President's DNA Initiative
-
Advancing Justice Through DNA Technology
DNA Analyst Training
previous pagenext page

Primer Design

Home > DNA Amplification > Overview > Primer Design

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

Consideration

Comment

Primer Length

18-30 bases

Primer Melting Temperature (Tm)

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%

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

< Previous Page  ::  Next Page >

© 2007 NFSTC Science Serving Justice®
NOTE TO USERS: The President’s DNA Initiative DNA Analyst Training program and assessment were completed and published in 2005, in cooperation with the National Institute of Justice. The science and techniques in the program are sound and proven, however, program content has not been updated to include tools and technologies developed and in use after 2005, including many kits and robots. Assessment questions address only content delivered in this program and may not contain the full range of tools in use in your laboratory.