Design principles of PCR primers - Database & Sql Blog Articles

Experimental procedure

First, the primers are closely complementary to the sequence of the template, and the primers cannot be triggered at the non-target site of the template.

DNA

Polymerization

(

Mismatch

)

Avoid again forming a stable dimer or hairpin structure between the primer and the primer. Primer design should pay attention to the following points:

1

. The length of the primer is generally

15-30 bp

Commonly used

18-27 bp

.

2

. Primer sequences should have no similarity within the template, especially

3

'Sequences with higher end similarity, otherwise easily lead to mismatch. Primer

3

'end appears

3

More than one continuous base, such as

GGG

or

CCC

It will also increase the probability of errors.

3

. Primer

3

'end base pair

Taq

Enzyme

DNA

Synthetic efficiency has a large impact. Different terminal bases result in different amplification efficiencies at the mismatch position, and the last base is

A

Mismatch efficiency is significantly higher than other

3

Bases, therefore should be avoided in the primers

3

'end use base

A

.

4

. Primer sequence

GC

The content is generally

40-60%

Too high or too low is not conducive to triggering the reaction. Upstream and downstream primers

GC

The content cannot differ too much.

5

. Another important parameter of the primer is the melting temperature (

Tm

). This is when

50

% of primers and complementary sequences appear as double strands

DNA

The temperature of the molecule. Reasonable annealing temperature from

55

°C to

70

°C. For best results, the two primers should have an approximate

Tm

value.

6

. Primer dimer or hairpin structure may also result

PCR

The reaction failed. Should be avoided as much as possible.