[The influential factors on DNA cycle sequencing and data evaluation of practical example].

OBJECTIVE

To study some factors that may influence DNA sequencing by analyzing 2040 sequencing samples retrospectively.

METHODS

The effects of DNA templates, primers, cycle sequencing reactions as well as purification methods were comparatively analyzed.

RESULTS

70 percent of sequencing failure is caused by DNA template. Characteristics of data from poor template preparation include top-heavy with a short read length, acceptable signal with some noise peaks (Ns), weak signal, overall noise and flat lines or no signal. The ineffective primers can cause 15.4 percent sequencing failure. Possible explanations include primer mismatches and low Tm value.

CONCLUSIONS

The quality of template DNA can have a major impact on the quality of the DNA sequencing. Purification of the extension products is absolutely necessary in DNA sequencing, and isopropanol precipitation is highly recommended for this procedure.