Optimization of Polymerase Chain Reaction for Inter Simple Sequence Repeat Technique for Four Species of Plants

Abstract

Polymerase chain reaction optimization for inter simple sequence repeat primers is a key factor to obtain accurate and reproducible results for gene mapping, studying the genetic structure of populations, plant passporting, phylogenetic analysis. Changing temperature conditions, the amount of amplification cycles and concentration of reaction mixture components is allowed to vary the number of bands obtained by this method. This article is result of preliminary research of method selection for molecular analysis. It is aimed to show how to adjust the profile of inter simple sequence repeat fragments by polymerase chain reaction for four model species Stipa lessingiana, Poa intricata, Equisetum fluviatile and Pteridium aquilinum. The working concentrations of magnesium chloride for primer ((СТС)3GC) and ((АС)8YG) were 2.5 mM for 0.63 units of Taq DNA polymerase and for primer ((СА)6GG) it was 4.5 mM for 1.25 units. Sharply defined banding was observed from the minimal amount of DNA 5 ng per reaction, with primer concentration from 10 to 80 pmol and dNTPs concentration 0.2 mM. Optimal hybridization temperatures were 51.9 °C for primers ((АС)8YG), ((СА)6GG) and 50.0 °C for ((СТС)3GC). The best imaging results were obtained when setting up electrophoresis in 1.9% agarose gel

Full text

Optimization of Polymerase Chain Reaction for Inter Simple Sequence
Repeat Technique for Four Species of Plants
GUDKOVA Polinaa, BAYAHMETOV Eugeneb
National Research Tomsk State University, 36 Lenina ave., Tomsk, 634050, Russia
a polina-shavrova@yandex.ru
b eugenebayahmetov@gmail.com
Keywords: Herbarium material, ISSR, ISSR primers, molecular markers, PCR, reproducibility.
Abstract. Polymerase chain reaction optimization for inter simple sequence repeat primers is a key
factor to obtain accurate and reproducible results for gene mapping, studying the genetic structure
of populations, plant passporting, phylogenetic analysis. Changing temperature conditions, the
amount of amplification cycles and concentration of reaction mixture components is allowed to
vary the number of bands obtained by this method. This article is result of preliminary research of
method selection for molecular analysis. It is aimed to show how to adjust the profile of inter simple
sequence repeat fragments by polymerase chain reaction for four model species Stipa lessingiana,
Poa intricata, Equisetum fluviatile and Pteridium aquilinum. The working concentrations of
magnesium chloride for primer ((СТС)3GC) and ((АС)8YG) were 2.5 mM for 0.63 units of Taq
DNA polymerase and for primer ((СА)6GG) it was 4.5 mM for 1.25 units. Sharply defined banding
was observed from the minimal amount of DNA 5 ng per reaction, with primer concentration from
10 to 80 pmol and dNTPs concentration 0.2 mM. Optimal hybridization temperatures were 51.9 °C
for primers ((АС)8YG), ((СА)6GG) and 50.0 °C for ((СТС)3GC). The best imaging results were
obtained when setting up electrophoresis in 1.9% agarose gel.
Introduction
ISSR technique (Inter Simple Sequence Repeat) has been known for more than 20 years [1, 2].
Along with RAPD (Random Amplified Polymorphic DNA) [3], SSR (Simple Sequence Repeats)
[4] and AFLP (Amplified Fragment Length Polymorphism) [5], ISSR allows analyzing genome
polymorphism. The markers based on intermicrosatellite sequences have several advantages – easy
handling, inexpensiveness and small amount of starting material required for genetic amplification
as well as higher reproducibility and specificity in comparison with other methods [6, 7, 8, 9].
It was established that all kinds of microsatellites (from mono to hexanucleotide repeats) are
abundant in the non-coding regions of plants, animals and other eukaryotic organisms [10, 11, 12].
This explains the widespread use of ISSR technique for genome mapping, studying of the genetic
structure of populations, plant passporting, as well as in phylogenetic analysis [13, 14]. It should be
noted that high rates of evolutionary change limit the applicability of multilocus markers for
researching of phylogenetic relationships for taxonomic rank higher than genus [15].
ISSR primers consist of short tandem repeats of two to four base pair motifs, the total length of
15-24 nucleotides and one selective nucleotide at the 3' end [16], wherein the annealing
temperature, dependent on GC-content, is generally within the range of 45-65 °C.
This paper is devoted to the influence of various parameters of the polymerase chain reaction to
reproducibility of amplification based on the Stipa lessingiana Trin. et Rupr., Poa intricata Wein
(Poaceae), Equisetum fluviatile (Equisetaceae) and Pteridium aquilinum (Dennstaedtiaceae).
Materials and methods
The samples of Stipa lessingiana, Poa intricate, Equisetum fluviatile and Pteridium aquilinum
collected in summer 2014 were selected as a model. Isolation of genomic DNA was performed
according to the protocol of a commercial kit DiamondDNA Plant Kit D (DiamondDNA, Russia).
Key Engineering Materials Vol. 683 (2016) pp 511-518 Submitted: 2015-04-27
© (2016) Trans Tech Publications, Switzerland Revised: 2015-05-16
doi:10.4028/www.scientific.net/KEM.683.511 Accepted: 2015-06-19
Online: 2016-02-16
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans
Tech Publications, www.ttp.net. (ID: 92.63.71.38-15/12/15,13:29:10)

