Investigation of optimum concentrations of betaine for improving the resolution of sequencing G-C rich DNA with trinucleotide repeats.
- VernacularTitle:应用甜菜碱增加富含三核苷酸重复的高G-C含量DNA片段测序辨识度的最佳浓度探讨
- Author:
Dan WANG
1
;
Hao CAI
;
Long YU
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Betaine; pharmacology; Huntingtin Protein; Molecular Sequence Data; Nerve Tissue Proteins; genetics; Sequence Analysis, DNA; methods; Trinucleotide Repeats
- From: Chinese Journal of Medical Genetics 2014;31(2):163-169
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSConcentration 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.
RESULTSThe 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.
CONCLUSIONG-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.
