First, the knowledge background:
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1. Gene expression: DNA → RNA → Protein
There is a step-by-step amplification mechanism for single-copy gene expression, such as a silk fibroin gene → 104 fibroin mRNA (each filament can survive for 4 days, 105 silk fibroin can be synthesized) → a total of 109 fibroin is synthesized. Therefore, the mRNA expression level of a single copy gene is very important for the regulation of its functional level. Shanghai Chuangsai Technology provides Pfu (high fidelity DNA polymerase), BR, commodity number: D16-1027382-100U, the price is 230 yuan.
2, PCR technology (Polymerase chain reaction): the polymerase chain reaction.
The enzymatic reaction of DNA polymerase is dependent on the presence of the template, primers and four deoxynucleotides, the specificity of which is determined by two synthetic primer sequences. Shanghai Chuangsai Technology provides 143491-57-0|emtricitabin Emtricitabine>99%, nucleoside reverse transcriptase (NRTI) inhibitor, commodity number: C84-8090-1G, price 361 yuan.
The reaction is divided into three steps:
A, denaturation: the hydrogen bond of the DNA double helix is ​​broken by heating to form single-stranded DNA;
B. Annealing: The reaction mixture is cooled to a temperature to allow the primer to bind to the template.
C. Extension: The annealing primer extends in the 5' → 3' direction in the presence of DNA polymerase and dNTPs and Mg2+.
The above three steps are a loop, and so on.
3. Reverse transcriptase and RT-PCR
A reverse transcriptase is an RNA-dependent DNA polymerase present in an RNA virus and has at least three activities:
1. RNA-dependent DNA polymerase activity: synthesize the first strand of cDNA using RNA as a template;
2. Rnase hydrolysis activity: hydrolysis of RNA in RNA:DNA hybrids;
3. DNA-dependent DNA polymerase activity: synthesize complementary double-stranded cDNA using the first DNA strand as a template.
Second, the preparation of RT-PCR:
1. Primer design and principles:
1) The specificity of the primer determines the specificity of the PCR reaction. Therefore, whether the primer design is reasonable or not has a crucial impact on the whole experiment. Primers should be designed with full consideration of possible homologous sequences, different subtypes of the same protein, different mRNA splicing patterns, and the possible effects of hnRNA on the specificity of the primers. Try to choose primers that cover the two introns, or introns that are specific in the expression of the protein of interest but not in other subtypes.
2) Grasping the design principles of primers: Primer design principles include
a, primer length: generally 15 ~ 30bp, primers too short will affect the specificity of PCR, primers too long PCR optimal extension temperature will exceed the optimal temperature of Taq enzyme, but also affect the specificity of the reaction.
b. Base distribution: The four bases should preferably be randomly distributed to avoid the accumulation of purine or pyrimidine, especially the continuous appearance of more than three single bases. GC content (Tm value): 40% to 60%, and the renaturation temperature of PCR amplification is generally a lower Tm value minus 5 to 10 degrees.
c, 3' end requirements: The 3' end must be strictly complementary to the template, no modification can be made, and there is no possibility of forming any secondary structure. When the last base is A, the mismatching efficiency is the lowest, and G and C are in the middle. Therefore, it is preferable to use A, G, and C at the 3' end of the primer to avoid two or more consecutive Ts as much as possible.
d. Secondary structure of the primer itself: The primer itself should not have a complementary sequence, otherwise it will fold into a hairpin-like structure or the primer itself will be renatured.
e. Secondary structure between primers: there should be no more than 4 consecutive bases complementary between the two primers, and no more than 2 at the 3' end.
f. Homologous sequence: The homology of the primer to the non-specific amplified sequence should be less than the existence of 8 consecutive complementary bases.
There is no strict restriction on the g and 5' ends: the 5' terminal base can be free, but preferably G or C, to stabilize the terminal binding of the PCR product. Specific modifications (markers, restriction sites, etc.) and the like can also be performed.
2, consumables:
The consumables used for the contact samples used in the experiment, such as cryotubes, tips, EP tubes, etc., were previously subjected to 0.1% DEPC water soaking to remove RNase to prevent RNA degradation during the operation. It is then inactivated by high pressure (sterilization and inactivation of DEPC).
3. Reagent preparation:
Denaturant, water saturated phenol, sodium acetate, chloroform, isopropanol, 75% alcohol, treated with DEPC and high pressure water.
Third, the extraction method of RNA:
The quality of RNA isolated from cells in RT-PCR is critical, including the purity and integrity of the RNA. The most critical factor in RNA isolation is to minimize RNase contamination. However, RNase activity is very stable and widely distributed. In addition to cellular endogenous RNase, a large number of RNases are also present in the environment. Therefore, when extracting RNA, try to create an RNase-free environment, including removing exogenous RNase contamination and inhibiting endogenous RNase activity, mainly using diethylpyrocarbonate (DEPC) to remove exogenous RNase. Endogenous RNase is inhibited by the RNase repressor protein Rnasin and a potent protein denaturant such as guanidinium isothiocyanate. Shanghai Chuangsai Technology provides RNase inhibitor, RNasin Inhibitor, product number: B11000504-1000u, the price is 118.3 yuan.
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February 23, 2023