Isolated DNA was dissolved in 100 µl of TE buffer (DiamondDNA, Russia). Concentration and
quality of the isolated DNA was assessed using the spectrophotometric ratio of light absorbance at
wavelengths of 230, 260 and 280 nm using spectrophotometer P330 (Implen, Germany).
Three ISSR primers (Medigen, Russia) were used for amplification – M2 ((АС)8YG), НВ14
((СТС)3GC) and 17899В ((СА)6GG). Several PCR parameters were tested during the optimization
of conditions: number of cycles (20, 30, 35, 40), primer annealing temperature (from 48 to 58 °C, 8
variations), and the reaction mixture components: DNA (from 0.63 to 200 ng per reaction, 9
variations), Taq-polymerase (from 0.31 to 2.5 units per reaction, 4 variations), primer (from 2.5 to
480 pmol per reaction, 9 variations), MgCl2 (concentration from 1.5 mM to 8.5 mM, 8 variations),
dNTPs (from 0.03 to 6.4 mM, 9 variations). Reactions without DNA were used as a negative
control. The reliability of DNA spectra was tested three times using PCR.
Thermo Scientific’s reagents (USA) were used for polymerase chain reaction. Total volume of
the final reaction mixture was 15 µl. The amount and concentration of regents for first PCR was:
buffer – 1.5 µl (10x with 500 mM of KCl, 15 mM of MgCl2, 100 mM of Tris-HCl and 0.8%
Nonidet P40); dNTPs – 0.12 µl (25 mM); MgCl2 – 0.6 µl (25 mM); Taq-polymerase – 0.25 µl (5
u/µl); ISSR primer – 1 µl (10 mM); dH2O – 9.53 µl; DNA sample – 1 µl (10 ng).
PCR was performed in a programmable thermocycler Thermal Cycler S1000 (Bio-Rad, USA).
The amplification conditions: initial DNA denaturation – 3 min at 95 °C, then 35 cycles comprising
three steps: 30 sec at 95 °C, 30 sec at 50 °C, 1 min at 72 °C; final elongation – 10 min at 72 °C,
followed by cooling to 4 °C.
Electrophoretic separation of PCR products had been being carried out for 3 hours at a voltage of
70 V using horizontal chambers SE-1 (Helicon, Russia) and SE-2 (Helicon, Russia) in 1.5%, 1.9%
and 2.25% agarose gels with ethidium bromide. 1x TAE was used as a buffer solution. Subsequent
imaging was carried out using gel documentation system Universal Hood II (Bio-Rad, USA).
Results and discussion
Several polymerase chain reaction parameters capable of affecting the reproducibility and quality
of ISSR DNA fragments profile were investigated (data shown only for Stipa lessingiana specimen
except Fig. 11).
First, for all the three primers (M2, НВ 14 and 17899B) it was established that the number and
sharpness of bands is best controlled by changing the concentrations of Taq-polymerase and MgCl2
(Fig. 1, 2, 3). Second, we analyzed the effect of annealing temperatures and number of cycles (Fig.
4, 5, 6). When selecting the optimal annealing temperature, it should be noted that nonspecific
amplification increases under the low-temperature annealing, which leads to stripe artifacts when
imaging [17, 18].
Furthermore it was established that the initial amount of DNA nanomatrix had the smallest effect
on the spectrum of PCR products (Fig. 7). We have also shown influence of primer concentration
(Fig. 8) and dNTPs concentration (Fig. 9) on the relative intensities and number of bands.
512 Multifunctional Materials: Development and Application

Fig. 1. Influence of the amount of Taq-polymerase (from 0.31 to 2.5 units) and the
concentrations of MgCl2 (from 1.5 mM to 8.5 mM) upon pattern quality amplified with primer
HB14.
Fig. 2. Influence of the amount of Taq-polymerase (from 0.31 to 2.5 units) and the
concentrations of MgCl2 (from 1.5 mM to 8.5 mM) upon pattern quality amplified with primer M2.
Key Engineering Materials Vol. 683 513

