Real time PCR principle
Real-time fluorescence quantitative PCR is a method for measuring the total amount of products after each cycle of polymerase chain reaction (PCR) using fluorescent chemicals in a DNA amplification reaction. A method for quantitative analysis of specific DNA sequences in a sample to be tested by internal or external reference methods.
Real-time PCR is the real-time detection of the PCR process through the fluorescent signal during the PCR amplification process. Due to the linear relationship between the Ct value of the template and the initial copy number of the template during the exponential period of PCR amplification, it becomes the basis for quantification.
The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence.
PCR kit including purified nucleic acid template, deoxyribonucleotide (dNTP), oligonucleotide primers and DNA polymerase. Nucleic acid purification and quality assessment are important steps in the PCR experiment process. It is important to use appropriate techniques to isolate and purify nucleic acids from raw materials, as this step will affect the amplification reaction.
DNA polymerase catalyzes the addition of dNTPs to extended DNA strands by adding bases that are complementary to the template strand. The identification of thermostable DNA polymerases and subsequent modifications to these enzymes plays a key role in the development and widespread adoption of PCR reaction.
Five elements of PCR reaction: There are five kinds of substances that participate in the PCR reaction: primers (PCR primers are DNA fragments, and the primers for DNA replication in the cell are an RNA strand), enzymes, dNTPs, templates and buffers (of which Mg2+ is required).
The standard PCR process is divided into three steps:
DNA denaturation: (90℃-96℃): Double-stranded DNA template under the action of heat, hydrogen bond breaks to form single-stranded DNA.
Annealing: (60℃-65℃): The temperature of the system is reduced, and the primers are combined with the DNA template to form a local double strand.
Extension: (70℃-75℃): Under the action of Taq enzyme (about 72℃, the best activity), dNTP is used as the raw material, starting from the 3′ end of the primer and extending from the 5′→3′ end. Synthesize DNA strands complementary to the template.
Each cycle undergoes denaturation, annealing and extension, and the DNA content doubles. Now some PCRs have a short amplification region, and even if the Taq enzyme activity is not optimal, they can be replicated in a short period of time, so it can be changed to a two-step method, that is, annealing and extension are carried out at 60℃-65℃ at the same time. In order to reduce the temperature rise and fall process, the reaction speed is increased.
PCR results and PCR analysis
1. If there is a bai standard curve, calculate according to the standard curve. du
2. Generally, the relative quantity is calculated by the delta delta CT method.
3. Examples are as follows: the CT value of gene A in the control dao group is 20, and the CT value of internal control (such as βactin) is 15. The CT value of gene A in the experimental group was 18, and the CT value of internal reference was 14.
4. Calculate the sample volume first: delta CT=15-14=1. The power of 2 is 2. In other words, the sample volume of the experimental group is twice that of the control group. Gene A: delta CT=20-18=2. The power of 2 is 4. In other words, the amount of gene A in the experimental group was 4 times that of the control group. However, because the amount of sample added is twice, so 4 = 2 = 2, the final relative amount is 2 times.
5. A few points: The specificity of the amplification must be determined. Only the CT value of the same target can be subtracted (the amplification efficiency may be different). The power of 2 is only the theoretical value, and the actual amplification efficiency is less than 2. Syber Green.
PCR is usually applied to the following aspects:
1. Basic research on nucleic acid: genome cloning
2. Asymmetric PCR to prepare du single-stranded DNA for DNA sequencing
3. Reverse PCR to determine unknown DNA regions
4. Reverse transcription daoPCR (RT-PCR) is used to detect the gene expression level in cells, the amount of RNA virus and directly clone the cDNA of specific genes
5. Fluorescence quantitative PCR is used for real-time monitoring of PCR products
6. Rapid amplification of cDNA ends
7. Detection of gene expression
8. Medical applications: detecting bacterial and viral diseases; diagnosing genetic diseases; diagnosing tumors; applying to forensics.
PCR machine is also called PCR gene amplifier, PCR nucleic acid amplifier, polymerase chain reaction nucleic acid amplifier, it is a kind of instrument and equipment that uses PCR (Polymerase chain reaction, polymerase chain reaction) technology to amplify specific DNA It is widely used in medical and biological laboratories, for example, to determine whether the sample will show a genetic disease map, infectious disease diagnosis, gene replication, and paternity testing.
Major Companies Profiled in the Global Diagnostics PCR Market are: Abbott Laboratories,
Agilent Technologies Inc.,
Alere Inc., Asuragen Inc.,
Biocartis Group Nv, Biofire Diagnostics LLC. (Acquired By Biomerieux),
Bio-Rad Laboratories Inc.,
Cepheid (A Danaher Company),
Genmark Diagnostics Inc.,
Hologic Inc., Luminex Corporation,
Meridian Bioscience Inc.,