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Perhaps the most vital step in the development of a PCR method is the design of suitable primers. A PCR primer consists of two oligonucleotides that hybridize to complementary stands of the DNA template, and thus identify the region to be copied. A set of primers is used to amplify each DNA target region identified for the reaction. The following are considerations for optimal primer design. Each item will be discussed in more detail throughout this module.02-08
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Primer Design Considerations |
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|
Consideration |
Comment |
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Primer Length |
18-30 bases |
|
55°-72°C |
|
|
Primer Annealing Temperature (Ta) |
~5°C < the lowest Tm of the of primers |
|
Tm difference between forward and reverse primers |
≤ 5°C |
|
Max 3' Stability |
∆G value for five bases from 3' end |
|
Percentage GC content |
40-60% |
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No Secondary Structures |
Identify primer pairs which do not assume secondary structure |
|
No self-complementarity |
< 4 contiguous bases |
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No complementarity to other primer(s) |
< 4 contiguous bases |
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No long runs with the same base |
< 4 contiguous bases |
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Distance between two primers on target sequence |
< 2000 bases apart |
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Plateau Effect |
accumulation of product ≤0.3 to 1 pmol |
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