Musca domestica sequence-related amplified

polymorphism amplification system Establishment

and Optimization of
Posted:2010-2-7 9:17:00 Browse:9784 Times

Author: Liu Hong Mei, Fang Xiaobo, ZHENG Qin Ni, Hu Shiming, Jian-Wei
Wu
[Abstract] Objective: To establish and optimize the housefly sequence-related amplified
polymorphism (SRAP-PCR) amplification system. Methods: The domestication and houseflies as
material, through a single factor experiment studied the template DNA, Mg2, dNTPs, primer
concentration and the amount of Taq DNA polymerase housefly SRAP molecular marker system
and the impact of amplification, and amplification system was optimization. Results: The optimized
total volume of reaction system 30 �� l, containing 50 ng template DNA, 1.5 mmol / L Mg2, 0. 20
mmol / L dNTPs, 0. 15 �� mol / L primer and 0. 50 U Taq DNA polymerase. Conclusion: The
house fly SRAP-PCR amplification of the establishment of optimized system for future use of genetic
diversity of Musca domestica SRAP technology analysis, laid the foundation for gene mapping.
[Key words] Muscidae; random amplified polymorphic DNA technique; genetic marker
[Abstract] Objective: To establish and optimize SRAP-PCR reaction system of housefly.
Methods: Effects of concentrations of template DNA, Mg2, dNTPs, primers and amount of Taq DNA
polymerase on SRAP molecular marker amplification system of Musca domestica L were studied in
monofactorial experiment, and the amplification system was optimized. Results: Total volume of the
optimal system was 30�� l containing 50 ng of template DNA, 1.5mmol / L of Mg2, 0. 20 mmol / L
of dNTPs, 0. 15�� mol / L of primers and 0. 50 U of Taq DNA polymerase. Conclusion: This
system provides a useful tool for analysis of genetic diversity and gene location of Musca domestica
L.
[Key words] Muscidae; random amplified polymorphic DNA technique; genetic markers
Housefly (Musca domestica L) is an important medical vector insects can spread dysentery,
typhoid, cholera, polio and other diseases. In recent years, with the discovery of antimicrobial
peptides in vivo housefly, Musca domestica in the industrial raw materials, medical care and natural
materials such as energy cycle have an important application value. Because of the housefly
prejudice at home and abroad from the molecular level is still relatively few reports of Musca
domestica, and only limited to the use of RAPD technique [1]. Sequence-related amplified
polymorphism (Sequence-related amplified polymorphism, SRAP) is a new theory based on PCR
technology, DNA molecular marker, its technology is the core of PCR amplification, PCR
amplification of the factors that influence will affect the success or failure of SRAP . Therefore, Taq
enzyme, Mg2, the template DNA, dNTPs, primers that five factors to optimize the establishment of a
suitable housefly DNA, SRAP-PCR amplification system for the SRAP system in the housefly and
even insects in the study identified the foundation.
1 Materials and methods
1.1 Materials
1.1.1 for the test materials
Musca domestica from domesticated Parasitology, Guiyang Medical College, established in
2000 populations, and many generations in the laboratory breeding populations. Larvae to wheat
bran, maggot paste, flour and water mixture as feed, rearing temperature (25 �� �� 1) �� ,
relative humidity 50% ~ 60%, light 12 h. Experiments chosen as the mast of the 3rd instar larvae of
material.
1.1.2 equipment, reagents
PCR amplification experiments using gradient PCR instrument for the Eppendorf 5331
instrument; 10 * PCR buffer, MgCl2, dNTPs, Taq DNA polymerase, agarose, and DL2000 (Marker)
Dengjun purchased from Dalian Takara (treasure biological) Engineering Limited. SRAP primers
from Bio-Technology Co., Ltd. Shanghai Ding An synthesis, primer name and sequence in Table 1.
Table 1 SRAP primers and sequence (abbreviated)
1.2 Methods
1.2.1 Genomic DNA extraction of Musca domestica
With Xu Shu-Hua et al [2] The method to extract 3-instar larvae of Musca domestica genomic
DNA, DNA by UV spectrophotometer and 0.8% agarose gel electrophoresis detection and
quantification of the purity, low impurity samples stored at -20 �� used for PCR amplification.
1.2.2 PCR systems, procedures and testing
Refer to Li et al [3] The amplification reaction system. Standard PCR reaction system in the
template DNA 100 ng, dNTPs 0.2 mmol / L, on the upstream and downstream primers 0.2 �� mol /
L, 1.5 mmol / L MgCl2, 1U Taq enzyme, total volume of 30 �� l. Amplification process using a
complex of variable-temperature method: Start 95 �� denaturation 2min, response to the first 5
cycles at 94 �� 1 min, 35 �� 1 min, 72 �� 2 min under the conditions of operation; after 35
cycles of 94 �� 1 min, 50 �� 1 min, 72 �� 2 min; the last 72 �� extension 7 min. 2% agarose
gel electrophoresis, the electrode buffer, using 0.5 * TBE, steady voltage of 80 V, 30 min. Ethidium
bromide staining, gel imaging system to observe and take photographs.
1.2.3 SRAP primer screening
Obtained using 1.2.2 under the SRAP-PCR amplification system and expansion of procedures
in Table 1 between the combination of forward primer and reverse primer combinations and between
the forward and reverse primer combinations of selection for expansion of effective, multi-band , and
reproducible primer combinations used for amplification system optimization.
1.2.4 SRAP-PCR amplification reaction system optimization
To Musca domestica genomic DNA as a template, SRAP-PCR amplification of the five major
influence factors on five levels of single-factor gradient settings, in Table 2. Table 2 Musca
domestica SRAP-PCR amplification of the gradient of the effects of each factor setting (a little)
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2 Results

