Did you choose the right PCR consumables?
What is the polymerase chain reaction?
Polymerase chain reaction (PCR) is a molecular biology technique used to amplify a specific segment of DNA to a sufficient amount for structural and functional analysis with the involvement of a DNA polymerase and a nucleotide substrate.
One of the factors affecting PCR experiments --Consumables
In PCR experiments, there are many factors that affect the amplification effect. We can think of issues such as the quality and concentration of dNTP, the amount and purification of template nucleic acid, the design of primers, the concentration of DNA polymerase, and the amplification length.
In fact, the consumables used may also affect the experimental results, which is often overlooked by us. Therefore, we also need to understand the characteristics and terminology related to PCR consumables before selecting them for experiments.
How to choose the right PCR consumables?
1.Raw material
PCR/qPCR consumables are generally made of polypropylene (PP).
Because it is biologically inert material, the surface is not easy to adhere to biomolecules, and has good chemical resistance, and temperature tolerance (can be 121 ℃ autoclaved, can also withstand temperature changes during the thermal cycle). These materials are usually in direct contact with reagents or samples, and therefore require high quality materials and good processing in the production and preparation process.
2. Choose the right volume
Most of the volume specifications of PCR tubes can meet the requirements of PCR reactions. However, on the basis of meeting the experimental requirements, it is recommended to use low volume tubes. Because the low volume reaction tube / plate has a smaller space above, can improve the thermal conductivity and reduce evaporation. Moreover, when adding samples, it is necessary to avoid adding too much or too little sample. Overdosing can lead to reduced thermal conductivity, spillage and cross-contamination, while underdosing may result in loss of sample evaporation.
You can choose the right product according to your specific experimental requirements.
3.The choice of PCR tube cover
Flat cap and convex cap have their own advantages.
Flat cap can provide accurate fluorescence signal transmission for qPCR; easy to write markers; easy to close the cap, good sealing, easy to operate.
The convex cap is in contact with the hot cover of the PCR instrument, reducing pressure-induced deformation of the reaction tube and better preventing liquid evaporation. However, it will affect the fluorescence signal transmission and cannot be applied to qPCR experiments.
4.The choice of 96-well plate skirt
No skirt plate: can be applied to most of the PCR instrument or qPCR instrument, but not for automation, applications. Not high stability during pipetting, need to be used with plate tray.
Semi-skirt plate: can be adapted to labels or application barcodes, suitable for automated applications and has good pipetting stability.
Fully skirted plates: very suitable for automated experimental applications, also adaptable to labels and applied barcodes. Good mechanical strength for PCR instruments with protruding modules and high stability during pipetting.
5.Choose consumables according to the experiment
According to the different detection methods, the choice of consumables is also different.
PCR experiments are divided into ordinary PCR method and fluorescence quantitative PCR method. Ordinary PCR method is generally the end point PCR running gel method determination, so the thermal conductivity and sealing of the consumables used and the experimental results are inextricably linked; while fluorescent quantitative PCR is real-time detection of fluorescent signals, in addition to thermal conductivity and sealing, light transmission and light protection are also required.
In short, the consumables used for fluorescence PCR can also be used for general PCR, but not vice versa.
Thermal conductivity
The better the thermal conductivity and the more uniform it is, the faster and more accurate the PCR will be.
Sealing
The better the sealing, the less the product volatilization, the more the product obtained, and the less the aerosol generation, the less the contamination for the next round of experiment, which means the lower the false positive generation rate.
Light avoidance
This is for fluorescence quantification. If we do not pay attention to light avoidance during the whole experiment, and the reagents with fluorescence are excessively consumed, it will directly affect the amplification efficiency of our experiment and lead to inaccurate results.
Light transmission
This is also true for fluorescence quantification, as we know it is a real-time quantification method, then the light transmission of the consumables itself is directly related to the amount of fluorescence signal we can detect. If we choose consumables with good light transmission, the signal that needs to be detected will become weak, and that will cause us to misjudge the results. Therefore, for fluorescence quantification, white PCR plates are better than transparent PCR plates.
Clinical applications of PCR technology
While monoclonal antibody technology has led to new breakthroughs in the theory and practice of immunology, PCR technology has led to a methodological revolution in molecular biology and related disciplines in recent years. In less than 10 years, PCR technology has gained practical value in the following fields, and its application is expanding.
1. Prenatal diagnosis of genetic diseases
Using fetal amniotic cells, amniotic fluid or even maternal blood can check the sex of the fetus, which is necessary in the diagnosis of genetic diseases associated with sex chromosomes. For the high incidence of genetic diseases, such as thalassemia, sickle cell anemia, coagulation factor deficiency, DMD, etc. have been in clinical use for many years, contributing to eugenics, and for diseases with genetic predisposition, especially diseases of old age, such as diabetes, hyperlipidemia, and even a part of tumors, are the focus of current research, and a breakthrough is expected in the near future.
2.Detection of disease-causing pathogens
These exogenous invasive genes, once the partial nucleic acid sequence is elucidated, you can design primers or probes, PCR, PT-PCR or hybridization methods to detect the range of bacteria, viruses, protozoa and parasites, mycobacteria, rickettsia, chlamydia and mycoplasma and all other microorganisms, PCR diagnosis is characterized by the selection of conserved regions in their genes for general detection, but also selected for greater differences in It is also possible to select genetic sites for typing. It is possible to do a special test for one pathogen, and also to do a multiplex test for different species of viruses and bacteria. The sensitivity and specificity of the test are much higher than the current immunological methods, and the time required has reached the clinical requirements, which is particularly suitable for viruses (hepatitis B), bacteria (such as tuberculosis, anaerobic bacteria) and protozoa (such as syphilis spirochetes), which are difficult to culture.
3. Oncogene detection and diagnosis
Although most of the research on oncogenes is still at the basic stage, the basic fact that carcinogenesis is caused by genetic mutations is indisputable. inactivation, and activation and expression of N-myc genes in neurostromal tumors. The implantation of specific oncogenes and anti-metastatic genes by in situ hybridization and the blocking of strongly expressed oncogenes by antisense oligonucleotides have become the focus of recent gene therapy.
4.DNA fingerprinting, individual identification, paternity identification and forensic physical evidence
This subject, which is of interest to the public, the prosecution and the law, has already gained legal recognition in some countries. Differences in peptidability and number of repetitions of myoglobin minisatellite gene, β-bead protein gene, ApoB gene, etc. have been applied for identification, and their sensitivity has reached a hair, a cell, a sperm to obtain individual characteristic profiles, and this field has also been developed to bone marrow or organ transplantation mating and animal germline research.
5.Animal and plant quarantine
Sensitive, specific, rapid diagnostic testing methods are China's import and export ports of entry and exit gatekeeper, check whether the entry and exit of personnel, animals, plants (breeding animals, seeds), etc. carry virulent infectious diseases (AIDS, animal viruses, plant viruses, etc.) pathogens, food, feed, etc. with Salmonella, etc. need genetic diagnostic means will be these germs outside the gate, is necessary to improve our comprehensive national power to ensure.
6.Application in the field of high-tech biomedicine
PCR technology can be applied to gene splicing, sequencing and other fields.
