What is extended from the 5 ends and 3 ends of the primer during the PCR technique , Xie

There are upstream primers and downstream primers, the 5 ends of the two primers are bound to the 3 ends of the template, and the Taq enzyme used in PCR is polymerized from the 5 ends 3, so the PCR process is the extension of the 5 ends and 3 ends of the primers.

The DNA polymerase Taq, which is commonly used in PCR, only has a polymerization activity of 5'->3' (of course, exonuclease activity, which is not considered here).

Eukaryotic cells have 5 types of DNA polymerases, namely DNA polymerase (localized to the nucleus, involved in replication initiation, and does not have 5'－3'exonuclease activity), localized in the nucleus, involved in repair, and does not have 5'－3'exonuclease activity), localized to mitochondria, involved in mitochondrial replication, and does not possess5'－3', there are 3'－5'exonucular activity), δ localized nucleus, involved in replication, has 3'－5', not with 5'－3'Exonologically active), localized to the nucleus, involved in damage repair, has 3'－5', not with 5'－3'Exonoactive activity.

The Taq enzyme we used was Taq DNA Polymerase, isolated from the Yt1 strain of Thermalasia aquaticus, and was the most active of the thermostable DNA polymerases found, reaching 200,000 mg units. With 5'-3'Exonuclease activity, but not with 3'-5'exonuclease activity, so that there is no correction function for some single nucleotide mismatches in synthesis.

The PCR reaction is from the primers.

The 3' end of the new DNA strand begins to be synthesized. Therefore, in the newly synthesized DNA strand, the original primer is at the 5' end, and the newly added base is at the 3' end.

The 3' end of the primer is the key base, which is the starting end of the PCR extension, and can not be modified in any way, nor can it be possible to form a secondary structure by slipping the code, and the 3' end can not be mismatched, and the pairing should be strictly required to avoid PCR failure due to the mismatching of the terminal bases.

There is no strict restriction on the 5' end, which only plays the role of limiting the length of the PCR product and is specific for amplification.

The effect is not large, so it is often used to introduce modification sites or markers.

There may be a suitable digestion site in the primers, and the amplified target sequence should have a suitable digestion site, which is very beneficial for enzyme digestion analysis or molecular cloning.

The concentration of each primer is 1umol or 10 100 pmol, which is better to produce the desired result with the lowest primer amount, as high primer concentrations can cause mismatches and non-specific amplification, and can increase the formation of dimers between primers.

opportunities.

The role of primers in PCR is to act as a binding site for DNA polymerase at the beginning of DNA replication, and under extracellular conditions, DNA can only begin to replicate through primers.

A synthetic two-segment oligonucleotide sequence, with one primer complementing one strand of DNA template at one end of the region of interest and the other primer complementing another strand of DNA template at the other end of the region of interest.

There are two primers in the PCR reaction, the 5-terminal primer and the 3-primer. Primers are designed based on a single strand of DNA (often based on the information strand), and the 5-end primer is the same as a small piece of DNA on the 5-end of the fragment to be amplified; The 3-end primer is complementary to a small piece of DNA sequence located at the 3rd end of the fragment to be amplified.

In general, primers exist in pairs, either at the 5' end or at the 3' end.

Typically, cloning a piece of gene requires a pair of primers.

PCR technique from primers 3'The end begins to extend.

PCR is a gene amplification technique that enables DNA samples to be proliferated and prepared from them enough DNA templates for subsequent molecular biology experiments. PCR is a specific DNA amplification technique that consists of DNA polymerase, source DNA template, primers, and nucleotides. Among them, primers are a key component of PCR amplification, and the selection and design of primers determine the specificity, selectivity, and amplification of PCR reactions.

The design of primers needs to take into account many factors, such as primer sequence, length, double-stranded structure stability, TM value, etc., as well as the needs of PCR amplification, such as the selection of primers to make the amplified fragment appropriate and avoid amplification of non-specific products. During the PCR reaction, primers are complementarially bound to the DNA template, followed by DNA polymerase, DNA strand extension, and amplification. Primer 3'The ends are "glued" to the complementary sequence of the DNA template and then to 5'The end is extended, so the PCR reaction is removed from primer 3'The end begins to extend.

PCR technology has a wide range of applications, and its importance is reflected in molecular biology, medical diagnosis, biological research, and other fields. In the experiment, the correct primer design and selection is the key to PCR amplification, and all parameters need to be strictly controlled in the experimental design to ensure the success and stability of the PCR reaction. In addition to primers, there are many other factors that can affect the success rate of PCR technology, such as reaction system, amplification enzyme, reaction temperature, and conditioning.

At the same time, in the future development of PCR technology, the emergence of new technologies and new methods will further promote the development and application of PCR technology. <>