| 摘要: |
| 根据D03等11个微卫星引物单位点PCR扩增时优化的扩增条件(退火温度、MgCl2等)和11个微卫星引物所带有的荧光颜色、等位基因大小变异范围及反应的灵敏度,可将11个微卫星引物分成A组(D03、G04、B04和H08)、B组(A09、H11、A08和G01)和C组(CmrHr1.24、CmrHr2.30和CmrHr2.23)进行多元PCR扩增,结果显示各组内位点之间的分离清晰,这表明多元PCR可以大大提高微卫星检测仪器的使用效率和提高实验效果,在微卫星研究中是一种快速便捷的方法。 |
| 关键词: 微卫星 黑鲍 多元PCR TouchDown PCR |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金资助项目,30231013号;福建省自然科学基金资助项目,B0110036号 |
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| MULTIPLEX PCR APPLICATION IN MICROSATELLITE OF HALIOTIS RUBRA |
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LI Zhong-Bao,Appleyard Sharon A,Elliott Nicholas G
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1.Fisheries College, Jimei University, Xiamen, 361021;2.CSIRO Marine Research, GPO Box 1538, Hobart, Tasmania 7001, Australia
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| Abstract: |
| Multiplex polymerase chain reaction (multiplex PCR) is a variant of PCR in which two or more loci are simultaneously amplified in a same reaction. The application of it would enhance largely the genetic research on fishery resource and management.
An efficient multiplex PCR requires generally a strategy to optimize reaction conditions. For a successful multiplex PCR assay, it is important that the cycling temperatures, concentration of MgCl2, relative concentration of the primers, concentration of dNTPs, concentration of PCR buffer, and the amount of template DNA and Taq DNA polymerase.
Eleven microsatellite loci screened (H08, G04, D03, B04, A08, A09, G01, H11, CmrHr2.23, CmrHr1.24 and CmrHr2.30) were used. Single-locus PCR protocol is the same as multiplex PCR protocol described below, except that it uses only a single primer pair. Loci for multiplexing have consistent amplification conditions (annealing temperature and MgCl2 concentration), allele length distributions that do not overlap, similar product intensity, and different fluorescent colour of their primers. We divided eleven screened microsatellite loci into three groups for multiplex PCR. The first group contained four loci (H08, G04, D03 and B04); the second group had four loci (A08, A09, G01 and H11) and the third one, three loci (CmrHr2.23, CmrHr1.24 and CmrHr2.30).
Multiplex PCR amplifications were performed in a total volume of 25μl. All loci were amplified in a mutiplex PCR reaction containing 2.5mmol/L MgCl2, 0.4mmol/L dNTPs, 2.5μl 10×PCR gold buffer (which contains 15mmol/L Tris-HC1, 50mmol/L KCl, pH 8.0), 0.25 units Taq DNA polymerase, 0.8μmol/L primer mixed forward and backward for the first and second, or 0.6μmol/L primer mixed forward and backward for group three, and 2μl 1/10 dilution of template DNA.
All PCRs were conducted in GeneAmp PCR System 9600 thermal cycler. For group three, the multiplex PCR conditions began with an initial denaturation step of 10 min at 94°C. Amplification included 10 cycles of denaturation at 94°C for 30s; annealing at 60–55°C for 30s, dropping 0.5°C per cycle; and extending at 72°C for 1min, followed by 25 more cycles of denaturation at 94°C for 30s; annealing at 55°C for 30s, and extending at 72°C for 1min. The PCR conditions ended with a final extension step of 10min at 72°C. For the first and second groups, the multiplex PCR conditions began with an initial denaturation step of 10min at 93°C. Amplification included 35 cycles of denaturation at 93°C for 30s, annealing at 55°C for 30s, and extending at 72°C for 1min. The PCR conditions ended with a final extension step of 10min at 72°C.
Samples were run on an ABI PRISMTM 377 DNA Sequencer, and genotypes were determined using Software GenoScanTM. The results showed that loci were separated well in three groups: from top to bottom, for Group one: H08: blue; B04: yellow; D03: green; G04: blue; as well as for Group two members Group two: A08: blue; A09: green; H11: yellow; G01: yellow, and Group three 2.30: green; 2.23: blue; 1.24: yellow.
Multiplex PCR is a rapid and convenient method for wide application in genetic studies. In general, multiplex PCR is an advanced technique that can significantly reduce the time and cost associated with microsatellites analyses in abalone in our case, and also increase the efficiency of apparatus associated with microsatellites analyses and improve the quality and quantity of loci coamplified. |
| Key words: Microsatellites, Haliotis rubra, Multiplex PCR, TouchDown PCR |
