| 摘要 |
#CMT# #/CMT# Determination of frame-shift mutations in coding target nucleic acids comprises: (i) providing a host cell comprising a double-stranded nucleic acid, which comprises a target nucleic acid and de-encode to a complementary nucleic acid encoding opposite strand, in which the opposite strand via a linker nucleic acid with a reporter gene in 3'-position is associated; (ii) effecting the expression of the opposite strand nucleic acid; and (iii) determining whether the host cell is an expression of the reporter gene, which indicates that the target nucleic acid has no frame-shift mutation-points. #CMT# : #/CMT# Determination of frame-shift mutations in coding target nucleic acids comprises: (i) providing a host cell comprising a double-stranded nucleic acid, which comprises a target nucleic acid and de-encode to a complementary nucleic acid encoding opposite strand, in which the opposite strand via a linker nucleic acid with a reporter gene in 3'-position is associated; (ii) effecting the expression of the opposite strand nucleic acid; and (iii) determining whether the host cell is an expression of the reporter gene, where expression of the reporter indicates that the target nucleic acid is no frame-shift mutation-points, the linker sequence comprises a translational coupler, the one stop codon in frame with the reading frame of the opposite strand nucleic acid comprises a start codon, and the reporter is in frame to the start codon. #CMT#USE : #/CMT# The method is useful for the determination of frame-shift mutations in coding target nucleic acids. #CMT#ADVANTAGE : #/CMT# The method provides improved identification of the correct reading frame. #CMT#BIOTECHNOLOGY : #/CMT# Preferred Components: The linker in the reading frame is shifted by +1 and -1 upstream of the stop codon has the translational coupler sequence located further stop codon. The distance from the other stop codon to the start codon of the translational coupler sequence is selected so that no translational coupling takes place, is at least 30 base pairs, preferably at least 50 base pairs. The start and stop codon directly connect the translational coupler sequence to each other. The start and stop codon overlap the translational coupler sequence to each other. The start and stop codons of the translational coupler sequence have a distance (preferably a distance of not more than 10 base pairs), which is chosen so that a translational coupling is made possible. The opposite strand nucleic acid remains in operative linkage with an expression control sequence is present in 5'-position. The opposite strand nucleic acid is present in the expression vector in operative linkage with a control expressions sequence in 5'-position. The nucleic acid sequence is: genomic sequences, cDNA sequences or cDNA-fragments; and a library containing a collection of sequences. The target nucleic acid sequence is a normalized cDNA library. In the method, a vector is used as an expression plasmid. The host cell is eukaryotic cell. The reporter gene contains a gene for coding detectable gene. The reporter gene comprises an antibiotic resistance such as kanamycin resistance. The detectable gene product is made of fluorescent proteins such as preferably green fluorescent protein (GFP). The detectable gene product catalyzes a measurable colorimetric reaction. Preferred Method: The step (i) comprises introducing an expression vector comprising the double-stranded nucleic acid, which includes a coding target nucleic acid and one to complementary coding opposite strand nucleic acid in a host cell, where the opposite strand nucleic acid in the expression vector is linked via a linker to a reporter gene in the 3'-position. The step (i) comprises using an expression vector, at least one selection marker gene. The selection marker gene is expressed constitutively. The opposite strand nucleic acid is optimized so that it contains no stop codons. The introduction of the expression vector is carried out by calcium phosphate co-precipitation, lipofection, electroporation, particle bombardment or viral infection. The determination in step (iii) is carried out by cytometry or fluorescence imaging assays. |
