1.The reliability of cloning-sequencing to detect the number of trinucleotide repeats
Qiaojuan ZHENG ; Shirui GAN ; Ning WANG ; Zhiying WU
Chinese Journal of Neurology 2010;43(9):659-663
Objective Cloning-sequencing is a common method to detect the number of trinucleotide repeats.The aim of the present study is to discuss its reliability.Methods One clinically diagnosed SCA1 patient was recruited in the study.The numbers of CAG repeats in ATXN1 gene were estimated via polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis (DPAGE).To verify accuracy of CAG numbers estimated, the PCR products were electrophoresed on a 2.5% agarose ge] and separated bands were excised for direct sequencing.Also, the longer separated band underwent cloning-sequencing using a TA cloning kit.Results The patient was identified as SCA1 by DPAGE.After direct sequencing, the numbers of CAG repeats were 26 and 47 in the shorter and longer bands, respectively.However, after cloning-sequencing of the longer band, there are 10 different numbers of CAG repeats, including 50, 47, 46, 41,32, 28, 27, 26, 25 and 24.Furthermore, there are other kinds of trinucleotide repeats, such as CCG, CGG, CTG, CAA and TAT scattered among the CAG repeats.Conclusions It is not reliable to identify the number of trinucleotide repeats by cloning-sequencing alone.To improve the reliability, it is better to combine cloning-sequencing with other methods.
2.Investigation of survival motor neuron gene deletion in Chinese patients with sporadic amyotrophic lateral sclerosis
Zongquan SU ; Shirui GAN ; Zhiying WU ; Wanjin CHEN ; Yan CHEN ; Ning WANG ; Shenxing MURONG ; Chuanzhen Lü
Chinese Journal of Neurology 2009;42(4):245-247
Objective To investigate the correlation between survival motor neuron (SMN) gene deletion and Chinese patients with sporadic amyotrophic lateral sclerosis (SALS).Methods A total of 141SALS patients and 134 unrelated controls were recruited from the Chinese population.Polymerase chain reaction (PCR) and restriction fragment length polymorphisro (RFLP) analysis were performed to screen SMN gene deletion.Frequencies of deletion were coropared by Chi-square test.Results Four patients and 3 controls were detected to have horoozygous SMN2 deletion.The frequencies of SMN2 deletion were 2.84%(4/141) and 2.24% (3/134), respectively, which was not significantly different (χ2= 0.0001, P =1.000).No subjects were found to have homozygous SMN1 deletion.Condusion There is no correlation between SMN gene deletion and Chinese patients with SALS.