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

  • Tomsk State University, Altay State University

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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
Content may be subject to copyright.
Optimization of Polymerase Chain Reaction for Inter Simple Sequence
Repeat Technique for Four Species of Plants
National Research Tomsk State University, 36 Lenina ave., Tomsk, 634050, Russia
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.
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/ 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, (ID:,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
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
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
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
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
, [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
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]
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.
[1] W. Meyer, T.G. Mitchell, E.Z. Freedman, R. Vilgalys, Hybridization probes for conventional
DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of
Cryptococcus neoformans, J. Clin. Microbiol. 31 (1993) 2274-2280.
[2] E. Zietkiewicz, A. Rafalski, D. Labuda, Genome fingerprinting by simple sequence repeat
(SSR) – anchored polymerase chain reaction amplification, Genomics. 20 (1994) 176-183.
[3] J.G.K. Williams, A.R. Kubelik, K.J. Livak, J.A. Rafalski, S.V. Tingey, DNA polymorphisms
amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res. 18 (1990) 6531-
[4] D. Tautz, Hypervariability of simple sequences as a general source for polymorphic DNA
markers, Nucleic Acids Res. 17 (1989) 6463-6471.
Key Engineering Materials Vol. 683 517
[5] P. Vos, R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J.
Peleman, M. Kuiper, M. Zabeau, AFLP: a new technique for DNA fingerprinting, Nucleic Acids
Res. 11 (1995) 4407-4414.
[6] M.Z. Galvan, B. Bornet, P.A. Balatti, M. Branchard, Inter simple sequence repeat (ISSR)
markers as a tool for the assessment of both genetic diversity and gene pool origin in common bean
(Phaseolus vulgaris L.), Euphytica. 132 (2003) 297-301.
[7] M. Korbin, A. Kuras, E. Urawicz, Fruit plant germplasm characterisation using molecular
markers generated in RAPD and ISSR PCR, Cell Mol. Biol. Lett. 7, 2B (2002) 785-794.
[8] W.P. Yang, A.C. Oliveira, I. Godwin, K. Schertz, J.L. Bennetzen, Comparison of DNA marker
technologies in characterizing plant genome diversity: variability in Chinese sorghums, Crop Sci.
36 (1996) 1669-1676.
[9] S. Cichorz, M. Goska, A. Litwiniec, Miscanthus: genetic diversity and genotype identification
using ISSR and RAPD marker, Mol. Biotechnol. 56, 10 (2014) 911-924.
[10] D. Metzgar, J. Bytof, C. Wills, Selection against frameshift mutations limits microsatellite
expansion in coding DNA, Genome Res. 10 (2000) 72-80.
[11] Y. Wang, M. Chen, H. Wang, J. Wang, D. Bao, Microsatellites in the Genome of the Edible
Mushroom, Volvariella volvacea, BioMed Res. Int., 2014, Available at:
[12] J. Huang, Y. Li, L. Du, B. Yang, F. Shen, H. Zhang, Z. Zhang, X. Zhang, B. Yue, Genome-
wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus
on the applications of a novel microsatellite marker system, BMC Genomics, 2015, Available at:
[13] M. A. Latif, M. M. Rahman, M. E. Ali, S. Ashkani, M.Y. Rafii, Inheritance studies of SSR and
ISSR molecular markers and phylogenetic relationship of rice genotypes resistant to tungro virus,
CR. Biol. 336, 3 (2013) 125-133.
[14] T. J. Givnish, G. J. Bean, M. Ames, S. P. Lyon, K. J. Sytsma, Phylogeny, floral evolution, and
inter-island dispersal in Hawaiian Clermontia (Campanulaceae) based on ISSR variation and plastid
spacer sequences, PLoS One, 2013, Available at:
[15] L. Zande, R. Bijlsma, Limitation of the RAPD technique in phylogeny reconstruction in
Drosophila, J. Evolution. Biol. 8 (1995) 645-656.
[16] R. Kalendar, T. Grob, M. Regina, A. Suoniem, A. Schulman, IRAP and REMAP: Two new
retrotransposon-based DNA fingerprinting techniques, Theor. Appl. Genet. 98 (1999) 704-711.
