Chemistry
The cycle sequencing chemistry currently used by Micromon is the
Applied Biosystems PRISM BigDye Terminator Mix - versions 3.1 and 1.1, both incorporating the key enzyme
AmpliTaq DNA polymerase, FS. Version 3.1 is the mix recommended for most sequencing applications and
we can supply version 3.1 directly to our customers.
While our sequence runs generate in excess of 1000 basecalls, up to 900-1000 bases are routinely called with quality values exceeding
QV=20 (error probability of 1.0% or less), the majority exceeding
QV=50 (error probability of 0.001% or less) using the BigDye v3.1 chemistry.
The quality of the basecalls or resolution, is significantly dependent on the
purity of the starting DNA template. A reduction in final resolution and signal quality can be caused by the presence of RNA, salt, protein and other contaminating chemicals. This can be eliminated by effective template purification prior to cycle sequencing and thorough reaction clean up, post-reaction. These are both key factors in the generation of long, highly resolved reads.
Many useful user documents can be downloaded from the
Applied Biosystems Genetic Analysis web site. The
AB 3730 Sequencing Chemistry Guide is an extensive resource covering all aspects of cycle sequencing and has an excellent troubleshooting section.
