Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.
Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.
The development of PCR-based markers has revolutionized the science of plant genetics and breeding1. Simple sequence repeat (SSR) markers are among the most commonly used and most versatile DNA markers. Their broad genome coverage, abundance, genome specificity, and repeatability are some of the merits of SSR markers in addition to their codominant inheritance for the detection of heterozygous genotypes2. Several studies have used SSR markers to investigate genetic diversity, track ancestry, construct genetic linkage maps, and map genes for economically important traits3,4.
PCR products of SSR markers are commonly separated using agarose or polyacrylamide gel electrophoresis and then visualized with silver staining or under UV light after staining with ethidium bromide. Silver staining of DNA fragments in polyacrylamide gels is more sensitive than the other staining methods5,6 and has been widely used to detect DNA fragments such as SSR markers7.
Like many biological laboratory techniques, silver staining of polyacrylamide gels has steadily improved since its first being reported as a fragment visualization technique in 19798. The technique was initially modified for detection of DNA fragments by Bassam et al.6 in 1991 and then improved by Sanguinetti and coworkers9 in 1994. The method has been further optimized in the last few decades6,7,9,10,11,12,13,14,15. However, most of these updated versions of the protocols still have some drawbacks such as high technical demand and long processing time for fixation and mounting6, that limit the application of these protocols7,11. An optimal protocol that combines low-cost with high efficiency of DNA fragment detection is urgently needed for routine application of silver staining in biological research.
In addition, polyacrylamide gel can be divided into denaturing and non-denaturing polyacrylamide gels, and both can be used for the detection of SSR markers using the silver staining method. The effect and resolution of which do not significantly differ, but non-denaturing polyacrylamide gels are easier to process and take less time16.
On the basis of the previous research15, the aim of the current study is to describe an optimized silver staining protocol in detail for quick, easy and low-cost detection of SSR markers in a non-denaturing polyacrylamide gel.
1. Preparation of PCR Products of SSR Markers
2. Preparation of Solutions for Polyacrylamide Gels Casting
3. Preparation of Polyacrylamide Gels for Electrophoresis
4. Running Gels
5. Silver Staining for Detecting SSR Markers in a Non-Polyacrylamide Gel
The PCR amplicons were produced using the corresponding SSR primer pairs in flowering Chinese cabbage and tobacco. After electrophoresis, the polyacrylamide gels were stained using the above silver staining protocol, which unambiguously detected the banding patterns of SSR markers (Figure 1).
To compare the detection efficiency of different silver staining protocols, PCR products of SSR markers in tobacco and flowering Chinese cabbage were separated using polyacrylamide gel electrophoresis and visualized using five published silver staining protocols9,11,12,13,14 and the new protocol. All six protocols detected SSR banding patterns for tobacco and flowering Chinese cabbage genotypes (Figure 2), but the present protocol had the lowest background noise and highest contrast of DNA fragments, such that it produced the highest picture clarity for the unambiguous screening of SSR polymorphisms. The running time and number of the main steps and reagents used to complete the silver staining process are listed in Table 1 for each protocol, the new protocol takes the least time and requires fewer chemical reagents and process steps.
Figure 3 shows the sensitivity of the protocol as measured by 50-2,000 bp DNA marker on a non-denaturing polyacrylamide gel.
Figure 1: Detection of SSR banding patterns. Detection of SSR banding patterns for genotypes of flowering Chinese cabbage (A) and tobacco (B) using the new silver staining protocol. The PCR products were separated on 6% non-denaturing polyacrylamide gels and stained as per the above-reported silver staining protocol. Lane M is DNA size marker, lanes 1 to 62 represent the amplicons of 62 genotypes amplified by SSR primer pairs of "CX-157" (the sequences of forward and reverse primers are 5'-TCGACGCTGACTTCACTGAC-3' and 5'-GGACAGCTTCACACATTTGC-3', respectively) in flowering Chinese cabbage (Figure 1A) and "PT51333" (the sequences of forward and reverse primers are 5'-GCACCTTTGGTTATCCGACA-3' and 5'-TGCTTTAAGTCATGTACCAAATTGA-3', respectively) in tobacco (Figure 1B). The arrows point to the target SSR bands. Please click here to view a larger version of this figure.