2.1 SRAP primer screening

Designed for the detection of primers (Table 1) the quality and the male and female Musca
domestica genomic DNA amplification effect, the Table 1 5 forward primer, 6 reverse primer
between groups with the group of primer combinations screening, using agarose gel electrophoresis
to detect the amplification effect of primer combinations (see Figure 1). Table 3 for the amplification
effect of a good primer combinations. Experiments were selected 21 pairs of amplification primer
combinations with good effects. Forward primer combinations amplified with good effects 3 pairs, 6
reverse primer combinations amplified with good effects are nine pairs of the remaining nine pairs of
primers for the forward and reverse primer between the combination. The results showed that SRAP
primers designed not only between the forward and reverse primer pair combinations, and the
forward primer, the reverse primer can also be combined between the pair for PCR amplification.
Optimization of the template DNA concentration of 2.2 housefly PCR amplification reaction, the
template amount too much or too little will affect the amplification efficiency. Too came from a DNA
template volume, amplification efficiency is low, templates and primers can not be effectively paired
with no amplification or amplification with less; as excessive when the DNA appears diffuse
background, and with no amplification products. Can be seen from Figure 1, template DNA 10 ~ 200
ng were amplified when the band shows that SRAP-PCR system, the amount of the amount of
template DNA not ask for much. When the amount of template DNA by adding 10 ng, the amplified
bands are very weak, in the more than 100 ng Shi You began to diffuse background. 50 ng template
DNA quantity when the best, shown in Figure 2.

