[The influence of some factors on DNA cycle sequencing].
OBJECTIVE
To study the influence of some factors on DNA cycle sequencing.


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


RESULTS
When the DNA concentration was low, the nucleotide curve showed low ratio of signal to noise, even there were no fluorescent signal. While DNA concentration was too high, the readable length of nucleotides was short. Ions in the DNA template could result in bad sequencing reaction. The Tm value, length and G+C content of primers had no obvious influences on sequencing reaction. The alteration of the denaturing, annealing and extension temperature, or the addition of dimethl sulfoxide or glycerin facilitated sequencing some DNA templates. The fluorescent residues in the purified products of the sequence reaction could interfere the automatic reading of the sequencer, but did not influence the manually proofreading of the sequence.


CONCLUSIONS
The purity and concentration of DNA templates are closely related to sequence data quality. The modification of reaction parameters and usage of additives can help to obtain good result of sequencing some DNA with certain structure. The use of 70% ethanol is recommended to precipitate the extension product.
He
Y
Y
National Laboratory of Medical Genetics, Hunan Medical University, Changsha, Hunan, 410078 P.R.China. nlmglcy@public.cs.hn.cn.
Fang
H
H
Xia
K
K
Xia
J
J
D017422:Sequence Analysis, DNA / Q000379:methods*