[17] D.L. Hyndman, M. Mitsuhashi, PCR primer design, Methods Mol. Biol. 226 (2003) 81-88.
[18] K.H. Roux, Optimization and troubleshooting in PCR, PCR Meth. Appl. 4 (1995) 185-194.
518 Multifunctional Materials: Development and Application
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Using bioinformatics software and database, we have characterized the microsatellite pattern in the V. volvacea genome and compared it with microsatellite patterns found in the genomes of four other edible fungi: Coprinopsis cinerea, Schizophyllum commune, Agaricus bisporus, and Pleurotus ostreatus. A total of 1346 microsatellites have been identified, with mono-nucleotides being the most frequent motif. The relative abundance of microsatellites was lower in coding regions with 21 No./Mb. However, the microsatellites in the V. volvacea gene models showed a greater tendency to be located in the CDS regions. There was also a higher preponderance of trinucleotide repeats, especially in the kinase genes, which implied a possible role in phenotypic variation. Among the five fungal genomes, microsatellite abundance appeared to be unrelated to genome size. Furthermore, the short motifs (mono- to tri-nucleotides) outnumbered other categories although these differed in proportion. Data analysis indicated a possible relationship between the most frequent microsatellite types and the genetic distance between the five fungal genomes.
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Chinese sorghums [Sorghum bicolor (L.) Moench] are reputed to have a narrow genetic base, resulting from infrequent introduction of exotic germplasm into China. We have used several different molecular approaches to evaluate diversity and relatedness in a selection of 34 Chinese sorghums. The results indicated that different DNA marker technologies for germplasm assessment yield comparable results, but that a relatively new technique termed inter-simple sequence repeat amplification (ISSR) was relatively rapid, reproducible, and inexpensive. Extensive diversity was observed within the Chinese sorghums, and all lines could be easily differentiated. Different lines collected from the same locality were found to exhibit particularly high levels of relatedness. Contrary to expectations, improved varieties were found to contain more diversity and to be more different from each other than were the Chinese landraces studied, suggesting recent introduction of non-Chinese germplasm into these improved materials.
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Previous studies based on DNA restriction-site and sequence variation have shown that the Hawaiian lobeliads are monophyletic and that the two largest genera, Cyanea and Clermontia, diverged from each other ca. 9.7 Mya. Sequence divergence among species of Clermontia is quite limited, however, and extensive hybridization is suspected, which has interfered with production of a well-resolved molecular phylogeny for the genus. Clermontia is of considerable interest because several species posses petal-like sepals, raising the question of whether such a homeotic mutation has arisen once or several times. In addition, morphological and molecular studies have implied different patterns of inter-island dispersal within the genus. Here we use nuclear ISSRs (inter-simple sequence repeat polymorphisms) and five plastid non-coding sequences to derive biparental and maternal phylogenies for Clermontia. Our findings imply that (1) Clermontia is not monophyletic, with Cl. pyrularia nested within Cyanea and apparently an intergeneric hybrid; (2) the earliest divergent clades within Clermontia are native to Kauài, then Òahu, then Maui, supporting the progression rule of dispersal down the chain toward progressively younger islands, although that rule is violated in later-evolving taxa in the ISSR tree; (3) almost no sequence divergence among several Clermontia species in 4.5 kb of rapidly evolving plastid DNA; (4) several apparent cases of hybridization/introgression or incomplete lineage sorting (i.e., Cl. oblongifolia, peleana, persicifolia, pyrularia, samuelii, tuberculata), based on extensive conflict between the ISSR and plastid phylogenies; and (5) two origins and two losses of petaloid sepals, or-perhaps more plausibly-a single origin and two losses of this homeotic mutation, with its introgression into Cl. persicifolia. Our phylogenies are better resolved and geographically more informative than others based on ITS and 5S-NTS sequences and nuclear SNPs, but agree with them in supporting Clermontia's origin on Kauài or some older island and dispersal down the chain subsequently.