2.3 Mg2 concentration of Mg2 concentration affects not only the activity, but also affects primer
annealing, templates, and intermediate products of the dissociation temperature, product-specific,
primer dimer formation, so the concentration of Mg2 on the PCR amplification reactions to a
significant impact on . The concentration is too low, no amplification product; the concentration is too
high, easy to produce non-specific amplification, there diffuse background. Mg2 concentration of 1.5
mmol / L amplification effects (brightness and abundance) as compared with other concentrations is
good, shown in Figure 3.
Table 3 housefly SRAP primer screening results (omitted)
2.4 Taq enzyme concentration of Taq DNA polymerase in the PCR reaction volume by the
amount, enzyme activity, enzyme heat-resistance and other factors. Taq enzyme PCR reaction with
the less easily lead to reduced efficiency, resulting in less amplification products. The use of a higher
concentration of Taq enzyme easily lead to non-specific amplification, there diffuse background, but
no amplified bands, resulting in drug waste increase costs. Can be seen from Figure 4, in 30 �� l
of the reaction system, Taq enzyme dosage preferably 0.5 ~ 1.0 U, Chao Guo 1.5 U, there will be
diffuse background, but no amplified bands. So, from economic considerations, 30 �� l reaction in
Taq enzyme suitable for the dosage of 0. 5 U.
2.5 dNTPs concentration of dNTPs is the PCR amplification reaction of raw materials, must
reach a certain concentration in order to meet demand for PCR amplification. If the dNTPs
concentration is too low, will affect the reaction efficiency, and even premature due to the
consumption of dNTPs Ershi PCR amplification products of single-chain, thereby affecting the
effectiveness of PCR amplification; but will be too high to compete with the Taq DNA polymerase
Mg2, leading to polymerase incorporation errors, the same is not conducive to PCR amplification.
Can be seen from Figure 5, different concentrations of dNTPs amplification results vary widely. In
the 0.05 ~ 0.10mmol / L when the PCR product bands is weak, at 0.15 and 0.20mmol / L when the
amplification effect of better, in the 0.30 mmol / L was no amplified bands. From the amplified
brightness considerations, dNTPs concentration determined to be 0.20mmol / L.
2.6 primer concentration will affect the concentration of primer specificity. The best primer
concentration is generally 0.1 ~ 0.5�� mol / L. Primers volume is too low, then the product of the
amount of reduced or no amplification product; high concentration of primers would be misleading to
promote primer synthesis of non-specific products, but also increase the formation of primer dimers.
Non-specific products and primer-dimer PCR reaction is also a substrate to compete with the target
sequence DNA polymerase and dNTP substrate, thereby reducing the amount of target sequence
amplification. From Figure 6 shows that primer concentration of 0. 15 �� mol / L and 0.25 �� mol
/ L, the PCR products of gel electrophoresis bands of clear and stable, taking into account the
excessive drug use, as well as easy to form primer primer dimers, so to determine the concentration
of primers to 0. 15 �� mol / L.
3 Discussion
SRAP marker was summing up the advantages and disadvantages of existing DNA molecular
markers developed on the basis of a new PCR-based DNA molecular markers. SRAP marker to
overcome the random amplified polymorphic DNA (RAPD) reproducibility, poor stability of the
shortcomings; do not like the restriction fragment length polymorphism (RFLP) marked as requiring
high purity and high concentrations of DNA and the use of radioisotopes, or required, as amplified
fragment length polymorphism (AFLP), as pre-amplification and connections need to cumbersome
operations, thereby reducing the need for manpower and material needs and dependence.
Importantly, through the SRAP is simple, efficient, high co-dominant, reproducible and easy to
sequence, etc., in particular, that it can detect genes that can be translated reading frames (ORFs)
regions, resulting in improved results and performance-based amplification the correlation [4]. At
present, DNA molecular marker technology in house fly is mainly RAPD, this experiment will SRAP
technology to the study of housefly, Musca domestica in the molecular research in the field provides
the possibility of genetic variation. Experiments show that the use of high concentrations of agarose
gel electrophoresis, can also better identify polymorphic bands, avoiding the use of cumbersome
polyacrylamide gel electrophoresis to detect PCR product. It was found that enzyme concentration
and Mg2 pairs of SRAP-PCR amplification results of a greater impact on small changes can cause a
lot of changes, and Guan Cheng-nan et al [5] were the findings of difference is that the system
requirements on the template concentration is not high, it is necessary for different species to carry
out SRAP-PCR amplification system optimization. Primers, dNTPs concentration changes in a
relatively small impact on the system, the concentration of each component system based on
specific experimental conditions can be adjusted. These results suggest that, Musca domestica
SRAP-PCR reaction system suitable for: in 30 �� l reaction containing 50 ng template DNA, 1.5
mmol / L Mg2, 0.20 mmol / L dNTPs, 0.15 �� mol / L primers and 0.50 U Taq DNA polymer
enzyme.
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on the free Paper Download Center http://www.hi138.com