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Enhanced PCR amplification incorporating 5’ primer flap sequences and free energy values near equilibrium
Oklahoma State University, National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Department of Entomology and Plant Pathology, 127 Noble Research Center, Stillwater, OK 74078.
M. Arif, I. Oikonomakos, D. J. Caasi and F. M. Ochoa-Corona
• The objective of this study is to enhance the sensitivity of PCR-based detection by
combining primer design, optimal thermodynamics and 5’ AT-rich non-complementarysequences.
• Primers with AT-rich non-complementary sequences, i.e. 5′-AATAAATCATAA, located at
the 5’ terminal position were reported to increase PCR amplification yield (1).• Primers with ΔG value circa zero perform well in broad gradients of melting temperature
(Tm) (2).• Primer quality can be qualitatively assessed using reference ∆G values and
bioinformatics tools such as mFOLD and Primer3 during primer design (3).• The polymerase chain reaction (PCR) is a cornerstone technique in molecular biology,
with extended use and application in microbial forensics and agricultural biosecurity.
INTRODUCTION
MATERIALS AND METHODS1. Optimal and suboptimal primers were designed based on the sequence of the
movement protein gene of Tobacco mosaic virus (TMV) and the rDNA subunit ofPythium cryptoirregulare (Pc), using the Web interface software pathway Primer3-mFOLD-BLASTn (Fig.1), (3).
2. Two pairs are optimal, ‘TMV-2’ (ΔG = 0; product size 195 bp) & Pc-1 (ΔG = 0; productsize 434 bp) , and two other pairs are suboptimal ‘TMV-3’ (ΔG = ~1; product size 192
bp) & ‘Pc-2’ (ΔG = ~1; product size 454 bp).
(A)
RESULTSE-mail: [email protected] and [email protected]
Primer Set
TMV-3;
ΔG = ~1
TMV-2;
ΔG = 0
Increase in PCR
yield (ng/µl)
PCR yield (ng/µl)
without 5’ flap
20.70 37.35 16.65 (80.4%)
PCR yield (ng/µl)
with 5’ flap
23.80 42.35 18.55 (77.9%)
Table 1. DNA quantification of one-step RT-PCR products from
eluted bands using Nanodrop®
Primers having optimal ∆G and 5’ AT-rich non-complementary nucleotides widened the range of
effective annealing temperatures of PCR assays (Fig. 4), improving also sensitivity (Fig. 5), andPCR- product yields (Table 1 & Fig. 6).
Fig. 4. TMV-gradient one step RT-PCR amplification. Tms ranged from 48⁰⁰⁰⁰C to 66⁰⁰⁰⁰C (A & B) and 48⁰⁰⁰⁰C to 62⁰⁰⁰⁰C (C & D). TMV-2 optimal primer set, (A)
without and (B) with 5’ AT-rich non-complementary nucleotides sequences. TMV-3 suboptimal primer set, (C) without and (D) with 5’ AT-rich non-
complementary nucleotides sequences.
(C)
(D)
(A)
(B)
1 Kb 48 48.5 49.6 50.9 53.0 55.6 58.6 61.2 63.2 64.6 65.6 66.0
Temperature in ºC
1 Kb 48 48.4 49.2 50.3 51.9 54.0 56.3 58.3 59.9 60.9 61.7 62.0
Temperature in ºC
200bp
100bp
100bp
200bp100bp200bp
100bp200bp
(A)
(B)
(C)
(D)
100bp200bp
100bp200bp
100bp200bp
100bp200bp
RNA template in ng/reaction
M 500 5 0 5 0.5 0.05 0.005 0.0005 0.00005
RNA template in ng/reaction
M 500 50 5 0.5 0.05 0.005 0.0005 0.00005
(A)
(B)
Fig. 1. (A) Location of TMV-2 optimal (blue) and TMV-3 suboptimal (red) primers in the TMV genome. (B) Location of Pc-1 optimal and
Pc-2 suboptimal primers in the P. cryptoirregulare rDNA subunit.
3. The effect of combining free energy values (ΔG) selected during primer design (Fig. 2)
with and without the addition of 5’ AT-rich non-complementary nucleotides was
measured by one step RT-PCR (for TMV; Fig. 3) and end point PCR (for Pc; notshown) of serially diluted RNA/DNA, and by quantifying the DNA yield using a
Nanodrop® (O.D.260 nm) after elution of excised bands. The effect on DNA yields wasalso quantified by SYBR® Green real time RT-PCR assays.
with 5’ flap
Increase in PCR
yield (ng/µl)
3.10
(15.0%)
5.0 (13.4%) 21.65 (104.6%)
CONCLUSIONS
1. Designing primers having ∆G = 0 and 5’ AT-rich non-complementary nucleotides increased DNA
yields and the sensitivity of one step RT-PCR and PCR detection.2. The addition of 5’ AT-rich non-complementary nucleotides to suboptimal primer assays has the
potential to improve DNA yields.
Fig. 3. RNA isolation from infected tobacco leaves and 10 fold serial dilution for sensitivity assays.
