What software is available for primer design? What are the advantages and disadvantages?

The advantages and disadvantages are as follows:

1. Software design.

In addition, the design of primers, PCR amplification is largely related to the amplified enzyme, especially the fragment with special structure, the GC content is too high or too low, which will lead to a decrease in amplification efficiency, and it is necessary to choose the right enzyme. Needless to say, the brand of enzyme must be better in Europe and the United States, and some brands of enzymes can also be diluted to 30% to increase ordinary fragments.

2. Handmade design.

When encountering software design that can't be done, or the need to amplify the full length, you can only design it manually, and experienced people can take a rough look at it to know which paragraph to choose.

Note: 1. Don't be entangled in the annealing temperature, the calculation method is different, and the annealing temperature is also different, as long as it is about the same, generally between 55-65 degrees. For the same pair of macro sheds, I have also encountered a very good amplification of 55-65 degrees.

So, don't ask about the primer annealing temperature in the future, it's not a question worth tasking about.

2. The fluorescence quantitative primer design of mRNA, the forward primer is designed at the tail of the coding region, and the reverse primer is designed at the non-coding region near the tail end, so that the DNA cannot be amplified, and the amplified ones are mRNA.

The advantages and disadvantages of designing primers for manual slow hail are as follows, advantages: fast, automated. Disadvantages: After the design is completed, it needs to be supplemented by manual analysis of the virtual sail. Web design directly obtains the designed primers through a specific web page.

Forward primer, reverse primer.

There should be no complementary sequences between the primer itself and the primer loss. There should be fewer than 4 consecutive complementary bases. The primer should not have a high g-value, the higher the g-value, the more stable the duplex.

Primer length and GC content should be moderate. The length of the primer is about 18 28 bp, but it should not be greater than 38 bp, too short the primer will affect the specificity of the amplification, and if it is too long, the extension temperature will be greater than 74, which is not conducive to the catalytic reaction of Taq enzyme. If the amplification product is 4 5 kb, the primer should not be less than 24 bp.

As for the design of primers, the general principles are as follows:

Sequence selection should be in the conserved segment of the gene.

Avoid the formation of 4 or more consecutive pairs between the primers themselves or with the primers, and avoid the formation of a ring-shaped hairpin structure by the primers themselves.

Typical primers are 18 to 24 nucleoside long. The primers need to be long enough to guarantee sequence uniqueness and reduce the likelihood that the sequence will be present at a non-target sequence site. But primers longer than 24 nucleosides do not imply higher specificity.

Longer sequences may hybridize to mispaired sequences, reducing specificity, and are slower than short sequence hybridization, resulting in lower yields.

The TM value is 55-65 (because 60 exonuclease activity is the highest) and the GC content is between 40% and 60%.

Avoid a TM difference of more than 2 between primers

Avoid using base A at the 3' end of the primer and 3 or more consecutive identical bases at the 3' end of the primer.

To avoid amplification, primer design is best across two exons.

TaqMan probe PCR requires fragment lengths of 80 bp to 150 bp

Primer ends (last 5 nucleotides) cannot have more than 2 g and c.

A primer is a small piece of single-stranded DNA or RNA that serves as a starting point for DNA replication and allows the DNA polymerase to attach deoxynucleotides from the 3' end of the primer. (DNA polymerase can only extend the DNA strand from the 3' end). Primers include DNA replication primers (RNA primers) from organisms found in nature and primers synthesized by polymerase chain reaction (PCR) (usually DNA primers).

Generally speaking, primers refer to DNA primers.

Primers are synthesized two synthetic compounds generally composed of 15 30 nucleotides linked by 3,5-phosphodiester bonds. One primer is complementary to one end of the DNA template strand, and the other primer is complementary to the opposite end of the other template strand.

There are some basic principles in the design of primers, such as the GC content of the primers is 40% 60%, the melting temperature is preferably close to 72 degrees, the bases should be randomly distributed, and there can be no complementary sequences between the primers, etc., in addition to special computer software, there are also special designers in special companies can undertake this work.

The easiest way is to take about 20 nt on the head and 20 nt on the tail of the sequence you are interested in. Note that the GC content is approximately the same.

For a more professional one, you can use software design, such as vector nti

Most of them are part of your purpose clip. There are many aspects that need to be paid attention to when designing, which are introduced on the Internet, but it should be noted that the 20 or so bases he said do not include the number of enzyme cleavage sites and protective bases.

1. The length of the primer is generally 15-30 bp, and the commonly used one is 18-27 bp, but it should not be greater than 38, because too long will cause its elongation temperature to be greater than 74, which is not suitable for Taq DNA polymerase reaction.

2. The primer sequence should not have high similarity in the template, especially the sequence with high similarity at the 3' end, otherwise it is easy to lead to mismatch. The presence of more than 3 consecutive bases at the 3' end of the primer, such as GGG or CCC, also increases the chance of false triggering.

3. The last base at the 3' end of the primer has a great influence on the DNA synthesis efficiency of Taq enzyme. Different last bases lead to different amplification efficiencies at the mismatched positions, and the mismatch efficiency of the last base A is significantly higher than that of the other 3 bases, so base A at the 3' end of the primer should be avoided.

Primers should be at least 3 nucleotides beyond the restriction enzyme recognition site.

Avoid dimers and self-complementation: Primers need to be designed to avoid the formation of dimers or self-complementarity between the two primers, which can affect PCR efficiency and specificity. Perform specific tests:

Use a software tool to perform specific assays to ensure that the primers selected amplify only the target DNA sequence and not other non-specific DNA.

The TM value of the template sequence corresponding to the primer should be around 72. A tm value of around 72 can make the refolding conditions optimal, at least between 55-80.

The length of the primer is generally 15-30 bp, and the commonly used one is 18-27 bp, but it should not be greater than 38, because too long will cause it to extend to the temperature of Luhezi greater than 74, which is not suitable for TaqDNA polymerase reaction.