c)








4 Conclusion




Acknowledgments


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















uore

scen

ce (465

ndash510

)

652928

592928

532928

472928

412928

352928

292928

232928

112928

172928

52928

minus7072

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

[Na+] 0125M[Na+] 025M

[Na+] 05M[Na+] 1M

I

II

III

IV

V VII

VIII

VI

(a)

Fluo

resc

ence

(465

ndash510

)

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

[Na+] 0125M[Na+] 025M

[Na+] 05M[Na+] 1M

I

II

III

IV

V

VII

VIII

VI

886793

801793

716793

631793

546793

461793

291793

376793

206793

121793

36793

(b)










Fluo

resc

ence

(465

ndash510

)

508100

463100

418100

373100

328100

283100

238100

193100

148100

103100

58100

13100

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIIIVI

(a)



Fluo

resc

ence

(465

ndash510

) 1756916069

14569

13069

11569

10069

8569

7069

5569

4069

2569

1069

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIII

VI

(b)


Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T

A


(a)

Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T


(b)



Fluo

resc

ence

inte

nsity

700

500

300

100


minus110

110

310

5


(a) (b) (c)








4 Conclusion




Acknowledgments


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Fluo

resc

ence

(465

ndash510

)

508100

463100

418100

373100

328100

283100

238100

193100

148100

103100

58100

13100

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIIIVI

(a)



Fluo

resc

ence

(465

ndash510

) 1756916069

14569

13069

11569

10069

8569

7069

5569

4069

2569

1069

Temperature (∘C)20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

I

II

III

IV

VVII

VIII

VI

(b)


Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T

A


(a)

Tem

plat

e-C

Tem

plat

e-T

Tem

plat

e-A

Tem

plat

e-C

TTe

mpl

ate-

CA

Tem

plat

e-T


(b)



Fluo

resc

ence

inte

nsity

700

500

300

100


minus110

110

310

5


(a) (b) (c)








4 Conclusion




Acknowledgments


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Fluo

resc

ence

inte

nsity

700

500

300

100


minus110

110

310

5


(a) (b) (c)








4 Conclusion




Acknowledgments


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Conclusion




Acknowledgments


References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article A Locked Nucleic Acid Probe Based on ...

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