1 µl
9 µl
1 µl 1 µl 1 µl 1 µl 1 µl 1 µl
9 µl 9 µl 9 µl 9 µl 9 µl 9 µl
Serial dilutionRNA isolation
RNeasy plant mini kit
(QIAGEN)
Fig. 2. Optimal energy values of each oligonucleotide primer calculated and plotted using mFOLD. The black dots represent all
the optimal foldings. The colored, dots represent the superposition of all possible suboptimal foldings within p% deviation from
the minimum free energy, the red and green, representing foldings within p3% and p2% respectively and the yellow dots
represent base pairs least likely to form. X and Y axis are the nucleotide position in primer sequence.
TMV-2 Forward Primer TMV-2 Reverse Primer TMV-3 Forward Primer TMV-3 Reverse Primer
(4871)TGATAAAGTTCTTTTTAGAAGTTTGTTTATAGATGGCTCTAGTTGTTAAAGGAAAAGTGAATATCAATGAGTTTATCGACCTGACAAAAATGGAGAAGATCTTACCGTCGATGTTTACCCCTGTAAAGAGT
GTTATGTGTTCCAAAGTTGATAAAATAATGGTTCATGAGAATGAGTCATTGTCAGAGGTGAACCTTCTTAAAGGAGTTAAGCTTATTGATAGTGGATACGTCTGTTTAGCCGGTTTGGTCGTCACGGGCGAGTG
GAACTTGCCTGACAATTGCAGAGGAGGTGTGAGCGTGTGTCTGGTGGACAAAAGGATGGAAAGAGCCGACGAGGCCACTCTCGATCTTACTACACAGCAGCTGCAAAGAAAAGATTTCAGTTCAAGGTCGTT
CCCAATTATGCTATAACCACCCAGGACGCGATGAAAAACGTCTGGCAAGTTTTAGTTAATATTAGAAATGTGAAGATGTCAGCGGGTTTCTGTCCGCTTTCTCTGGAGTTTGTGTCGGTGTGTATTGTTTAT (5401)
1. RT-PCR amplification was carried out in 20 µl containing 10µL 2X reaction mix, 0.5µL RNaseOut, 2µL of each forward and reverseprimer (5µM), 0.4µL SSIII/Platinum Taq, 0.5µL BSA (10ug/ul), 2µL RNA template and nuclease free water 1.6 µL. Real time RT-PCRreactions were also in 20µL volume containing 10µL RT SYBR Green qPCR master mix, 6.4µL nuclease free water, 0.8µL of eachforward and reverse primer (5µM) and 2µL of cDNA.
2. PCR cycling conditions were 50⁰C, 30 min. (cDNA synthesis), initial denaturation 94⁰C, 2 min. for 40 cycles at 94⁰C, 30 sec.,annealing, 61⁰C (TMV-2) 30 sec. & 54⁰C (TMV-3). Polymerization, 30 sec. at 72⁰C and 7 min. final extension. For real time PCR,95⁰C, 10 min. initial denaturation 94⁰C, 2 min. for 40 cycles at 94⁰C, 30 sec., annealing, 54⁰C. Polymerization, 30 sec. at 72⁰C and
7 min. final extension.
LITERATURE CITED
LAB PROTOCOLS
1. Afonina, I., Ankoudinova, I., Mills, A., Lokhov, S., Huynh, P. and Mahoney, W. 2007. Primers with 5′ flaps improve real-time
PCR. Biotechniques 43 (6): 770-774.2. Ochoa Corona, F.M., Locali-Fabris, E. C., and Freitas-Astua, J. 2009. An RT-PCR procedure for detection and surveillance of
Citrus leprosis virus C (CiLV-C) in post-entry quarantine stocks of citrus. Phytopathology 99:S96.3. Ochoa Corona, F.M., Tang, J., Lebas, B.S.M., and Alexander, B. 2007. Validation of primer design for plant virus diagnostics
using the Web-interface pathway Primer3-mFOLD-BLASTn. Phytopathology 97: (7) S86.
Fig. 5. Sensitivity assay of one step RT-PCR. TMV-2 optimal primer set, (A) without and
(B) with 5’ AT-rich non-complementary nucleotides sequences. TMV-3 suboptimal primer
set, (C) without and (D) with 5’ AT-rich non-complementary nucleotides sequences. Lane
M: 1kb ladder, Lane 1-8: 10-fold serial dilutions of isolated TMV RNA starting from 500
ng/reaction.
Major rRNA Transcript
Internal Transcribed Spacer (ITS) Region Intergenic Spacer (IGS) Region
SSU (18S) RNA LSU (28S) RNA5.8S RNA
Region used for the primer design
9 µl
Fig. 6. Real Time PCR amplification of optimal TMV-2 and suboptimal TMV-3 with and without 5’ A/T rich flaps .
(A) TMV-2 & TMV-3
without 5’ flap(B) TMV-3 with &
without 5’ flap
(C) TMV-2 with &
without 5’ flap
TMV-2 without 5’ flap1 ng/rx
TMV-3 without 5’ flap1 ng/rx TMV-3 without 5’ flap
1 ng/rx
TMV-3 with 5’ flap1 ng/rx
TMV-2 without 5’ flap1 ng/rx
TMV-2 with 5’ flap1 ng/rx
TMV-2 without 5’ flap0.1 ng/rx
TMV-3 with 5’ flap0.1 ng/rx TMV-2 with 5’ flap
0.1 ng/rx