[Investigation of optimum concentrations of betaine for improving the resolution of sequencing G-C rich DNA with trinucleotide repeats].
OBJECTIVE
To develop an optimal sequencing system which can improve the resolution of sequencing G-C rich DNA with abundant trinucleotide repeats by applying concentration gradients of betaine to the Sanger sequencing system.


METHODS
Concentration gradients of betaine were introduced into the sequencing system by taking the 5' terminal of Nogo-B cDNA (Am-Nogo-B) (G-C%=72%, without trinucleotide repeats) and 5' terminal of Huntingtin cDNA (Am-HTT) (G-C%=74%, with abundant CAG and CCG repeats) the results of sequencing were compared.


RESULTS
The optimum concentration of betaine for sequencing Am-Nogo-B has differed from that for Am-HTT. Result of sequencing Am-Nogo-B has achieved the best quality when the concentration of betaine was at 0.8-1.2 mol/L, whereas the result of sequencing Am-HTT obtained the best quality when the concentration of betaine was at 1.6 -2.4 mol/L. The results were reproducible.


CONCLUSIONS
G-C rich DNA with similar G-C% required different concentrations of betaine in the sequencing system due to base pair compositions. The sequencing system developed for improving the resolution of sequencing of G-C rich DNA with abundant trinucleotide repeats can be used as a reference for similar studies.
State Key Laboratory of Genetic Engineering, Fudan University, Shanghai 200433, P. R. China. longyu@fudan.edu.cn.
Dan
D
Wang
Hao
H
Cai
Long
L
Yu
D001483:Base Sequence; D001622:Betaine / Q000494:pharmacology*; D000071058:Huntingtin Protein; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D018911:Trinucleotide Repeats
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D001622:Betaine