Figure 2: Comparison of the detection efficiency for SSR markers in tobacco and flowering Chinese cabbage genotypes using different silver staining protocols. The PCR products were separated in 6% of non-denaturing polyacrylamide gels and stained using five published silver staining protocols9,11,12,13,14 and the new protocol. Lane M is DNA size marker. Lanes 1 to 4 represent the amplicons of SSR primers of "PT50903" (the sequences of forward and reverse primers are 5'-AAATTTCTTTCGGTGTGATAACTG-3' and 5'-AGAGACTGCCGTTTGATTTGA-3', respectively) in four tobacco genotypes; lanes 5 to 8 indicate the amplicons of SSR primers of "CX-43" (the sequences of forward and reverse primers are 5'-TGGGGATGTGAGCTTCTTCT-3' and 5'-AGGGTTCCTTTGGGGTGATA-3', respectively) in four flowering Chinese cabbage genotypes. The target SSR bands are indicated with arrows. This figure has been modified from the Figure 2 of Liu et al.8 Please click here to view a larger version of this figure.
Figure 3: Sensitivity of the protocol as measured by 50-2000 bp DNA marker on a non-denaturing polyacrylamide gel. The first lane was loaded with 1µL of DNA marker with fragment sizes of 50, 100, 200, 300, 400, 500, 600, 700, 1000, 1500 and 2000 bp, each at 10 ng/µL. The remaining samples were loaded with a 1:2 serial dilution in preceding lanes. The minimum detectable amount for the protocol was 9.8 pg in the 11th lane (2.2 pg/mm2 in a 4.5 mm2 well). This figure has been modified from the Figure 1 of Liu et al.8. Please click here to view a larger version of this figure.
Item | Sanguinetti et al.9 | Qu et al.11 | An et al.12 | Byun et al.13 | Kumar et al.14 | New protocol |
Running time (min) | 30 | 12–25 | 8–9 | 9–31 | 42 | 6–7 |
Number of steps | 5 | 4 | 3 | 3 | 3 | 2 |
Number of reagents | 5 | 6 | 6 | 5 | 7 | 3 |
Table 1: Running time, number of key steps and reagents used in the different silver staining protocols.
The washing of gel after impregnation is a critical step. Insufficient washing time and water volume may cause the incomplete removal of impregnation solution on the surface of the plate and the gel, and result in a dark background. The appropriate developing time is another key step, over-development can result in a dark-brown background with low contrast image of DNA fragments. In addition, the impregnation step significantly affects staining efficiency of DNA fragments. Although extending the impregnation time over 5 min or increasing AgNO3 amount in the impregnation solution do not significantly improve the staining quality, a reducing the impregnation time to less than 3 min or decreasing the amount of AgNO3 (<1 g) can result in grey or shallow black DNA fragments.
A quick and easy operation for efficient DNA detection is desirable for a silver staining method. The new protocol developed in this study avoids the fixing, stopping and several washing steps described in other protocols6,9,10,11,12,13,14 without affecting the staining quality (Figure 1 & Figure 2), and only requires two simple steps of impregnation and development that take 7 min. Therefore, the new protocol is faster than all the other staining protocols current available6,7,9,10,11,12,13,14. In addition, the new protocol only requires three chemicals, i.e. AgNO3, formaldehyde, and NaOH, at similar amounts as that used in other protocols6,7,9,10,11,12,13,14, therefore, the new protocol is more economical and generates less chemical hazards, which not only reduces the chemical cost but also minimizes the hazardous material handling process of extra chemicals in the laboratory.
