Multi-PCR (Polymerase Chain Reaction) primer design method based on Primer 3

Abstract

The invention provides a multi-PCR (Polymerase Chain Reaction) primer design method based on Primer 3. The method comprises the following steps: S1, acquiring an original sequence of a target DNA (Deoxyribonucleic Acid) sequence; S2, performing PCR primer design on the target sequence by using Primer 3, and generating candidate primers; S3, evaluating a multi-PCR primer by using a PE (Polyethylene) model or an SE model, and screening a qualified multi-PCR primer; and S4, changing primer screening parameters, and designing and screening target DNA sequences that no primer is designed, thereby finally obtaining multi-PCR primers of all target DNA sequences. By adopting the method, target sequences in a relatively close distance can be recognized, degenerate primers can be also designed, andfurthermore non-specific amplification caused by mutual interference of primers of target sequences in a relatively short distance is reduced; the specificity of the designed primers is systematicallyevaluated, amplification failure caused by factors such as non-specific amplification, primer dimer and hairpin structures can be reduced, and multi-PCR primers with relatively high specificity are designed for multi-PCR experiments.

Description

technical field [0001] The invention relates to a multiple PCR primer design method based on Primer3. Background technique [0002] Polymerase chain reaction (polymerase chain reaction, PCR) technology, also known as in vitro gene amplification technology, is a widely used molecular biology technology. It uses DNA polymerase to synthesize a large amount of specific genes in vitro or in test tubes. technology. Its basic working principle is to use the DNA molecule to be amplified as a template, use a pair of oligonucleotide fragments that are complementary to the template as primers, and under the action of DNA polymerase, extend along the template chain according to the principle of complementary base pairing. until new DNA synthesis is complete. Repeating this process continuously can amplify the target DNA fragment. Along with the development of PCR technology, a variety of related technologies have been produced, among which multiplex PCR (Multiplex PCR) was used as ea...

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Problems solved by technology

Its most basic functions include designing PCR primers and designing hybridization probes. Although it has the advantages of free, open source, and cross-platform, it can only design primers for one target sequence at a time, and it cannot avoid single nucleotides with high frequency and multiple It cannot evaluate the specificity of primers and calculate the calculation of degenerate bases; it cannot identify target sequences with close sequences and design degenerate primers; it cannot design reasonable multiplex PCR for multiple target gene sequences Primer

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