Fig. 3. Influence of the amount of Taq-polymerase (from 0.31 to 2.5 units) and the
concentrations of MgCl2 (from 1.5 mM to 8.5 mM) upon pattern quality amplified with primer
17899В.
Fig. 4. Variations of the number of PCR cycles (from 20 to 40) and the annealing temperature
(from 48 to 58 °C) for primer HB14.
Fig. 5. Variations of the number of PCR cycles (from 30 to 35) and the annealing temperature
(from 48 to 58 °C) for primer 17899B.
514 Multifunctional Materials: Development and Application

Fig. 6. Variations of the number of PCR cycles (from 30 to 35) and the annealing temperature
(from 48 to 58 °C) for primer M2.
Fig. 7. Variations of amount of DNA (from 0.63 to 200 ng/reaction) for primer HB14.
Fig. 8. Variations of the primer HB14 concentration (from 2.5 to 480 pmol/reaction).
Fig. 9. Variations of the dNTPs concentration (from 0.03 to 6.4 mM) for primer HB14.
Key Engineering Materials Vol. 683 515

The obtained results show that reproducible ISSR DNA fragments profiles can be observed
under a wide range of experimental conditions.
For primer HB14 working concentrations of magnesium chloride were 2.5 mM for 0.63 units of
Taq DNA Polymerase, 2.5-4.5 mM for 1.25 units and 2.5-5.5 for 2.5 units. For primer M2 – 3.5
mM for 0.31 units, 2.5 mM for 0.63 units, 2.5-3.5 mM for 1.25 units and 2.5 for 2.5 units. For
primer 17899B – 2.5 mM for 0.31 units, 2.5-3.5 mM for 0.63 units, 4.5 mM for 1.25 units and 4.5-
5.5 for 2.5 units.
Optimal hybridization temperatures were between 48 °C and 51.9 °C for primer HB14, between
51.9 °C and 54.3 °C for 17899B and 51.9 °C for M2. For acceptable banding patterns is sufficient
for at least 30 cycles of amplification for all three primers.
Sharply defined banding was observed from the minimal amount of DNA 5 ng per reaction, with
primer concentration from 10 to 80 pmol and dNTPs concentration 0.2 mM.
To test the general applicability of the ISSR protocol, we performed amplifications three times.
In all cases, the amplification gave clear and reproducible banding patterns. The best imaging
results were obtained when setting up electrophoresis in 1.9% agarose gel and the range of obtained
bands was from 200 to 1500 bp depending on the primer (Fig. 10, 11).
Fig. 10. Reproducibility of fingerprints for primers HB14, M2 and 17899B.
Fig. 11. Reproducibility of fingerprints for primers HB14, M2 and 17899B: a) Pteridium
aquilinum; b) Equisetum fluviatile; c) Poa intricata.
516 Multifunctional Materials: Development and Application

Conclusions
Thus, PCR optimization for ISSR primers НВ14, M2 and 17899В have shown optimal amount
and concentration of reagents (Table 1) and PCR conditions that are presented in Table 2.
Table 1 – Optimal amount and concentration of reagents for PCR with ISSR primers HB14, M2
and 17899B
PCR reaction
components
ISSR primer
HB14 M2 17899B
Buffer 1x 1x 1x
DNA, [ng] 5 5 5
Primer, [pmol] 30 30 30
dNTPs, [mM] 0.2 0.2 0.2
MgCl
2
, [mM] 2.5 2.5 4.5
Taq polymerase, [u] 0.63 0.63 1.25
Table 2 - Thermocycling PCR conditions for primers HB14, M2 and 17899B
PCR conditions
ISSR primer
HB14 M2 17899B
Initial denaturation 95 °C – 3 [min] 95 °C – 3 [min] 95 °C – 3 [min]
30 cycles
95 °C – 30 [sec] 95 °C – 30 [sec] 95 °C – 30 [sec]
50 °C – 30 [sec] 51.9 °C – 30 [sec] 51.9 °C – 30
[sec]
72 °C – 1 [min] 72 °C – 1 [min] 72 °C – 1 [min]
Final extension 72 °C – 10 [min] 72 °C – 10 [min] 72 °C – 10 [min]
Hold 4 [°C] 4 [°C] 4 [°C]
Acknowledgements
This study was supported by the D.I. Mendeleev Scientific Fund Program of Tomsk State
University and by grant of President of Russian Federation for State Support for young Russian
scientists-candidates of science – MK-2722.2014.4, grant of President of Russian Federation for
State Support for young Russian scientists-candidates of science – MK-3862.2015.4, grant of
President of Russian Federation for State Support of Leading Scientific School – No. 324.2014.4.
The reported study was partially supported by RFBR, research project No. 13-04-01715, by RFBR,
research project No. 14-04-31962, by RFBR, research project No. 15-34-20513.
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