Problems with pcr, cDNA primers

Student, from the situation you asked, I guess you may have some basic questions that are very vague.

I'll try to analyze it.

1.First of all, PCR is a technique for amplifying a large amount of target DNA in vitro.

You ask what the template DNA is, which is not needed, what is the gene of interest that you want to amplify, what is the template. If you use cDNA as a PCR template, of course, it is a cDNA double-stranded. (must be double-stranded).

cDNA is used as a template, and the primer is of course not cDNA, but a segment of oligonucleotides that can be complementarially paired with a single stranded cDNA. (Primers are designed).

To sum it up. mRNA is a single strand of cDNA that has been removed from the intron to synthesize a double-stranded cDNA PCR, and the resulting large amount of cDNA product is equivalent to amplification of useful information about eukaryotic DNA (the information we want to study --- exons).

2 mRNA reverse transcription does not lose fragments. The primary product of eukaryotic DNA transcription is with introns, and mRNA is obtained by joining exons after excision of introns.

3. Direct extraction of eukaryotic DNA for PCR, the obtained is a complete DNA product. As for how you asked how to get the gene of interest, I don't quite understand. First of all, you need to know what your target gene is, your target gene is the complete DNA of eukaryotes, and use DNA for PCR; If the gene of interest is cDNA, use cDNA for PCR.

Figure out the PCR process, the principle, and some terms, and then look at your problem, and you will find that it is not a problem at all.

I didn't know how to send a message to the station.

Hope it helps.

When you transcribe the eukaryotic extracted DNA into RNA, the introns are eliminated, and then the DNA can be reversed.

The first question is, of course, the template is cDNA, since yours are all reverse transcription products, it must be cDNA for PCR amplification.

The second question is, if you use the DNA of the rhucleoid cell for PCR, if you want to get the target gene, there is an intron sequence.

So do PCR, and use the cDNA obtained from reverse transcription as primers"Your understanding is incorrect, cDNA is used as a PCR template;

When mRNA is reversed, there will be fragment breaks or incomplete, but there are hundreds of thousands or even tens of millions of mRNA copies, and not every mRNA copy will be broken or degraded at the target gene you amplified.

In addition, when extracting RMA, there is generally no DNA contamination, and even if there is, we can avoid amplification of genomic DNA through the design of primers. Design primers so that they are at the junction of two exons so that genomic DNA can be amplified.

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.

The role of primers in PCR: to find a suitable pair of nucleotide fragments that allow them to efficiently amplify the template DNA sequence.

Primers are artificially synthesized two-segment oligonucleotide sequences, one primer that complements one strand of DNA template at one end of the gene of interest, and the other primer that complements another strand of DNA template at the other end of the gene of interest.

In PCR (polymerase chain reaction) technology, the nucleotide sequence of a target gene is known, and primers are synthesized according to this sequence, and the DNA of the target gene is denatured by heat and denatured into a single strand, and the primers are combined with the corresponding complementary sequence of the single strand, and then extended under the action of DNA polymerase, so that the cycle is repeated, and the product obtained after extension can also be combined with the primers.

The essence of the primer is the nucleotide sequence, which is used as the binding site of DNA polymerase at the beginning of DNA replication, and the specific PCR principle is to use DNA molecules to denature and unwind into a single strand at a high temperature of 95 in vitro, and then when it is cooled to about 60 °C, the primer will be combined with single-stranded DNA according to the principle of base complementarity, and then the temperature is raised to about 72 °C, that is, the optimal reaction temperature of DNA polymerase, and DNA polymerase from 3'The end begins to synthesize the corresponding complementary strand in the direction of the new nucleic acid strand, and so on to achieve the purpose of DNA molecule amplification.

Generally, PCR uses two primers, one before and one before and after, and after binding to the template DNA, the primers before and after form a replication start site, which is jointly amplified to the middle until it is connected, and the amplification is completed at one time.

The main steps of PCR technology are as follows:

1. DNA denaturation: (90 -96): Under the action of heat, the hydrogen bonds of the double-stranded DNA template are broken, and the single-stranded DNA is traveled.

2. Annealing: (60 -65): The system temperature decreases, and the primers combine with the DNA template to form local double strands.

3. Extension: (70 -75: With DNTP as raw material, under the action of Taq enzyme (about 72), with DNTP as the substrate, starting from the 3 ends of the primer, extending from 5 3 ends, and synthesizing complementary DNA strands by template method.

Each cycle is denatured, annealed, and prolonged, doubling the DNA content. There are now PCR PCs that replicate in a very short period of time even if the Taq enzyme activity is not optimal because the amplified region is very short.

Therefore, it can be changed to a two-step method, i.e., annealing and elongation at 60 -65 at the same time, to reduce the primary heating and cooling process and increase the reaction speed.

Basic principles of primer design.

1. Primer length: 15-30bp, generally about 20bp.

2. Primer base: the content of G+C is 40-60%, too little G+C, the amplification effect is not good, and too much G+C is prone to non-specific bands. ATGC should be randomized to avoid more than 5 purines or pyrimidine nucleotides.

3. There should be no complementary sequences in the primers.

4. There should be no complementary sequence between the two primers, especially to avoid complementary overlap at the 3 ends.

5. The homology between the primer and the non-specific amplification region should not exceed 70%. The 8 bases at the 3rd end of the primer should not have a complete complementary sequence outside the region to be amplified, otherwise it is easy to lead to non-specific amplification.

6. Primer 3'The bases of the ends, especially the last two bases, should be closely matched. The best options are G and C.

7, the primer of 5'The end can be modified. For example, restriction enzyme sites are added, mutation sites are introduced, labeled with biotin, fluorescent species, digoxin, other short sequences are added, including start codons, stop codons, etc.

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 primarily of DNA polymerase, source DNA templates, 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 base pairing, DNA strand extension, and amplification by DNA polymerase. 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 the fields of molecular biology, medical diagnosis, and biological research. 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 regulation.

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. <>

Hello, PCR technology is a commonly used technique in molecular biology to amplify DNA sequences through the action of primers. Primers are nucleic acid fragments involved in amplification in a PCR reaction and consist of two parts:5'End and 3'End.

Primer of 5'The end sequence is complementarially paired with one base on the template DNA strand, 3'The end sequence is complementarially paired with an adjacent base on the template DNA strand. This allows primers to bind to the template DNA in a targeted manner and provides a starting point for the TPI polymerase to begin amplifying the DNA sequence. In PCR reactions, primer selection is very important, which directly affects the specificity of the amplification product, the amplification efficiency, and the occurrence of hybridization reactions.

Therefore, when designing PCR experiments, the appropriate selection of primers is crucial for the accuracy and reliability of the experimental results.

The ,,, DNA polymerase that initiates DNA replication does not have the function of initiating replication, but only the function of extending replication, so a primer is required to initiate replication.

Pairs with single-stranded DNA to form a replication start point.

Pair them up first, and then start synthesizing gradually.