5 results about "Pcr sequencing" patented technology

The invention relates to the technical field of gene engineering technology, in particular to a preparation method of an efficient and stable gene knockout cell line. According to the AKAP11 gene knockout cell line provided by the invention, the 6th exon of the AKAP11 gene in the cell line is subjected to 3395bp frameshift deletion mutation, so that a coding frame is shifted, and protein translation is terminated in advance; after continuous subculture, the homozygous knockout phenotype is still maintained through PCR sequencing verification. A CRISPR / Cas9 system is combined with puromycin screening, the positive cloning rate reaches 30% or above, the construction period is shortened to 45 days, the constructed cell line can be directly used for AKAP11 related cell signal path research, disease model construction and targeted drug screening, and a reliable tool is provided for basic research and clinical transformation.

The application discloses a kind of based on target specificity competition internal standard and concentration gradient system's super-multiplex PCR sequencing absolute quantification method and application, the method includes: synthesis N different artificial internal standard sequence;Configuration is different M group internal standard mixed solution of absolute concentration;The DNA of sample to be measured and M group internal standard mixed solution are mixed, obtain the DNA of sample to be measured internal standard mixed solution, and the DNA of sample to be measured internal standard mixed solution is carried out super-multiplex PCR amplification processing;The internal standard sequencing reads number and target sequencing reads number are counted;Internal standard sequencing reads number, target sequencing reads number and M absolute concentration are as model data set, and overall curve fitting is carried out, and the standard relationship model of target absolute concentration-sequence reads number is established;All target sequencing reads number is substituted therein, and the absolute concentration corresponding to N target gene sequences in sample to be measured is calculated.This application can realize the synchronous absolute quantification of dozens to hundreds of target genes.

The present invention provides a PCR sequencing method, which by using of the combination of primer index + DNA incomplete shearing strategy + the second sequencing technique (Pair-End sequencing technique) and taking advantage of the high-throughput and low-cost features of the second generation sequencer, makes the sequenceble length of PCR product obtained is over the sequenceing length of the sequencer, so as to extends the use of it. The present invention also provides the primer indexes for the above PCR sequencing method, and the use of the method for genotyping, particularly HLA analysis.

The invention discloses a chicken mitochondrial genome high-throughput sequencing method based on multiple PCR (Polymerase Chain Reaction). The method comprises the following steps: (1) extracting genome DNA (Deoxyribose Nucleic Acid) of a chicken to be detected; (2) carrying out PCR (Polymerase Chain Reaction) amplification through the primer set; (3) carrying out high-throughput sequencing; and (4) detecting to obtain mutation types and haplotypes. The invention provides application of the PCR primer or the multiple PCR sequencing method for the chicken mitochondrial genome to detection of different mutation types or haplotype groups.

The utility model provides PCR sequencing product purification equipment which comprises an equipment shell, a sliding cylinder is arranged on the inner side wall of the equipment shell, the output end of the sliding cylinder is in sliding connection with a sliding rail block, the bottom of the sliding rail block is in sliding connection with a sterilization assembly, the bottom of the sliding rail block is in sliding connection with a clamping assembly, and the side wall of the equipment shell is in sliding connection with a PCR plate storage drawer. According to the utility model, the PCR plate can be disinfected and sterilized in all directions through the disinfecting and sterilizing assembly, and the clamping assembly can be used for grabbing the PCR plate without contacting with the PCR plate, so that the PCR plate is moved to the upper part of the oscillation assembly, and the PCR plate can be stored in the equipment shell through the oscillation assembly. The temperature of liquid in the PCR plate for sequencing is controlled, so that the liquid is adjusted to the required reaction temperature, and the liquid is fully mixed under vibration, so that not only is the accuracy of sequencing improved, but also the risk of being polluted is remarkably reduced.