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DNA Amplification for Forensic Analysts

Primer Design

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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

Primer Design Considerations



Primer Length

18-30 bases

Primer Melting Temperature (Tm)


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


No Secondary Structures

Identify primer pairs which do not assume secondary structure

No self-complementarity

< 4 contiguous bases

No complementarity to other primer(s)

< 4 contiguous bases

No long runs with the same base

< 4 contiguous bases

Distance between two primers on target sequence

< 2000 bases apart

Plateau Effect

accumulation of product ≤0.3 to 1 pmol

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