Keywords: point mutation; Polymerase; Hereditary thalassemia; Gene mutation screening
China Library Classification Number: Q3 19.3 1 Document Identification Number: A Document Number:1007-7847 (2007) 04-0295-06.
β thalassemia (β thalassemia) is an autosomal recessive genetic disease. Because of the mutation or deletion of β globin gene, the peptide chain synthesis of globin HbA(α2β2) is reduced or cannot be synthesized, which leads to the imbalance between α and β peptide chain synthesis. If the fetus receives two β thalassemia genes from its parents at the same time, it is a mutant homozygote, and most of them show severe β thalassemia. There is no effective treatment at present. These patients often die young or are maintained by blood transfusion.
Point mutation is the most common mutation in β -thalassemia gene. The point mutation of β -globin gene CDl7(A→T) is one of the earliest reported point mutations related to β -thalassemia. The traditional detection method of β -thalassemia gene mutation is based on gel electrophoresis. The operation is complicated and time-consuming. At present, the commonly used diagnostic method is polymerase chain reaction combined with dot blot hybridization, which has high accuracy, but requires radioisotope labeling and cumbersome molecular hybridization techniques. Pun et al. reported a new genotyping technique of denaturing high performance liquid chromatography, which does not need radioisotope labeling, but this method is complicated in operation, expensive in instrument and long in experimental period. The newly developed real-time fluorescence PCR technology has been used to detect the point mutation of HFE gene in hereditary hemochromatosis, and the operation is simple, but the design difficulty of the probe and the high cost of the detection instrument limit its wide application.
Based on the principle that double-stranded binding dyes are embedded in double-stranded DNA to emit fluorescence, we have established a simple method for real-time detection of polymerase activity at room temperature. On this basis, based on polymerase extension technology and the characteristics of double-stranded specific embedded dyes, a fast and simple real-time detection method of point mutation was developed, which realized point mutation detection by embedding dyes and changing fluorescence signals without expensive instruments. The operation is convenient and simple, and complicated gel electrophoresis operation and labeling are not needed. The mutation of β thalassemia related gene CD 17 was detected by this method, which provided a rapid, simple and effective method for the detection of gene point mutation and single nucleotide polymorphism, and provided a new means and idea for drug screening, disease prevention and haplotype planning.
1 experiment
1. 1 reagents and instruments
Reagents: Primers and oligonucleotides were synthesized by Takara, and the sequences are shown in table 1. Klenow Fragment(exo-) polymerase (KF- enzyme, Fermentas), SYBR Green I (Shanghai Science and Technology Development Co., Ltd.), TakaRa Ex taq enzyme (TakaRa), DNTP mixture (Takara), Tris (Sigma), dithiothreitol DTT(Bebco) and other reagents are all analytically pure in China, and the experimental water is filtered. The instruments are Perkin Elmer LS-55 fluorescence spectrophotometer, Amersham constant temperature water bath and Ce-neAmPTM PCR System 2700 of ABI company.
1.2 experimental method
1.2 1 fluorescence determination
In this study, SYBRGreen I, a double-stranded specific binding dye, was used to measure the fluorescence intensity with 497 nm light and detected at 520nm. The incident slit and emission slit of the instrument are both set to 2.5nm. After the fluorescence value is stable, the sample is measured at a constant temperature of 37℃ for 65438±00min, and the final volume of the sample solution is 200 μ L. ..
1.2.2 point mutation detection
Prepare the basic solution of 1 (primer N3 100 nmol/L, 50 mmol/L LTRIS-HCl (pH 8.0), 5mmol/L MgCl2,1mmol/l ldtr.50μ mol/l dntp mixture,1l dntp mixture. Add n 1 and N2 with the final concentration of 100 nmol/L to mix 1mmo|/L DTF, 50μmol/L dNTP mixture (same proportion), 1×SYBR GreenI) and the final concentration is100 nmol. After the fluorescence intensity stabilized, 2.5U KF enzyme was added, and the change of fluorescence intensity was continuously monitored.
1.2.3 denaturing polyacrylamide gel electrophoresis
The traditional electrophoresis method was used to verify the detection results of point mutation. Sample solutions A, B and C were denatured at 95℃ for 5 minutes, then quenched at 0℃ for 5 seconds, and analyzed by 20% polyacrylamide gel electrophoresis with 7% urea and silver staining.
