Q5.  IS THERE ANYTHING WE SHOULD CONSIDER WHEN SELECTING

                          PRIMERS FOR LA PCR

The specificity of the primer is the most important consideration. Although primers in the

size range of 30 to 35-mers are preferred, those of around 20 bases may amplify more

than 20 kbp fragments if their specificity is high. If the specificity of the primer is low,

amplification will result in extraneous bands.

Effects of primer sizes on LA PCR amplification

With primers of either heterogeneous pairs of 33 mer and 36 mer or homogeneous
20-mer pairs, the same region of -phage DNA was amplified to generate sizes of both

20 kbp and 28 kbp for performance comparison.

The study results indicate that the pair of a 33-mer and a 36-mer generated better

amplification than the 20-mer pair. However, under the same reaction conditions

described below, a different-20 mer primer pair could amplify DNA fragments in length

of more than 20 kbp.

Template:

DNA 2.5 ng/50  PCR

Amplified sizes:

20 kbp and 28 kbp