This modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C3, C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.
NHS based modifications are post synthesis conjugation performed using a primary amino group. The yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.
~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis
Conventional and popular dyes that are derivatives of fluoroscein (FAM, HEX and TET) and Cyanine dye derivatives (Cy3, Cy5, Cy5.5, Cy7 etc) are commonly used for fluorescently labeling oligos for use as molecular probes for real time PCR, FISH analysis and fragment analysis. For most purposes these provide a good range in wavelength and other optical properties and are available as amidites for direct coupling to oligos using automated chemistry. Other fluorescent dyes are available as N-hydroxysuccinimide (NHS) for conjugation using a primary amine group linked to the oligos. A new series of Atto dyes are now available that are are designed for high sensitivity applications, including single-molecule detection.