1.2.4 Influence of temperature on detection
At 25℃, 30℃, 34℃, 37℃, 42℃ and 47℃, 2.5U KF enzyme was added to sample solution A, and the change of fluorescence intensity was monitored. Taking the ratio (S/B) of fluorescence signal value of 1000s polymerization as the detection standard, the influence of temperature on the detection system was investigated.
1.2.5 Influence of metal ions on detection
The effects of four metal ions (M2+, Ca2+, K+ and Na+) on polymerase polymerization were studied, and the effects of Mg2+ were studied by adjusting the concentration of magnesium chloride in sample solution A.. Add CaCl2 _ 2, KCI and NaCl solution to sample A to adjust to different concentrations, calculate the initial reaction speed in reference [1 1], and investigate the influence of ions on polymerization.
1.2.6 preparation and detection of thalassemia genetic disease point mutation samples
We detected a CDl7(A→T) point mutation in thalassemia genetic disease. Sample 10 (3 homozygotes, 3 heterozygotes and 4 normal samples in our laboratory). Total genomic DNA of samples was extracted from white blood cells of patients and normal people according to standard DNA extraction technology, and primers (Table 65, 438+0) were according to primer 5. The PCR system (50νL) contains: 0.5U TakaRa Extaq enzyme, 0.2mmol/L dNTP mixture, 1×PCR buffer, 800nmol/L N6, 10nmol/L N7 and 50ng total DNA. The PCR reaction procedure is as follows: denaturation at 94℃ for 5 minutes, and then denaturation at 94℃. Annealing at 57℃ for 35s, extending at 72℃ for 30s, and cycling for 40 times. The PCR products were purified by standard gel electrophoresis, and the OD value was detected. In the bottom solution 3( 100 mmol/L primer N5, 50mmol/L Tris-HCl(pH 8.0), 5mmol/L MgCl2, 1mmol/LDTT, 50.
2 Results and discussion
2. 1 experimental principle
The experimental principle is shown in figure 1. A specific allele primer was designed, and its 3' end was terminated at the mutant (polymorphic) base site of the template. When the template is completely matched with the primer, the primer is extended to form a double strand under the action of polymerase, and the double-strand specific dye is inserted into the newly-born double-strand region, and the fluorescence intensity is enhanced. However, when the template and the primer do not match at their 3' ends, the primer cannot be effectively extended and the fluorescence signal does not change. By observing the change of fluorescence signal. Realize point mutation detection.
2.2 Point mutation detection method
According to the literature [13 ~ 15], we used synthetic oligonucleotides to simulate the point mutation target sequence of thalassemia genetic disease CD 17 (table 1), where N 1 and N2 respectively represent two homozygous genotypes of point mutation. 50% mixed samples of N 1/N2 simulated heterozygote genotype. Fig. 2 shows the results of point mutation detection using specific allele primers. The 3' end of primer N3 is terminated at the mutation base site, and it is completely matched with the homozygote of N 1. Under the action of polymerase, complementary dNTP was added to the 3'-OH end of the primer one by one to form a double-stranded structure, and the dye SYBR Green I was embedded in the double-stranded structure to generate fluorescence. When the template is N2 homozygous, the 3' end of primer N3 does not match it, so the polymerization extension reaction cannot occur effectively and the fluorescence signal does not increase significantly (curve 3). When the template is heterozygous, the fluorescence signal is also significantly enhanced (curve 2), but its fluorescence growth rate is obviously lower than curve 1. Through the change of fluorescence, three genotypes can be effectively distinguished. In order to better detect the point mutation, we also used the primer N4 which is completely matched with N2 homozygote for detection. When the template is N 1, add polymerase. Because of primer N4' s 3, when the template is N2, the fluorescence signal increases rapidly with the addition of DNA polymerase, and when the template is heterozygous, the fluorescence also increases, but the growth degree is less than that of the perfectly matched template N2. The detection result is the same as that when using the N3 primer (not shown in the figure).