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In this study, we report the use of ISSR to assess genetic diversity and to determine the relationships among ten cultivars of common bean developed in Argentina and three materials from France. ISSR markers resolved two major groups corresponding to the Andean and Mesoamerican gene pools of common bean. We compared the results of previous analysis, performed with RAPD markers (Galván et al., 2001), with the results generated by means of ISSR. It appears that ISSR are better tools than RAPDs to identify beans by gene pool of origin though they did not revealed as many differences between individuals as RAPDs.
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 The BARE-1 retrotransposon is an active, dispersed, and highly abundant component of the genome of barley (Hordeum vulgare) and other species in its genus. Like all retrotransposons of its kind, BARE-1 is bounded by long terminal repeats (LTRs). We have developed two amplification-based marker methods based on the position of given LTRs within the genome. The IRAP (Inter-Retrotransposon Amplified Polymorphism) markers are generated by the proximity of two LTRs using outward-facing primers annealing to LTR target sequences. In REMAP (REtrotransposon-Microsatellite Amplified Polymorphism), amplification between LTRs proximal to simple sequence repeats such as constitute microsatellites produces markers. The methods can distinguish between barley varieties and produce fingerprint patterns for species across the genus. The patterns indicate that although the BARE-1 family of retrotransposons is disperse, these elements are locally clustered or nested and often found near tandem arrays of a simple sequence repeat.
Multivariate analyses were performed using 13 morphological traits and 13 molecular markers (10 SSRs and three ISSRs) to assess the phylogenetic relationship among tungro resistant genotypes. For morphological traits, the genotypes were grouped into six clusters, according to D(2) statistic and Canonical vector analysis. Plant height, days to flowering, days to maturity, panicle length, number of spikelet per panicle, number of unfilled grain per panicle and yield were important contributors to genetic divergence in 14 rice genotypes. Based on Nei's genetic distance for molecular studies, seven clusters were formed among the tungro resistant and susceptible genotypes. Mantel's test revealed a significant correlation (r=0.834*) between the morphological and molecular data. To develop high yielding tungro resistant varieties based on both morphological and molecular analyses, crosses could be made with susceptible (BR10 and BR11) genotypes with low yielding but highly resistant genotypes, Sonahidemota, Kumragoir, Nakuchimota, Khaiyamota, Khairymota and Kachamota. The chi-square analysis for seven alleles (RM11, RM17, RM20, RM23, RM80, RM108 and RM531) of SSR and five loci (RY1, MR1, MR2, MR4 and GF5) of three ISSR markers in F2 population of cross, BR11×Sonahidemota, showed a good fit to the expected segregation ratio (1:2:1) for a single gene model.
In this study the limitations of the RAPD technique for phylogenetic analysis of very closely related and less related species of Drosophila are examined. In addition, assumptions of positional homology of amplified fragments in different species are examined by cross-hybridization of RAPD fragments. It is demonstrated that in Drosophila the use of RAPD markers is very efficient in identification of species. For assessment of phylogenetic relationships, however, the method is limited to sibling species, and reliable measures for genetic distances cannot be obtained. Hybridization experiments demonstrate that fragments of similar length amplified from different species are not always derived from corresponding loci, and that not all RAPD fragments within the same amplification pattern are independent.
INTRODUCTION The use of polymerase chain reaction (PCR) to generate large amounts of a desired product can be a double-edged sword. Failure to amplify under optimum conditions can lead to the generation of multiple undefined and unwanted products, even to the exclusion of the desired product. At the other extreme, no product may be produced. A typical response at this point is to vary one or more of the many parameters that are known to contribute to primer-template fidelity and primer extension. High on the list of optimization variables are Mg ⁺⁺ concentrations, buffer pH, and cycling conditions. With regard to the last, the annealing temperature is most important. The situation is further complicated by the fact that some of the variables are quite interdependent. For example, because dNTPs directly chelate a proportional number of Mg ⁺⁺ ions, an increase in the concentration of dNTPs decreases the concentration of free Mg ⁺⁺ available to influence polymerase function. This article discusses various optimization strategies, including touchdown PCR and hot-start PCR.