Amplimers, kit and method for detecting PKD1 gene mutation

A kit and gene technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of cumbersome methods, too many primer sets, short read sequences, etc., and avoid pseudogene interference , simplification of experimental operation, and the effect of small GC bias

Active Publication Date: 2015-10-14
NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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Problems solved by technology

[0004] US7553644B2 also discloses a method for detecting PKD1 gene mutation, but in this method, too many primer sets are required in the process of amplifying the PKD1 gene, the method is too cumbersome, and the detection efficiency is low
CN104531883A discloses a kit and a detection method for detecting PKD1 gene mutations, which relate to a primer set containing 5 pairs of primers. In the detection method, the second-generation sequencing (NGS) technology is used for sequencing. The technology has the advantages of high throughput, rapidity, accuracy and low cost, and can realize simultaneous detection of multiple samples, multiple genes, and multiple exons. However, the NGS technology used in this method has a preference for GC content, especially PKD1 cannot be obtained. Valid data for exon 1
In addition, NGS technology has the characteristics of short read sequence. The amplified LR-PCR product must be fragmented to obtain a short sequence before building a library. In order to improve the throughput, each sample needs to build a library independently, which greatly increases the detection cost.
Due to the large length of the PKD1 gene and the limitations of the method itself, the method only detects the exons and part of the introns and introns of the PKD1 gene other than exon 1, which cannot completely cover the reported pathogenic factors. disease site; therefore the detection method and primer sequence set of CN104531883A cannot fully reflect all PKD1 gene mutations in the detected sample, and the possibility of false positive detection results and missed detection obtained by this method is relatively large
At present, there is no research on combining long-range PCR (Long-range PCR, LR-PCR) technology with PacBio SMRT sequencing technology and applying it to the genetic diagnosis of single-gene genetic diseases, let alone applying PacBioSMRT to autosomal display. Research in the Field of Genetic Diagnosis of Polycystic Kidney Disease

Method used

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  • Amplimers, kit and method for detecting PKD1 gene mutation
  • Amplimers, kit and method for detecting PKD1 gene mutation
  • Amplimers, kit and method for detecting PKD1 gene mutation

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Effect test

Embodiment 1

[0032] Genomic DNA Extraction

[0033] Collect 2ml of peripheral blood from the subject and place it in an EDTA anticoagulant tube. Take 0.2 ml of EDTA anticoagulated peripheral blood sample, and extract DNA according to the instructions of the whole blood DNA extraction kit. The DNA concentration and purity were analyzed by Qubit 2.0, and the extracted genomic DNA was ready to be used as the next PCR template. In this study, a total of 6 gene DNA samples were detected, of which 4 gene DNA samples had known pathogenic mutation sites, and 2 were normal human genomic DNA.

Embodiment 2

[0035] LR-PCR amplification

[0036] According to the PKD1 gene sequence and its homologous pseudogene sequence, 9 pairs of PKD1 gene-specific primers were designed and synthesized, and GeneAmp High Fidelity PCR System was used for LR-PCR to amplify 9 large fragment sequences, respectively 1, 2, 3, 4, 5, 6, 7, 8 and 9, the primer sequences and amplified product sizes are shown in Table 1. The GeneAmp High FidelityPCR System was used, and the reaction conditions were set to perform LR-PCR on a Veriti 96-well Thermal Cycler PCR instrument. The total volume of the PCR reaction is 50 μl, including 5 μl of 10×GeneAmp High Fidelity PCR buffer, 2 μl of 10 mmol / L dNTP, 1 μl of 10 μmol / L upstream and downstream primers, 10 μl of 5 mol / L betaine, and 2.5 μl of DMSO (for the amplification of fragment 1 Use 5 μl), 1 μl of 5U / μl polymerase mixture, 5 μl of 20 μg / μl DNA template, and make up to 50 μl with ultrapure water. PCR cycle parameters: deformation at 94°C for 3min, 94°C for 10sec,...

Embodiment 3

[0043] LR-PCR Amplification Based on Primer Index Technology

[0044] According to PacBio's Guidelines for Using PacBio Barcodes for SMRT Sequencing, asymmetric label primers were designed on the basis of the above Table 1. The label sequence was referred to the 16-base label (Barcode) sequence provided by PacBio, and the interaction between the primers was calculated. Function and analysis of the stability of the secondary structure of primer molecules, designed 6 sets of index primers containing Barcode, each set of 9 pairs of primers. Using these 6 sets of primers containing labels, the 6 DNA samples were amplified by LR-PCR respectively. For the three amplification reactions of fragments 1, 2, and 3, multi-tube amplification was used due to the low yield, and single-tube amplification was used for the others.

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Abstract

The present invention relates to a method for detecting PKD1 gene mutation. The method utilizes sequence information of the PKD1 gene, designs nine pairs of PCR tag primers, and uses a long-chain PCR technology for targeting amplification of PKD1 gene; while amplification products are labeled, PCR products are purified and mixed for direct construction of a single SMRTBell library; a PacBio RSII sequencer is employed for sequencing; and bioinformatic analysis is carried out to obtain PKD1 gene mutation information. The invention has the beneficial effects that a simplified pair of primers is designed and combined with tag primer technology to realize respective marking of PCR products of multiple DNA samples; the amplified PCR products do not need to perform knockout and can be directly used for the library construction, and the library construction does not need extra PCR, so that a plurality of samples are mixed into one library and processed simultaneously by a PacBio RSII sequencing library construction link, and the experimental operation is greatly simplified.

Description

technical field [0001] The invention belongs to medical in vitro diagnostic technology, and specifically relates to an amplification primer set and a kit for detecting the mutation of autosomal dominant polycystic kidney disease pathogenic gene PKD1 and a method for detecting PKD1 gene mutation using the primer set and kit. Background technique [0002] Adult polycystic kidney disease (polycystic kidney disease, PKD) is the most common hereditary kidney disease, the incidence rate of the population is about 1 / 400-1 / 1000, mostly autosomal dominant inheritance, a small part is autosomal recessive inheritance. Autosomal dominant polycystic kidney disease (ADPKD) is mainly manifested as multiple progressive vesicles in both kidneys, causing damage to kidney structure and function, and is the most common end-stage renal disease (ESRD) The genetic etiology of ESRD accounts for about 10% of all ESRD, and it can also affect multiple organs of the body, such as liver cysts, hypertens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2535/122
Inventor 马定远许争峰胡平
Owner NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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