2.3 polyacrylamide gel electrophoresis
We use the traditional denatured polyacrylamide gel electrophoresis to verify the point mutation detection results. In fig. 3, 1 ~ 3 channels are the bands of N 1, N2 and N3 respectively. Lane 4 ~ 6 are reaction sample solutions of sample solutions A ~ C, respectively. We found that in lane 4 (sample solution A), the primer N 1 band disappeared because the primer N 1 was used as a template for polymerization, but a band of polymerization products appeared above the template N 1 band (primer 5 was designed to distinguish the products produced by polymerization from the original template. Lane 5 (sample solution B) can't produce effective polymerization reaction, and template N2 and primer N3 bands continue to exist: Lane 6 is the reaction solution of sample solution C, and primer bands, template bands and polymerization product bands can be seen, but the brightness of polymerization product bands is weaker than that of Lane 4, indicating that polymerization products are less than that of Lane 4. The above electrophoresis results are consistent with the fluorescence experiment results, which shows that the fluorescence analysis method is a reliable and accurate genotyping method.
2.4 Influence of temperature on detection
Adjust the constant temperature water bath to control the temperature at 25℃, 30℃, 34℃, 37℃, 42℃ and 47℃ respectively, add polymerase into sample solution A, and monitor the change of fluorescence intensity. The result is shown in Figure 4. As can be seen from Figure 4, with the increase of temperature, the ratio (S/B) of fluorescence signal to fluorescence background increases without adding polymerase, but when the temperature exceeds 37℃, the S/B increases.
2.5 Influence of metal ions on detection
Point mutation detection is based on polymerase extension reaction. The polymerization efficiency of polymerase has an important influence on the signal generation and sensitivity of this method. We studied the effects of several important metal ions (Mg2+, Ca2+, K+, Na+) on the polymerization reaction (Figure 5). As can be seen from the figure, when M does not exist, the polymerization reaction cannot occur, and with the increase of Mg2+ concentration, the initial reaction speed is accelerated. When the concentration of Mg2+ reaches 10 ~ 15 mmol/L, the initial reaction speed is the fastest. When the concentration continues to increase, the activity of polymerase is inhibited to some extent, which shows that Mg2+ is essential as an activator of polymerase in the reaction. However, when the concentration of Ca2+ is greater than 30 mmol/L, the low concentration of Ca2+ (less than 20mmol/L) has little effect on the polymerase activity, and obviously inhibits the reaction rate. The existence of K+ and Na+ inhibited the activity of polymerase, and with the increase of concentration, the activity of polymerase was inhibited. When the concentration of K+ and Na+ is higher than 100mmol/L, the polymerization reaction is basically inhibited.
2.6 genotyping of thalassemia disease point mutation
Thalassemia is a hereditary blood disease. We detected a CDl7 point mutation type, and the result is shown in Figure 6. As can be seen from the figure, the fluorescence signals of the three genotypes are similar without polymerase, but there are obvious differences among the three genotypes after polymerase is added, and the fluorescence signal of the mutant homozygote perfectly matched primer (N5) increases sharply after polymerase is added (curve 1). After the wild-type homozygote was added with polymerase, the fluorescence signal did not change significantly (curve 3). However, after adding heterozygote to polymerase, the fluorescence signal is also greatly enhanced. But the fluorescence was significantly lower than that of mutant homozygotes (curve 2). By adding dye molecules and observing the fluorescence signal, the point mutation of CDl7(A→T) can be identified conveniently, quickly and accurately.
3 Conclusion
Based on polymerase extension technology, the genotyping results of point mutation were directly given by the change of fluorescence signal through the embedding of double-stranded specific fluorescent dyes. This method does not need radioisotope labeling or fluorescent labeling of primers and oligonucleotides. Low cost; The experimental operation is simple, without gel electrophoresis detection and complicated and expensive instruments, which provides a simple and quick method for the prevention and screening of genetic diseases caused by point mutation, and also provides a new idea for haplotype planning of single nucleotide polymorphism.
About the author: Meng Xiangxian (1972 ――), female, from Huarong, Hunan Province, is an associate professor at Hunan University, Ph.D., engaged in bio-analytical chemistry research at molecular level; Wang Kemin (1957-), male, from Yuanjiang, Hunan, professor, doctor, correspondent, engaged in the research of bio-analytical chemistry and nano-biotechnology at nano and molecular levels.