The new protocol produced clear images with low background noise for the unambiguous detection of SSR banding patterns in tobacco and flowering Chinese cabbage genotypes (Figure 1). Compared with other protocols, the new protocol produced the best staining effect with the lowest background noise and highest picture contrast (Figure 2). At the range of 50-500 bp, the sensitivity of the new protocol for DNA detection was less than 19.5 pg/µL (4.3 pg/ mm2) with a minimum concentration of 9.8 pg/µL (2.2 pg/mm2) (Figure 3), which is higher than that reported in other protocols7,12,13.
Although fluorescence-labeling technologies can provide high throughput and resolution detection of DNA amplicons, they require specialized equipment and more expensive reagents that may not be accessible for many biological laboratories, especially those in developing countries. The new staining protocol is simple and fast that can screen ~800 DNA samples per person per day, thus, is a good option for these laboratories to perform routine research.
In conclusion, the new protocol developed in this study is quicker to process, easier to implement, cheaper, and only uses three reagents—without compromising detection effect or picture clarity—than all other existing silver staining techniques. The new silver staining protocol has been used successfully in tobacco and flowering Chinese cabbage research and can be a valuable tool for successful SSR genotyping in other species.
The authors have nothing to disclose.
This work was funded by the Guangdong Natural Science Foundation of China (2015A030313500), the Provincial Key International Cooperative Research Platform and the Major Scientific Research Project of Guangdong Higher Education (2015KGJHZ015), the Science and Technology Plan of Guangdong Tobacco Monopoly Administration (201403, 201705), the Science and Technology Plan of Guangdong of China (2016B020201001), the National Innovation Training Project for Undergraduate Students (201711078001). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.
PCR master mix (Green Taq Mix) | Vazyme Biotech Co. Ltd, China | #P131-03 | |
50-2000 bp DNA Ladder | Bio-Rad, USA | #170-8200 | |
DL500 DNA marker | Takara Bio Inc., Japan | #3590A | |
Tris base | Sangon Biotech Shanghai, China | #77-86-1 | |
Boric acid | Sangon Biotech Shanghai, China | #10043-35-3 | |
EDTA-Na2 | Guangzhou Chemical Reagent Factory, China | #6381-92-6 | |
Acrylamide | Sangon Biotech Shanghai, China | #79-06-1 | |
N,N'-methylene-bis-acrylamide | Sangon Biotech Shanghai, China | #110-26-9 | |
N,N,N',N'-Tetramethylethylenediamine | Sangon Biotech Shanghai, China | #110-18-9 | |
Ammonium persulfate | Guangzhou Chemical Reagent Factory, China | #7727-54-0 | |
Bind-silane | Solarbio Beijing, China | #B8150 | |
AgNO3 | Sinopharm Chemical Reagent Beijing Co.,Ltd, China | #7761-88-8 | |
Formaldehyde | Tianjin DaMao Chemical Reagent Factory, China | #50-00-0 | |
NaOH | Guangzhou Chemical Reagent Factory, China | #1310-73-2 | |
Acetic acid | Guangzhou Chemical Reagent Factory, China | #64-19-7 | |
Na2CO3 | Tianjin DaMao Chemical Reagent Factory, China | #497-19-8 | |
Ethanol | Guangzhou Chemical Reagent Factory, China | #64-17-5 | |
HNO3 | Guangzhou Chemical Reagent Factory, China | #7697-37-2 | |
Na2S2O3.5H2O | Sinopharm Chemical Reagent Beijing Co.,Ltd, China | #10102-17-7 | |
Eriochrome black T(EBT) | Tianjin DaMao Chemical Reagent Factory, China | #1787-61-7 | |
Plastic tray | Shanghai Yi Chen Plastic Co., Ltd, China | – | |
TS-1 Shaker | Qilinbeter JiangSu, China | – | |
BenQ M800 Scanner | BenQ, China | – | |
DYY-6C Power supply | Beijing Liuyi Instrument Factory, China | – | |
High throughout vertical gel systems, JY-SCZF | Beijing Tunyi Electrophoresis Co., Ltd, China | – |